9th International MeetingOncology, Immunology and Cancer Biology

December 19 and 20, 2019

Lebanese University, Main Conference Hall, Rafic Hariri University

Campus Hadath

ABSTRACT BOOK

WELCOME NOTE

The Organizing Committee of BioBeirut is delighted to welcome you to the 9th International Meeting on Oncology, Immunology, and Cancer Biology “BioBeirut 9” which will be held in Beirut at the Lebanese University on December 19 and 20 2019.

BioBeirut International Meeting is an annual event held at the Lebanese University and designed by a faculty of international renowned experts. The Meeting provides rich information in the oncology field bringing new hope to cancer patients.

The Meeting is intended for basic, translational and clinical researchers, immunologists, radiobiologists, oncology clinicians, representatives of the pharmaceutical industry and other stakeholders, gathering a multidisciplinary audience to exchange latest updates and to promote a "Knowledge Sharing Culture". The scientific program of BioBeirut attracts a broad range of national and international scientists and clinicians and bridges basic, translational and clinical research.

Biobeirut International Meeting is becoming a dedicated platform in Lebanon keeping professionals and students up to date with the most recent and significant developments in this rapidly evolving field.

This year, the multidisciplinary scientific program of BioBeirut 9 focuses on the latest developments in cancer signaling pathways, therapeutic strategies and biomarkers.BioBeirut 9 will provide a broad dialogue platform and will facilitate the development of multidisciplinary collaboration and interaction.

We look forward to welcoming you in Lebanon in 2019.

Professor Bassam BADRAN,Dean of the Faculty of SciencesLebanese UniversityBioBeirut - Meeting Coordinator

SCIENTIFIC PROGRAM

Thursday, December 19, 2019

11:00-12:00 Welcome and Registration 12:00 Opening Ceremony13:00 Cocktail

14:00-15:30 Session 1 : Immuno-OncologyModerators: Walid Rachidi, Elie Hadchity, Hussein Fayyad-Kazan

14:00-14:30 Roberto Accolla - University of Insubria, ItalyThe MHC class II transactivator: a molecule intersecting intrinsic and adaptive immunity against human retroviruses during evolution

14:30-15:00 Ahmad Awada - Jules Bordet Institute, Free University of Brussels, Belgium Cancer treatment based on Molecular Biology advances: A revolution?

15:00-15:30 Luigi Buonaguro - Istituto Nazionale Tumori IRCCS "Fondazione G. Pascale", Napoli, ItalyRoad to development of cancer vaccine for liver cancer

15:30-16:00 Coffee Break

16:00-17:30 Session 2 : Cell Therapy – Biomarkers – Inflammation Moderators: Zahraa Khamis, Nader Hussein, Kazem Zibara

16:00-16:30 Gilles Lemaitre - Université d'Évry-Val-d'Essonne, Évry, FranceCell therapy of epidermis using pluripotent stem cells and application in aging

16:30-17:00 Stefania Mondello - University of Messina, ItalyEmerging biomarkers in TBI: From pathophysiological pathways to clinical application

17:00-17:30 Ghiwa Achkar – American University of Beirut, LebanonModulation of inflammation in Liver fibrosis

Friday, December 20, 2019

09:30-11:10 Session 3 : Cancer BiologyModerators: Eva Hamade, Haidar Akl, Nada Borghol, Mahmoud Homsi

09:30-09:55 Walid Rachidi - University Grenoble-Alpes, Grenoble, FranceRNAi and Chemical-Based High Content Screening for the Normalization of XPC Phenotype

09:55-10:20 Wissam Faour, Lebanese American University, LebanonIn Vitro and In Vivo Evaluation of the Anticancer and Anti-inflammatory Activities of 2-Himachelen-7-ol isolated from Cedrus Libani.

10:20-10:45 Ziad Ibrahim - University of Leicester, Leicester, United KingdomProtein disorder and phase separation in NUT Midline Carcinoma

10:45-11:10 Hussein Fayyad-Kazan - Lebanese University, Hadat, Lebanon Human CD8+CD25+CD127low Regulatory T cells: microRNA signature and impact on TGF-β and IL-10 expression

11:10-11:40 Coffee Break

11:40-12:55 Session 4 : Infections – Neurosciences - EpigeneticModerators: Nabil El Zein, Hala Chamiyeh, Rita Azzi Nabbout

11:40-12:05 Mireille Laforge, Paris-Descartes University, Paris, France.Role of the energetic metabolism on the pathogenesis of AIDS

12:05-12:30 Ali Shaib - Max Planck Institute of Experimental Medicine, Goettingen, GermanyHuman-Induced Pluripotent Stem Cells-Derived Neurons from 22q11.2DS Patients with Autism Spectrum Disorder Show Morphogenic and Fuctional Synaptic Anomalies

12:30-12:55 Sara Fneich - Paris Sud University, Paris, France–Lebanese University, LebanonThe epigenetic mechanisms underlying the long-term effects of maternal obesity and/or maternal separation in mice

13:00-14:30 Lunch BreakPoster Session

14:30-15:40 Session 5 : Biologics and alternative pharmaceutical therapy: Perspectives and Path forward

Moderators: Rita Karam, Mazen Kurdi 14:30-15:00 Susanne Ausborn - Regulatory Policy Lead EEMEA, Switzerland

Regulation of Biologics and Biosimilars in the EU and US - Extrapolation of indications for Biosimilars

15:00-15:30 Susanne Ausborn - Regulatory Policy Lead EEMEA, SwitzerlandInterchangeability: Switching and Substitution - Post marketing Surveillance: Pharmacovigilance and Risk management plan

15:40 Meeting conclusionsYoung Researcher Award 2019

The MHC class II transactivator: a molecule intersecting intrinsic and adaptive immunity against human retroviruses during evolution

Roberto S. Accolla

Department of Medicine and Surgery, School of Medicine, University of Insubria, Varese – ITALY

Abstract Between 1983 and 1986 we discovered and described the function of the major transcriptional activator of MHC class II gene expression, encoded by the Air-1 locus, also designed CIITA. This molecule acts as a complex regulator and coordinator of transcription of the all family of MHC class II genes, although it does not bind directly to the promoter of these genes. Moreover it participates to the regulation of other gens such as the Invariant chain gene and DO and DM genes, all involved in the biology of MHC class II molecule transport, peptide loading and cell surface expression. As such, CIITA is indeed the major controller of antigen presentation and activation of adaptive immunity. In recent years our laboratory has also discovered that CIITA is a crucial molecule in restricting the replication of human retroviruses, including HIV and HTLV-1, the latter being the only known retrovirus to directly cause cancer in humans. Thus CIITA represents the first described factor that controls both adaptive and intrinsic immunity against retroviruses. In my presentation I will describe the basic mechanism through which CIITA inhibits retrovirus replication and some related new findings on the pathogenesis of HTLV-1 infection and related diseases that may unveil novel strategies of therapeutical intervention.

Biography Prof. Accolla obtained is MD and PhD degrees from the University “La Sapienza” in Rome. He was post-doctoral fellow of the University of Pennsylvania in 75-77 and then associate member at the Ludwig Institute for Cancer Research in Lausanne from 1978-1988 where he discovered the gene Air-1 whose product regulates the expression of MHC class II genes thus controlling antigen presentation and triggering of adaptive immune responses. In 1989 he became associate professor and then in 1995 full professor of General Pathology and Immunology at the University of Insubria, Varese. His major research interest is focused on the basic mechanisms of immune response against cancer cells and in the interaction between host cell and human oncogenic retroviruses.

Cancer treatment based on Molecular Biology advances: A revolution?

Ahmad Awada

Free University of Brussels, Brussels, Belgium

The revolution in technology and informatics have been translated in huge advances in the understanding of the molecular biology of cancer cell. In addition to surgery, radiotherapy and chemotherapy, molecular-targeted therapies, antibody-drugs conjugates and more recently immunotherapy emerged as very effective therapy for solid cancers with significant improvement of outcome and in particular survival.These advances will continue as technology will continue to evolve. Surgery is more and more robotic. Radiotherapy is more focus on tumor with less doses on normal tissue and toxicity. Molecular-targeted therapies will continue to be developed in more specific cancer types or specific subgroups of common tumors such as breast and prostate cancers and finally new immunotherapy approaches are emerging such as CAR-T (cellular therapy). Liquid biopsy will be of great help at all levels of tumor evolution from screening to tumor metastases and will surely help to understand the genomic evolution of cancer and consequently disease management.Finally, “artificial intelligence” which is an emerging tool will probably be at the basis of better understanding of the whole disease and will surely guide cancer management strategies.

BiographyProfessor Ahmad Awada is Head of Medical Oncology Clinic at Jules Bordet Cancer Institute Brussels, Belgium. Dr Awada studied Medicine at the Université Libre de Bruxelles. During his specialisation, he followed trainings in the clinical development of new anticancer drugs. As a research fellow, he stayed in the Netherlands (New Drug Development Office, Free University, Amsterdam) and in San Antonio, USA (Institute for Drug Development). He focused on the clinical development of new anticancer agents.

Dr. Awada became Assistant Head of Medical Oncology Clinic, and Head of the New Drugs Development Unit at Jules Bordet Institute, Brussels. Since April 2005, he is the Head of the Medical Oncology Clinic and he was held Associate Head of Medicine Department. He has an important clinical activity in the treatment of solid tumors and in particular breast cancer. Dr. Awada took an active part in the development of new drugs (cytotoxics, molecular-targeted therapies, immunotherapy), some of them already widely used.

Dr. Awada is member of several international scientific societies (ASCO, EORTC, ESMO) and Professor of Clinical Medicine at the Université Libre de Bruxelles. He published 26 book chapters and 249 articles in international publications.

Road to development of cancer vaccine for liver cancer

Luigi Buonaguro

Cancer Immunoregulation Lab, Ist. Naz. Tumori "Fond. G. Pascale", Via Mariano Semmola, 1 80131 NAPLES - ITALY

Abstract Hepatocellular carcinoma (HCC) accounts for about 6% of all new cancer cases diagnosed worldwide (nearly 750,000 new cases/year). It is the third and the fifth leading cause of death from cancer globally in men and women, respectively. The age-standardized incidence rates (ASR) of HCC in men in Europe, adjusted to the European Standard Population, is about 8 per 100,000, with a peak in Southern Europe of 10.5 per 100,000 (http://globocan.iarc.fr/).The main risk factor for the development of HCC is the hepatitis B and C virus (HBV and HCV) chronic infection; non-viral causes (e.g. alcoholism and aflatoxin) are additional risk factors. A range of therapies are used in the management of HCC according to the extent and severity of liver disease, nevertheless the overall prognosis is poor and the overall 5-year survival rate is approximately 5-6%. In the early stages, surgery (i.e., tumor resection and liver transplantation) represents the standard treatment of HCC and a 5-year survival rate is achieved in 70% of treated patients.However, the majority of patients can be treated only with non-surgical loco-regional therapies (i.e. radiofrequency [RF], thermal and non-thermal ablation, trans-arterial chemoembolization [TACE]) with extremely variable 3 to 5-year survival rates and tumor recurrence in 50 to 80% of patients at 5 years after treatment. Sorafenib is the only systemic approved therapy in advanced unresectable HCC providing a limited increase in survival of 2.3–2.8 months.In this framework, a therapeutic cancer vaccine may represent an effective strategy to cure HCC. Unlike several other cancers, only few cancer vaccine trials for HCC have been conducted so far with yet modest results, indicating that improvements in several aspects need to be implemented. In particular, identification of novel specific tumor antigens, evaluation of delivery systems and combinatorial strategies to counteract the immunosuppressive microenvironment could result in unprecedented clinical outcomes with great beneficial effect for HCC patients.

Biography Dr. Buonaguro received his medical degree in 1988 from the Univ. of Naples, Italy “Federico II”. In 1992, he received his Microbiology and Virology specialty degree from the same University. After his Post-Doctoral training at the Lab. of Tumor Cell Biology, Natl. Cancer Inst. of Health, Bethesda MD, USA, he joined the Lab. of Viral Oncology at the National Cancer Institute “Fond. Pascale”, Naples – Italy, where he currently is the Head of the Cancer Immunoregulation Unit. From 2005, he has an appointment as Adjunct Associate Professor, Univ. of Maryland School of Medicine, at Institute of Human Virology, Baltimore (Director, Robert C. Gallo).Dr. Buonaguro is Member of the Italian Society for Virology; Italian Society for Medical Virology; American Society for Microbiology; American Association for Cancer Research and CIMT. He is member of the CVC Trials Network of the Cancer Research Institute. Dr. Buonaguro is Member of several Journal Editorial Boards. Dr. Buonaguro has published, as author and co-author, more than 155 papers in International Peer-Reviewes Journals. He is the major contributor in the literature on the development and evaluation of Virus-Like Particles as vaccine approach to HIV. He is currently coordinating the FP7-funded project “Cancer Vaccine development for Hepatocellular Carcinoma – HEPAVAC” (Grant Nr. 602893) as well as the TRANSCAN2-funded project “Mutated neo-antigens in hepatocellular carcinoma - “HEPAMUT”.

Cell therapy of epidermis using pluripotent stem cells and application in aging

Gilles Lemaître

Université d'Evry Val d'Essonne (UEVE), Paris-Saclay, INSERM U861, I-Stem, Corbeil-Essonnes France.

Abstract Pluripotent stem cells are extensively used in pathological modelling, could be a good biological model for drug screening and will be the future for regenerative medicine. These cells can be differentiated into several cell types and represent a powerful tools to explore function and fate of cells and organs. Skin aging is the base of pathologies such as the appearance of cutaneous ulcers. Such ulcers need new therapeutic treatments. One way will be the used of regenerative medicine by grafting pluripotent stem cells-derived engineering skin. Beside skin regenerative medicine, we develop disease modelling of Progeria that is an extremely rare disorder affecting 1/8 000 000 births. This genetic disease is characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations of the LMNA gene, leading to the production of a mutated form of lamin A precursor called progerin. Skin is affected in this pathology and this accelerated aging could be used to modelling normal skin aging. Concerning skin ulcers, we develop a GMP protocol in order to obtain pluripotent stem cells-derived keratinocytes. These cells are able to reconstruct an in vivo fully stratified epidermis. The production of a newly tissue engineering product is ongoing in the perspective of a first in man clinical trial to heal leg ulcers of sickle cell disease patients. In skin aging modelling program, we firstly used pluripotent stem cells to investigate the hypopigmentation phenotypes observed in patients with progeria. Accordingly, iPS cell lines were derived from patient’s cells and differentiated into melanocytes. Measurements of melanin content revealed a lower synthesis of melanin in progeria melanocytes as compared to non-pathologic cells. Analysis of the melanosome maturation process by electron microscopy revealed a lower percentage of mature, fully pigmented melanosomes. Pluripotent stem cells could be used to understand skin aging and also to cure some complications due to the fragility of this organ during normal or pathological skin aging.

BiographyGilles Lemaitre has been obtained his PhD in 2004 at the Department of Cell Biology and Functional Genomics of the CEA (French atomic energy agency), based at the Evry University. He identified markers of proliferation and differentiation of human epidermal keratinocytes using methods of large-scale analysis for proteins and cellular messengers. This study leads to the characterization of molecules involved in the differentiation of keratinocytes. By a pure proteomic approach, he also identified a new membrane marker of keratinocyte progenitors. Since his arrival at I-Stem, he is responsible to participate in the development of a protocol for differentiation of human embryonic stem cells into keratinocytes. Currently, he leads the translational project of "cell therapy" within the "genodermatoses" team. He has in charge of implement a fully clinical grade protocol to obtain keratinocytes from hESC. He elaborates a catalogue of quality controls done by an external provider certified for that purpose by the ANSM (French medicinal regulatory agency). Finally, he has in charge of preclinical safety assays focus on animal experiments (teratoma - biodistribution and grafting). Based on his knowledge on epidermal biology, he paves the way to regenerative medicine of skin injuries using pluripotent stem cells.

Emerging Biomarkers in TBI: From Pathophysiological Pathways to Clinical Application

Stefania Mondello

University of Messina, Italy

AbstractRapid technical advances have enabled reliable and affordable measurement of blood-based biomarker panels. The availability of these objective, sensitive, noninvasive tool to study TBI is radically changing diagnosis and patient characterization, enhancing our understanding of disease pathophysiology and opening up new avenues to the delivery of precision medicine for TBI. During my talk, I will present an overview of the most promising candidate molecular biomarkers with advanced analytical and clinical validity, emphasizing their diagnostic and prognostic performance in clinical studies. Furthermore, I will outline the potential role of blood-based tests in drug discovery and clinical trials, and provide perspectives on the validation of such markers for use in the clinic.

Biography Dr. Mondello is physician scientist (MD, MPH, PhD) with extensive experience in clinical neurotrauma, biomarker research and statistical analysis methods, coordinating multidisciplinary laboratories and transitional neuroscience research projects as principal investigator and co-investigator. Dr. Mondello has worked at Banyan Biomarkers (Florida, USA), Inc. as Director of Clinical Research for 5 years, and currently she has an appointment at the University of Messina (Italy) as Associate Professor. Her research focuses on the use and the clinical validation of novel biochemical markers of brain injury to improve diagnostic classification, clinical decision-making and inform a rational approach to personalized interventions. The pioneering work Dr. Mondello has been leading has contributed to the development of new biomarkers being used in clinical studies worldwide and recently cleared by FDA. Her experiences in industry and academia have shaped a unique skill set combining the best of two different worlds – “flavoring the beauty of science and pursuing novel ideas and opportunities, while maintaining a practical approach and an extreme rigor, which are needed to deliver a product to the patient’s bedside.”

Modulation of inflammation in Liver fibrosis

Ghewa A. El-Achlar, Rola Al Sayegh, Duaa Hatem, Abeer Ayoub, Eva Hamade, Aida Habib

American University of Beirut, Lebanon

AbstractStatins inhibit cholesterol and isoprenoid synthesis and have many beneficial pleiotropic roles including antioxidant and anti-inflammatory effects. In the present study, we investigated the effect of statins on inflammation in vivo on the zymosan-induced air pouch model, and we assessed their impact on the regression of fibrosis in chronic liver injury induced by carbon tetrachloride (CCL4). Cells harvested from the air pouch of C57/BL6J mice treated with statins alone displayed increased expression of HO-1, arginase-1, C-type lectin domain containing 7A, and mannose receptor C-type 1. Flow cytometry analysis revealed an increase in monocyte/macrophage cells expressing HO-1 in response to statins. Mice treated with statins showed a significant reduction in cell influx in response to zymosan, and in the expression of proinflammatory cytokines and chemokines such as interleukin-1α, monocyte chemoattractant protein-1 and prostaglandin E2. The inhibitory effect of statins on cell influx and proinflammatory markers was reversed when mice were treated with the HO inhibitor tin protoporphyrin IX (SnPPIX), suggesting a protective role of HO-1. On the other hand, statins reduced inflammation and fibrosis in CCL4-induced liver injury by decreasing alpha smooth muscle actin and interleukin-6 levels. Statins also accelerated the regression of fibrosis by increasing Ly-6C anti-inflammatory. Our results demonstrate that stains decrease fibrosis and accelerates regression through modulation of inflammation. Additional experiments are required to decipher the mechanism by which statins modulate both liver regression and inflammation and further emphasize the role of macrophages.

Biography Dr. Ghewa El Achkar is a research associate at the Faculty of Medicine, department of Biochemistry and Molecular Genetics at the American University of Beirut. She received her Ph.D from Universite Paris Est-Creteil in France in co-direction with the American University of Beirut, in 2015, where she studied the modulation of heme oxygenase-1 and cyclooxygenase-2 in the anti-inflammatory properties of statins, triterpenoids and thiazole derivatives.

RNAi and Chemical-Based High Content Screening for the Normalization of XPC Phenotype

Farah Kobaisi1,2,3,4, Eric Sulpice1, Caroline Barrette3, Bassam Badran4, Xavier Gidrol1, Hussein Fayyad-Kazan4, Walid Rachidi2

1 Biomics, BGE/IRIG/CEA, Grenoble France, 2 Laboratory of Nucleic Acid Lesions, SyMMES/INAC/CEA, Grenoble France, 3 Gen & Chem, BGE/IRIG/CEA, Grenoble France, 4 Laboratory of Cancer Biology and

Molecular Immunology, Faculty of Sciences I-Lebanese University, Lebanon

AbstractSkin's exposure to UV radiation generates DNA damages, mended by Nucleotide Excision Repair (NER). Mutations in NER DNA damage recognition protein, Xeroderma Pigmentosum protein C (XPC), hinders repair and increases skin cancer incidence. The phenotype of XPC cells encompasses photosensitivity and accumulation of DNA lesions. Therefore, we aim to develop and execute screening methods to identify RNAi and chemical products reversing this phenotype.Screening procedure consists of transfecting/treating XPC fibroblasts with either RNAi Kinome targeting library or Prestwick FDA chemical drug library. The cells will then be UVB irradiated to finally assess potential phenotypic reversal. Hits are molecules inducing both increased viability post UV quantified by Presto Blue and repair of DNA lesions measured by ICC using Cell insight single cell analysis compared to sham treated irradiated controls.24 hours post UV was chosen as readout time point after time varied UV dose-response curves and repair kinetics analysis. It should be noted that the transfection was carried out for 48hrs. to allow proper knock down and the chemical treatment was performed pre and post UV to classify the effect as preventative or therapeutic to the induced damage. After the validation of the screening methods using the controls, both screens were carried out. For the siRNA screen, 10 kinases were identified whose knock down induces an increase in viability over 25% with a Zscore above 1.8. As for the chemical screen, 16 chemical compounds enabled a ≥ 25% increase in viability with RZscore above 2.6. Via DNA damage analysis, only a few molecules enabled repair. Secondary screening and mechanism of action identification are currently under study. After the ruling out of false positives and possible off-target effects the hits will be tested on 3D reconstructed XPC skin model. Products that reduce DNA damage can decrease cancer incidence in XPC patients and possibly delay its onset in normal individuals.

BiographyDr Rachidi is a Professor at Grenoble Alpes University since September 2006. In January 2016, he was appointed Vice Dean of the Faculty of Pharmacy and president of the Research Commission. After a PhD training in the Oxidative Stress Laboratory of Prof Alain Favier (1999-2002) and a post-doctoral period in the University College Dublin (2002-2003) on the relationship between oxidative stress, neurodegeneration and the therapeutic effects of new family of antioxidants, he moved to the CEA EVRY. He developed a research work on the effect of ionizing radiation on the epidermal stem cells, using classical and new genomic approaches. He was among the first researchers demonstrated the radioresistance and the enhanced DNA repair capacity in epidermal stem cells. All of these research activities have been enhanced by the publication of more than 40 articles in peer-reviewed international journals, several European and international patents, and numerous oral communications and conferences as guest speaker at national and international congresses. He has more than 30 articles and has a good experience of collaborative work within national and international contracts. He received the scientific excellence PES award from Grenoble University, the Volvic research price, the French Medical Research Foundation the SFERETE society.

In Vitro and In Vivo Evaluation of the Anticancer and Anti-inflammatory Activities of 2-Himachelen-7-ol isolated from Cedrus Libani

Wissam Faour

School of Medicine, Lebanese American University, Lebanon.

Abstract Cedrus libani is a majestic evergreen tree native to the Mediterranean mountains of Lebanon, Syria and Turkey. In this study, the tree heart wood was extracted using hexane to produce C. libani oil extract (CLOE) as a dark oil. GCMS analysis of CLOE identified up to 30 compounds whereby 2-himachalen-7-ol (7-HC) was the most abundant (40%). 7-HC was isolated using column chromatography and the identity of the white crystalline solid was confirmed via NMR spectroscopy and X-Ray Crystallography. 7-HC demonstrated potent cytotoxic activity against several human cancer cell lines including brain (SF-268, IC50 8.1 μg/mL) and colon (HT-29, IC50

10.1 μg/mL; Caco-2, IC50 9.9 μg/mL) with ovarian (Sk-OV-3, IC50 > 50 μg/mL) cells being the most resistant. However, while HT-29 displayed resistance to Cisplatin, 7-HC was 8-10 folds more potent. Co-treatment with 7-HC and Cisplatin showed a significant synergistic anti-proliferative effect against SF-268, HT-29 and Caco-2 cells. 7-HC also exhibited significant anti-inflammatory effect in formalin-induced paw edema in rats. Western blot analysis revealed that 7-HC displayed dose dependent inhibition of LPS-induced COX-2 protein expression in isolated rat monocytes. The present study demonstrates that 7-HC possesses promising anticancer and anti-inflammatory activities, and may serve as a lead molecule in cancer therapy.

Biography Dr. Wissam Faour is currently an Associate Professor of Pharmacology/Physiology and the coordinator of the Biomedical Research Laboratories at the Gilbert & Rose-Marie Chagoury School of Medicine at the Lebanese American University. He also serves on multiple School and university committees involved in research. His teaching activities involves physiology and basic and clinical pharmacology for MED1, MED2 and Nursing students. Dr. Faour finished a PhD degree in molecular biology from the University of Montreal and a postdoc in cellular and molecular medicine at the Kidney Research Center/University of Ottawa.Dr Faour’s lab focuses on the role of inflammation in various metabolic chronic illnesses (diabetic and obesity induced renal and liver damage as well as stem cell research) as well as anti-inflammatory drug screening.Dr. Faour is currently an Associate Editor for BMC Pharmacology & Toxicology and BMC Complementary & Alternative Medicine, and an editorial board member of the European Journal of Pharmacology

Protein disorder and phase separation in NUT Midline Carcinoma

Ziad Ibrahim

University of Leicester, Leicester, United Kingdom

AbstractRegulation of gene expression requires that the transcription apparatus be efficiently assembled at genomic sites. DNA binding transcription factors (TFs) typically consist of one or more DNA-binding domains (DBDs) and one or more separate activation domains (AD). While the structure and function of TF DBDs are well documented, comparatively little is understood about the structure of ADs and how these interact with co-activators to drive gene expression in physiological state and in disease. Many malignancies bear fusion-protein translocations involving portions of TFs. These abnormal gene products often fuse a DNA- or chromatin-binding domain to a wide array of partners, many of which are intrinsically disordered regions (IDRs). The BRD4-NUT rearrangement in NUT Midline Carcinoma causes malignant transformation by recruitment of a disordered domain of NUT to the chromatin binding domain of the bromodomain-containing protein 4 (BRD4). The resulting BRD4-NUT fusion is oncogenic because it interacts with the p300 histone acetyl-transferase and stimulates its acetylation activity thus initiating cycles of BRD4-NUT/p300 chromatin recruitment and creating transcriptionally inactive hyperacetylated chromatin domains. We have identified the relevant interacting domains in p300 and NUT and have determined a structure of the complex with the aim to develop a new anti-NMC therapeutic strategies. Our structure has shown that the relevant domain of NUT is intrinsically disordered and undergoes disorder-order transition upon p300 binding. Using cell-based immunofluorescence assays we have shown that the BRD4-NUT fusion oncoprotein tends to form nuclear foci after interaction with p300 by stimulating its acetylation activity. By studying how BRD4-NUT engages and dysregulates p300 we will understand at a molecular level the mode of action of this oncogene. In addition, the model we are studying describes how phase separation provides a mechanism by which such gene products result in aberrant gene expression programs.

Biography Dr. Ziad Ibrahim received his MSc and his PhD in Molecular Biology from the Joseph Fourier University (UJF) in Grenoble, France. Since 2016, he is a postdoctoral researcher in molecular and structural biology in the group of Professor Daniel Panne at the European Molecular Biology Laboratory (EMBL) and later at the department of Molecular and Cell Biology at the University of Leicester (United Kingdom).His research focuses on understanding how signaling pathways control gene expression by epigenetic modulation of chromatin structure: trying to reveal mechanistic and structural insights into how the interplay between cellular signaling, transcription factors activation and co-assembly on transcriptional enhancers controls gene expression in physiological and pathological settings.

Human CD8+CD25+CD127low Regulatory T cells: microRNA signature and impact on TGF-β and IL-10 expression.

Redouane Rouas, Makram Merimi, Mehdi Najar, Mohammad Fayyad-Kazan, Nabil El Zein, Douaa Agha, Dominique Bron, Arsene Burny, Walid Rachidi, Bassam Badran, Philippe Lewalle

and Hussein Fayyad-Kazan

ABSTRACTRegulatory T cells (Tregs) are central for maintaining immune balance and their dysfunction drives the expansion of critical immunologic disorders. During the past decade, microRNAs (miRNAs) have emerged as potent regulators of gene expression among which immune related genes and their immunomodulatory properties have been associated with different immune-based diseases. The miRNA signature of human peripheral blood (PB) CD8+CD25+CD127low

Tregs has not been described yet. We thus identified, using TaqMan Low-Density Array (TLDA) technique followed by individual quantitative real-time PCR (qRT-PCR) confirmation, fourteen miRNAs, among which twelve were downregulated whilst two were upregulated in CD8+CD25+CD127low Tregs in comparison to CD8+CD25- T cells. In a next step, microRNA Data Integration Portal (mirDIP) was used to identify potential miRNA target sites in the 3’UTR of key Treg cell-immunomodulatory genes with special focus on IL-10 and TGF-β. Having identified potential miR target sites in the 3’UTR of IL-10 (miR-27b-3p, miR-340-5p) and TGF-β (miR-330-3p), we showed following transfection, and transduction assays that overexpression of two under-expressed miRNAs, miR-27b-3p and miR-340-5p, downregulates IL-10 expression upon targeting its 3’UTR. Similarly, overexpression of miR-330-3p negatively regulates TGF-β expression. These results highlight an important impact of the CD8+ Treg mirnome on the expression of genes with significant implication in immunosuppression. These observations could help in better understanding the mechanism orchestrating Treg immunosuppressive function towards unravelling new targets for treating auto- immune pathologies and cancer.

Biography Dr. Kazan is a full time Molecular Immunology Professor at the Lebanese University - Faculty of Science. He got his Bachelor degree in Biochemistry in 2005 from the Lebanese University-Faculty of Science. Later on, Dr. Kazan continued his studies in the Free University of Brussels (ULB) where he got a Master’s Degree in Molecular Biology and Biotechnology in 2007 and then a PhD in Science in December 2010. Thereafter, He did a postdoc in the Laboratory of Experimental Hematology - Jules Bordet Institute - ULB till September 2015. His research work is focused on Cancer Biology and Molecular Immunology.

Role of the energetic metabolism on the pathogenesis of AIDS

Soultawi Cynthia1, Saleh Rachelle1, Lombes Anne2, Bouillaud Frédéric2, Estaquier Jérôme3,4, Laforge Mireille1,5

1 Unité INSERM UMR1124, T3S, Université Paris-Descartes, Paris, France. 2 Institut Cochin, Unité U1016, INSERM, Université Paris-Descartes, Paris, France. 3 Unité INSERM UMR1124, Team 7, Université Paris-Descartes, Paris, France. 4 Centre de Recherche du CHU de Québec, Université Laval, Québec, QC, Canada. 5 Unité INSERM UMR1124, T3S, Université Paris-

Descartes, Paris, France,

Abstract Mitochondria are organelles essential to the life and the death of eukaryotic cells. They play a major role in the production of energy metabolism, providing ATP through different metabolism pathways such as glycolysis or oxidative phosphorylation. The majority of mitochondrial functions are closely related to its dynamics and morphogenesis and multiple researches point out that bioenergetics can impact and regulate immune response. As a result, disturbances and changes in mitochondrial morphogenesis impacting its bioenergetics could lead to defects and inefficiency of the immune response and even the collapse of the immune system during an infection such as the Human Immunodeficiency Virus (HIV) or the Simian Immunodeficiency Virus (SIV).The lab's work focuses on understanding the impact of mitochondrial dynamics and morphogenesis on the energy metabolism of CD4 T cells during AIDS. A defect in the morphology of the mitochondria can impact the energetic signature of a lymphocyte, which differs according to lymphocyte subpopulations. Naïve CD4 T and memory produce their energy mainly through oxidative phosphorylation and regulatory or effector T CD4 produce their energy through glycolysis. Reprogramming this energy signature through disruption of the oxidative phosphorylation/glycolysis balance may disrupt the function of a T cell and impact the quality of the immune response. AIDS is characterized by a loss of CD4 T lymphocytes following infection, but especially a poor quality of the immune response and a state of chronic activation of the immune system leading to its exhaustion. Understanding this energy reprogramming at the level of the different sub-populations of CD4 T cells will allow a better understanding of the poor quality of the immune response as well as the failure of the immune system during AIDS. Our recent results show that CD4 T cells isolated from SIV infected monkeys have a defect in oxidative phosphorylation compare to healthy monkeys. These cells do not consume oxygen because their respiratory chain which is the platform of oxidative phosphorylation is in major defect, forcing the cell to produce its energy through glycolytic fermentation. These energetic defects signature make the CD4 T unable to respond to stimulation or activation.

Biography Dr Laforge obtained her Master 1 in 1999 in Cellular Biology from the Faculty of Sciences of the Lebanese University and her PhD in 2006 in Immunology from Paris-Sud University with the highest honour distinction. In 2014, she obtained her HDR from Paris-Descartes University, and in 2016, Dr. Laforge becomes tenured Researcher at the CNRS. From January 2019, she is a principle investigator and recently a team leader of a group of research in Immunometabolism in infection diseases and inflammation at the University of Paris.Her research focused on infectious diseases and immunology, and especially on the elucidation of the mechanisms of programmed cell death and the role of lysosomes and mitochondria in the pathogenesis of HIV. She discovered and developed a new treatment against AIDS. She published more than 27 original papers, reviews and book chapters in the fields of Immunology, cancer and infectious diseases, and collaborates with several national and international researchers. She is author of two patents relating to preventing and treatment of viral infections, and have two awards of excellence. She is a member of the scientific Council of the UFR Biomedical of Paris-Descartes University, member of the council of the Inserm laboratory U1124, member of several Ph.D and HDR committee and an Expert member of HIV pathogenesis for the French national agency for research against Aids and Hepatitis (ANRS).

Human-Induced Pluripotent Stem Cells-Derived Neurons from 22q11.2DS Patients with Autism Spectrum Disorder Show Morphogenic and Fuctional Synaptic Anomalies.

Ali Shaib1, Hong Jun Rhee1, Chungku Lee1, Natalia Garcia1, Michael Peitz2,3, Oliver Brüstle2, Nils Brose1, and Jeong Seop Rhee1

1Max Planck Institute for Experimental Medicine, Department of Molecular Neurobiology, Göttingen, Germany 2Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty, Bonn, Germany 3Cell Programming Unit, German Center for Neurodegenerative Diseases (DZNE) and University of Bonn

Medical Center, Bonn, Germany

AbstractThe cerebral cortex almost constitutes three quarters of the mammalian central nervous system. It serves as the executive center in which functions such as perception, memory, association and sensation are integrated and processed allowing mammals to react accordingly. So disorders in cerebral cortex neuronal connectivity lead to mental disorders including autism spectrum disorder (ASD). ASD has been heavily studied over the course of half a century in murine neuronal models. As these models carry limitations, emerging alternative strategy involves switching to hIPSC cortical-derived neurons, which would provide a better and closer model to what actually happens within human cerebral cortex. We focused on three patients with 22q11.2 DiGeorge syndrome caused by the deletion of a small segment in chromosome 22. The patients had varying psychological disorders including intellectual disability, major depression, prodromal psychotic symptoms and ASD. Through small molecules, we classically derived hiPSC-cortical neurons from tissue samples from the patients in comparison to C62m healthy proband control; we cultured the neurons using our previously characterized autptatic culture system. Cortical neurons of autistic patients displayed varying phenotypes including anomalies in cell ID with increased proportion of inhibitory GABAergic neurons, neurite complexity, synaptogenesis and enhanced synaptic transmission and response rate. Our study provides first insights at investigating ASD in a human autaptic model system. A system that helps in understanding the basic electrical synaptic integrative mechanisms essential for the broader complex cognitive functions.

BiographyDr. Shaib has completed his masters in immunology at the Lebanese University with Prof. Bassam Badran and in 2012 he was awarded a PhD scholarship from the Lebanese Prime Minister which was turned down for PhD position in Prof. Jens Rettig lab in Homburg. Ali investigated synaptic transmission in dorsal root ganglion neurons during his PhD. In 2016, he got a Postdoc position at the Max-Planck Institute for Experimental Medicine in Gottingen. He is currently working together with Prof. Rhee and Prof. Brose and colleagues on establishing a human model to investigate Autism using electrophysiological and sub-resolution methods.

The epigenetic mechanisms underlying the long-term effects of maternal obesity and/or maternal separation in mice

Sara Fneich1,5, Lin Xia2, Cyrille Delpierre3, Claudine Junien1, Marlène Dufresne4, Jérôme Torrisani4, Muriel Darnaudéry2 & Anne Gabory1

1 UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France. 2 INRA, UMR 1286 Nutrition et Neurobiologie Intégrée, Université de Bordeaux, Bordeaux, F-33000, France. 3 INSERM,

UMR1027, Université Toulouse III Paul-Sabatier, F-31000 Toulouse, France. 4 INSERM, U1037, Cancer Research Center of Toulouse, France. 5 Laboratory of Cancer Biology and Molecular immunology, Faculty

of Sciences I, Lebanese University, Hadath, Beirut, Lebanon.

Abstract It is well-known that early life exposures to biological and/or social stressors may have a long-term impact on adult health. However, the biological mechanisms underlying the developmental origins of health and disease (DOHaD) are not fully understood. Epigenetic mechanisms are good candidates to explain how early events are memorized and induce a phenotype later in life. Various studies have thus shown in mammals that poor nutritional status of parents and low maternal care have an impact on the methylation of genes involved in physiology and in the hypothalamic-pituitary adrenal (HPA) axis in the offspring1,2,3. But the consequences of multiple adversities on epigenetic processes remain to be explored. In this context, our aim was to explore the epigenetic basis (DNA methylation) of maternal obesity combined with maternal separation stress in mice. We study the impact of a combination of maternal obesity and maternal separation on offspring’s physiology, behaviour (emotionality and motivation) and epigenome in C3H mice. Our results indicate that maternal obesity and maternal stress exacerbated anxiety-like behaviour in adult offspring. We hypothesised that epigenetic marks could be one of the molecular bases responsible for the embodiment of early exposure to stress. Currently, we use large-scale transcriptomic (RNA-seq) and DNA methylation (MeDIP-seq) analyses on adult offspring’s liver and nucleus accumbens, which are key organs for energy metabolism and food motivation. Preliminary results show that early life exposure to maternal obesity and stress affect metabolic pathways in liver at transcriptomic and epigenetic levels. Interestingly, we also reported a marked sexual dimorphism in the epigenetic effects of maternal obesity and stress. Our future work will focus on the heritability of these epigenetic marks across generations and their influence on observed phenotype.

Biography Dr. Sara FNEICH, PhD in Epigenetics, joined the Lebanese university as assistant professor in 2017 after spending two postdoc positions in France and UK. She obtained her PhD in 2014 at Perpignan, France after working on “the implication of epigenetic modifications in the phenotypic variants in a host/parasite model”. The first postdoc was between Paris, Bordeaux, and Cambridge where she worked on “the epigenetic mechanisms underlying the long-term effects of maternal stress in mice”. During the second postdoc at Perpignan, she developed the bioinformatics tools to analyse transcriptomic and epigenetic data. Her current research efforts focus on the study of epigenetic modifications in a breast cancer model.

Regulation of Biologics and Biosimilars in the EU and US - Extrapolation of indications for Biosimilars

Interchangeability: Switching and Substitution - Post marketing Surveillance: Pharmacovigilance and Risk management plan

Susanne Ausborn

Head International Regulatory Policy, F. Hoffmann – La Roche Ltd, Basel, Switzerland

AbstractThe WHO regulatory framework for Biotherapeutics covers the priniciples for development of recombinant-DNA products, development of similar biotherapeutic products (biosimilars) as well how to handle post-approval changes for products already on the market and can be considered as global standard for these type of products. Meanwhile many countries have implemented the WHO biosimilar guideline. Following global standards the “Totality of Evidence” has to be provided in order to bring biosimilars to the patient which are safe, efficacious and of high quality. Insights will be provided into the regulatory submission process for biosimilars in the US and Europe where biosimilars (as well as biotherapeutics in general) are approved under the centralized procedure.The global regulatory landscape regarding extrapolation of indications and interchangeability and the industry position on these topics will be presented.Proper Pharmacovigilance systems combined with a clear traceability of products need to be in place in each country in order to ensure patient safety.

BiographySusanne has almost 20 years of experience in technical regulatoryaffairs. After working in a small biotech company in the US Susannejoined Roche in 2001. There she held different positions with increased responsibilities and gained extensive experience with global filings of new drug submissions, clinical trial applications as well as post-approval changes. Knowing the challenges of operating globally she is now a strong advocate for global convergence of regulatory requirements and has been engaged in many international conferences, workshops and meetings with regulators from various emerging markets over the last years.Susanne is the vice chair of the EFPIA International Regulatory Expert Group, chair of the EFPIA Russia regulatory network, member of the Middle East regulatory network and the IFPMA expert group on post-approval changes. Susanne holds a M.Sci. in Analytical Chemistry from the HumboldtUniversity in Berlin, Germany and a PhD in Biophysical Chemistry from theBiozentrum of the University of Basel, Switzerland.

POSTER PRESENTATIONS

Interplay between snR190 small nucleolar RNA and RNA helicase Dbp7 in the compaction of the large ribosomal subunit RNA in yeast

Mariam Jaafar1,2, Julia Contreras3, Jesus de la Cruz3, Yves Henry1, Raghida Abou Merhi2 and Anthony K. Henras1

1 Laboratoire de Biologie Moléculaire Eucaryote, CBI, Université de Toulouse, CNRS, UPS, 31000 France, 2 Université Libanaise, campus Rafic Hariri, Faculté des Sciences, Département de Biologie, Hadath, 3

Departamento de Genética, Universidad de Sevilla, Seville, Spain.

Ribosome production begins with the synthesis by RNA polymerase I of a primary transcript, the pre-rRNA, that associates with ribosomal proteins and ribosome biogenesis factors (RBFs) to generate ribosome precursor particles. These particles undergo a complex maturation process to generate the final 40S and 60S ribosomal subunits. A large set of trans-acting factors chaperone this process, including small nucleolar ribonucleoprotein particles (snoRNPs) and RNA helicases. Box C/D snoRNPs are a large family of particles involved in the 2’-O-ribose methylation of rRNA nucleotides. Their snoRNA components base-pair with the pre-rRNA at the sites of modification thanks to complementarity regions called the antisense elements and thereby positions the methyl-transferase fibrillarin for the methylation reaction. Box C/D snoRNA snR190 has long been predicted as a methylation guide although the target methylation has never been detected. We showed recently that this snoRNA interacts with the Npa1p complex suggested to play a key role in the compaction of the central RNA core of the 25S rRNA within early pre-60S particles. We identified two antisense elements within the sequence of snR190 that have the potential to base pair with two distant domains of the 25S rRNA, suggesting that snR190 may cooperate with the Npa1 complex to chaperone the compaction of the 25S rRNA in early pre-60S particles. Using the CRISPR-Cas9 technique, we knocked out snR190 snoRNA from yeast cells and showed that snR190 is required for normal growth and efficient maturation of pre-60S particles. Interestingly, snR190 displays genetic interactions with the RNA helicase Dbp7 also involved in maturation of pre-60S particles, but whose precise molecular function remains unknown. Dbp7 is not essential in yeast but its depletion induces a strong growth defect. We show that Dbp7 knockout leads to an aberrant retention of snR190 into pre-60S particles. In addition, we found that mutation of the region of the 25S rRNA where snR190 base-pairs alleviates the growth defect caused by the absence of Dbp7. In the same line, snR190 knockout in cells lacking Dbp7 restores close to normal growth. These data suggest that snR190 knockout or mutations that weaken its interaction with the pre-rRNA allow to partially bypass Dbp7 function. We propose that one function of Dbp7 helicase is to unwind the base pairing between snR190 and the 25S rRNA within the earliest pre-60S particles.

Key words: Ribosome biogenesis, snoRNA chaperones, RNA Helicases.

G-quadruplexes in the Social Amoeba «Dictyostelium discoideum»

Mona SAAD1,2, Aurore GUEDIN2, Samir AMRANE2, Nicolas TOURASSE2 , Jean GUILLON2, Laurent LACROIX3, Jean-Louis MERGNY2 , Hussein FAYYAD-KAZAN1

1 Laboratory of Cancer Biology and Molecular Immunology, Faculty of Sciences I, Lebanese University, Beirut, Al-Hadath, Lebanon. 2 ARNA Laboratory, Inserm U1212, CNRS UMR 5320, Univ. Bordeaux,

IECB, F-33600, Pessac, France. 3 Inserm U1024, CNRS UMR 8197, IBENS, Paris, France.

G-quadruplexes (G4) are fascinating non-canonical DNA/RNA secondary structures that occur in genomic Guanine-rich regions. The over-representation of such structures in specific regions such as promoters of oncogenes and telomeres, suggests their involvement in key processes such as transcription, replication or RNA maturation. Putative G4 forming sequences (PQS) have been reported in Homo sapiens, yeast, bacteria, viruses and many others. However, one of the problems in studying G4 structures in the human genome is indeed the high number of putative G4 forming sequences (370,000 PQS according to Quadparser and over 1 million when using a threshold of 1.5 with G4Hunter). It is therefore difficult to deconvolute G4-related biological effects in human cells. For this, we chose Dictyostelium discoideum - a G4 poor genome - as a eukaryotic model to complement the human studies. By an in silico analysis of dicty genome with G4Hunter a home-made algorithm, we detected 249 (threshold=2) to 1055 (threshold=1.5) G4-prone motifs. Interestingly, despite an even lower GC content in comparison to the whole dicty genome, the density of G4 motifs in dicty promoters is significantly higher than in the rest of the genome. By using a combination of different biophysical and biochemical methods, we demonstrated that 14 dicty sequences located in key genes fold into stable G4 structures. In addition, five dicty genes containing G4-prone motifs in their promoters were studied for the effect of a new Porphyrin derivative on their expression. Our results demonstrated that the new ligand decreased the expression of the several dicty genes significantly. Overall, our results constitute the first step to adopt Dictyostelium discoideum as a model for G4 studies.

Keywords: G-quadruplex, Dictyostelium discoideum, G4Hunter, Genome analysis, G-quartet, gene expression.

The anti-cancer effect of a set of thiazole and benzothiazole derivatives on breast cancer cells

Khalife H1, Faraht D2,3,4,5, Fayyad-Kazan M1, Khalaf A6, Hussein N1,Liagre B7, Badran B1

1Laboratory of Cancer Biology and Molecular Immunology, Faculty of Sciences-I, Lebanese University, Hadath, Beirut, Lebanon. 2 Université Lyon 1, Lyon, France. 3 Inserm U1052, Centre de Recherche en

Cancérologie de Lyon (CRCL), Lyon, France. 4 CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon (CRCL), Lyon, France. 5 Department of Chemistry-Biochemistry, Laboratory of Cancer Biology and

Molecular 6 Immunology, EDST-PRASE, Lebanese University, Faculty of Sciences, Hadath- Beirut, Lebanon. Laboratory for Medicinal Chemistry and Natural Products, Faculty of Sciences (1), and PRASE-

EDST , Lebanese University , Hadath , Lebanon. Laboratoire PEIRENE EA 7500, Faculté de Pharmacie, Université de Limoges 2, Rue du Docteur Raymond Marcland, 87025 Limoges Cedex, France.

Breast cancer, the most commonly diagnosed malignancy in women, accounts for the highest cancer-related deaths worldwide. Triple negative breast cancer (TNBC), lacking the expression of oestrogen, progesterone and HER2 receptors, has an aggressive clinical phenotype and is susceptible to chemotherapy but not to hormonal or targeted immunotherapies. In an attempt to identify potent and selective anti-TNBC agents, a set of 15 thiazole and benzothiazole derivatives were screened for their cytotoxic activity against MDA-MB 231 cells, a TNBC cell line. Cell viability and proliferation were measured using MTT assay and IncuCyte-ZOOM, respectively. Interestingly, some of the tested compounds, which are compounds 1, 5, and 12, showed a cytotoxic effect with IC50 less than the one observed in case of cisplatin, a commonly used anti-cancer agent. Notably, and differently from cisplatin, the effect of these compounds was selective against MDA-MB 231 cells and did not harm MCF-10A cells, normal breast cells. Regarding the IncuCyte-ZOOM technique, the three selected compounds showed a significant decrease in cell proliferation in both MDA-MB-231 cells and MCF-7 (ER positive breast cancer cells). In the future steps, cell apoptosis will be studied using flow cytometry. Besides, the molecular mechanisms underlying this cytotoxic effect will be studied using Q-RTPCR and western blot technique. Altogether, the output of this project would provide better insights into novel therapeutic strategies against breast cancer.

Targeting Soluble CD146 in Glioblastoma: A New Step for Personalized Therapy

Ahmad Joshkon1, Wael Traboulsi1, Jimmy Stalin1, Richard Bachelier1, Aurelie Leroyer1, Francoise Dignat-George1, Nathalie Bardin1, Hussein Fayad-Kazan2 and Marcel Blot-Chabaud1

1 INSERM UMR-1263, Aix-Marseille University, UFR Pharmacy, Marseille, France2 Lebanese University, Faculty of Science, Hadath, Lebanon

Glioblastoma is a highly aggressive and malignant grade IV tumor that originates from astrocytes in the CNS. Despite the multifaceted therapeutic interventions, including chemo, radio, and immuno-therapies, it remains the most resistive and lethal tumor among primary brain tumors. Currently, Avastin “anti-VEGF mab” is the most common therapy prescribed for newly diagnosed patients. However, glioma tumor cells have developed manifold mechanisms to counteract the anti-angiogenic effect of this drug.U87 and U373 cells were grown in serum reduced DMEM for 48hrs. Soluble CD146 concentration in the media was measured by ELISA. Recombinant human sCD146 was used at a final concentration of 100ng/ml in all experiments. Cell Proliferation was assessed by WST-1 reagent. Mice were ectopically injected with U87 and tumor volume was monitored by calliper. Under serum starving condition, glioma cell lines, U87 and U373, shed the membrane isoform of MCAM (CD146) to produce soluble CD146, which directly induce their proliferation, migration, invasion, and metastasis in-vitro, and indirectly provide an alternative way for vascularization in response to anti-angiogenic therapies. We have also shown that the use of our patent fully humanized anti-sCD146 (H2L3) was able to reverse these effects both in-vitro and in-vivo in nude mice xenografted with U87. For the first time, we have demonstrated that sCD146 is secreted by Glioma U87 and U373 cell lines which plays a major role in resistant to current chemotherapies. Moreover, targeting sCD146 with H2L3 not only protects from the aforementioned effects, but also shows an additive therapeutic effect to Avastin, thus constituting a key breakthrough in Glioblastoma therapy.

Exosomal CD147 plays major roles in paracrine signaling in rhabdomyosarcoma

Farah Ramadan1, Assil Fahs1,2, Farah Ghamloush3, Bassam Badran4, Nader Hussein4, Raya Saab2,3, Sandra Ghayad1

1 Department of Biology, Faculty of Science II, Lebanese University, Fanar, Lebanon. 2 Department of Anatomy, Cell Biology and Physiology, American University of Beirut, Beirut, Lebanon. 3

Department of Pediatric and Adolescent Medicine, American University of Beirut, Beirut, Lebanon. 4 Laboratory of Cancer Biology Stem Cells and Molecular Immunology, Faculty of Science I, EDST,

Lebanese University, Hadath, Lebanon

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue sarcoma with two major histologic subtypes, the less clinically aggressive embryonal (ERMS) subtype and the alveolar (ARMS) subtype which may harbor the PAX3/7-FOXO1 translocation contributing to metastatic behavior. Exosomes are extracellular nanovesicles whose cargo influences changes within recipient cells and have been implicated in tumor progression. In a previous study, our team demonstrated the expression of 80 common proteins including CD147, a protein known to promote angiogenesis, extracellular matrix remodeling and metastatic behavior in various cancer types, within RMS-derived exosomes. We hypothesize that CD147 within these exosomes influences recipient cell behavior favoring RMS growth and metastasis. We transduced ERMS cells with shRNA targeting CD147 expression and isolated their secreted exosomes which exhibited efficient CD147 knockdown. We then treated recipient human fibroblasts with the isolated exosomes and found that CD147 inhibition was able to decrease the proliferation of recipient cells. We further assessed the effect on fibroblast migration and invasion in vitro by transwell migration and invasion assays and revealed a decrease in the migratory and invasive properties of the recipient cells. Invasion was also decreased in vivo as demonstrated by matrigel plug in assay. Our findings demonstrate that CD147 within RMS-derived exosomes influences the tumor microenvironment by enhancing recipient fibroblast growth and motility. This paves the way for the detection of CD147 within RMS-derived exosomes as a biomarker and potential therapeutic target for RMS treatment.

GNL3: a new protein involved in the protection of stalled replication forks

Rana Lebdy1,2, Julie Patouillard1, Soumaiah Abou Staiteieh2 , Marion Larroque3, Christian Larroque4, Hala Muhtasib5, Angelos Constantinou1, Raghida Abou Merhi2 and Cyril Ribeyre1

1 Institut de Génétique Humaine, Montpellier 2 Laboratoire de Génomique et Santé, Faculté de Science 1, Université Libanaise, Liban 3 Institut du cancer de Montpellier 4 Institut de Recherche en Cancérologie,

Montpellier 5 Department of Biology, American University of Beirut, Liban

During DNA replication a plethora of proteins is involved in protecting and maintaining the accuracy of this process against any obstacle that might perturb the progression of replication fork. The high proliferative capacity of cancer cells is accompanied with high levels of replicative stress which is a weakness exploited by chemotherapeutic agents. Although chemotherapies are efficient in most cases, some cancers might develop resistance against them. Therefore there is a need to identify new proteins functioning at the replication fork that may have a role in determining the response to these drugs. Using the iPOND (isolation of proteins on nascent DNA) technique coupled to mass spectrometry we uncovered new proteins associated with replication forks. We used a secondary screen to identify the best candidates and selected GNL3 (aka nucleostemin) for further analysis.GNL3 is involved in the maintenance of genomic integrity in stem cells but its precise role(s) in this process is poorly understood. We demonstrated that GNL3 is associated with ongoing replication forks using iPOND, Proximity Ligation Assay (PLA) and BioID. The depletion of GNL3 does not impair replication forks progression in basal conditions or in response to short treatments with camptothecin. However, prolonged treatment with hydroxyurea (HU), etoposide or camptothecin elevates the phosphorylation of RPA suggesting an increase in the frequency of collapsed forks. In concurrence with this, prolonged HU treatment resulted in a nucleases dependent DNA resection in absence of GNL3. On the other hand depletion of GNL3 in basal conditions changes the S-phase pattern and leads to an increase of chromatin bound CDC45. This suggests that GNL3 has a role in controlling replication origins firing and that increased resection in response to HU is an indirect effect of excessive origin firing. We propose that GNL3 is associated with the replisome to limit excessive origins firing in order to maintain the integrity of replicating DNA.

Keywords: DNA Damage Response, DNA Replication, Chemotherapy, iPOND, Mass Spectrometry

Cell death and signalization during erythroleukemia cells megakaryocytic differentiation

Dima DIAB1, Bertrand LIAGRE1, Rouba HAGE-SLEIMAN2, Mona DIAB-ASSAF2, David Yannick LEGER1

1 PEIRENE Laboratory EA 7500, Faculty of Pharmacy, 2 Dr Marcland Street, 87025 Limoges, France 2

Molecular Tumorigenesis and Anticancer Pharmacology, EDST, Lebanese University, Hadath, Lebanon

Differentiation therapy is one of two main strategies to stop or slow the proliferation of leukemia cells. It aims to reactivate the processes of hematopoietic differentiation that have been stopped during leukemic transformation, using chemical or natural differentiating agents. In this context, our laboratory succeeded to induce megakaryocytic differentiation of human erythroleukemia cells (HEL) after treatment by a natural steroid compound called diosgenin1. This compound induces megakaryocytic differentiation in HEL cells, which is characterized by morphological changes in cells, the induction of polyploidization, the expression of differentiation markers such as GpV and the fragmentation of differentiated cells thus releasing elements close to platelets1. Many teams, including our laboratory, have shown that apoptotic cell death is involved in megakaryocyte fragmentation and platelet release2-4. Nevertheless, there is currently very little data on the involvement of another death / cell survival pathway, called autophagy, during megakaryocytic differentiation5,6. We first showed that the autophagy pathway was activated early, starting after 48h treatment, during megakaryocytic differentiation of HEL cells and was characterized by increased expression of key autophagy actors such as beclin-1 , Atg proteins as well as the conversion of LC3-I to LC3-II. Relying on these results, we decided to modulate autophagy by using an inhibitor, 3-MA (3-Methyl Adenine) and an activator, Metformin. In order to target critical points during the differentiation process, we modulated autophagy before or after the induction of megakaryocytic differentiation. Our results show that the modulation of autophagy affects megakaryocytic differentiation but also that the timing of this modulation is as important.If autophagy is inhibited (3-MA), the megakaryocytic differentiation (polyploidization and GpV expression) after diosgenin treatment is decrease. However, the effect is much more pronounced if the inhibition occurs after the induction of differentiation (48h) than if the inhibitor is used before the induction of differentiation. On the other hand, if autophagy is activated (Metformin), the megakaryocytic differentiation after diosgenin treatment is increase. Apoptosis is a programmed cell death known for its importance in different type of leukemia differentiation7. In 2006, Leger et al demonstrated the involvement of this pathway during the HEL induced maturation and the pseudo-platelet release1. Moreover, a temporary increase in caspase 3 activity was reported at 48h of differentiation1. Basing on those previous data, we decided to detect the interconnection between apoptosis and autophagy during megakaryocytic differentiation. Surprisingly, the modulation of autophagy, whatever it is, increases apoptosis. Future work will aim to identify the molecular actors involved in megakaryocytic differentiation and which link apoptotic and autophagic processes.

Key words: Differentiation therapy, leukemia cells, megakaryocytic differentiation, autophagy apoptosis,

Effect of vitamin D deficiency and supplementation on abdominal aortic aneurysm pathogenesis

Afaf JREIJE1,2 Myrna MEDLEJ-HASHIM1, Lara KHOUZAMI1, Nassim FARES2

1 Cellular and Molecular Physiopathologies (CAMP) Laboratory, Faculty of Sciences II, Lebanese University, Fanar, BP 90656, Jdeidet El Metn, Lebanon

2 Laboratoire de Recherche en Physiologie et Physiopathologie (LRPP), Faculty of Medicine, Saint-Joseph University of Beirut, Damas Street, BP 115076, Beirut, Lebanon

Abdominal aortic aneurysm (AAA) is a permanent and irreversible localized dilatation of the aorta associated with alterations of the connective tissue in the aortic wall. It is typically asymptomatic until the catastrophic event of rupture and death. Vitamin D “25(OH) D” is mainly involved in calcium and phosphorus regulation, and bone mineralization. Recently, low vitamin D concentrations has been associated with the presence of AAA in older men. The present study aims to assess the molecular mechanisms underlying the effect of vitamin D deficiency and its supplementation on the pathogenesis of AAA in mice. BALB/c adult male mice were divided into four groups. G1 for normal mice, G2 for mice fed with ordinary diet and subject to AAA by angiotensin II and anti-TGFβ, G3 for mice with vitamin D deficiency with AAA, and G4 for mice with vitamin D supplementation and AAA. Blood pressure and echocardiography were monitored weekly until the sacrifice after seven weeks. Positive correlation was found between the severity of AAA and vitamin D deficiency. The ejection fraction and fraction of shortening were significantly lower in G2 and G3 compared to the other groups. In addition, TNFα and IL-1β plasma concentrations were elevated in G2, G3 and G4. However, IL-10 plasma concentration was significantly increased in G4 only. These preliminary results may suggest an anti-inflammatory effect of vitamin D in the pathogenesis of AAA.

Keywords: Abdominal aortic aneurysm; Vitamin D; Angiotensin II; TGFβ; Inflammation.

Fungal-mediated Biocatalysis of Bioactive Steroids, Exemestane and Desogestrel

Iman Ibrahim1,2,3, Atia-tul-Wahab 5, El Hassan Ajandouz 2, Akram Hijazi 3, Elias Baydoun1 and M. Iqbal Choudhary 4,5

1Department of biology, American University of Beirut, Beirut 1107 2020, Lebanon.2 Aix Marseille University, CNRS, Centrale Marseille, iSm2, Marseille, France

3 Doctoral School of Science and Technology, Research Platform for Environmental Science (PRASE), Lebanese University, Lebanon 4 H. E. J. Research Institute of Chemistry, International Center for

Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. 5Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences,

University of Karachi, Karachi-75270, Pakistan.

Biotransformation is one of the most important approaches for the stereo-, regio-, and chemo-selective/specific synthesis of almost all classes of organic compounds without applying protection, deprotection and functional group activation steps. This technique has been efficiently employed in green chemistry, i.e. discovery and development of pharmaceuticals, avoiding the use of expensive and toxic reagents and catalysts, and extreme reaction conditions, i.e. temperature, pressure and pH. During the current study fungal-mediated biotransformation of aromatase inhibitor, exemestane with Cunninghamella elegans yielded five known metabolites which showed potent inhibition against placental microsomes and pure aromatase in comparison to the substrate, and a biotransformation of contraceptive drug, desogestrel with C. blakesleeana yielded three new and one known metabolites which showed potent activity against Staphylococcus aureus EMRSA-17, S. aureus NCTC 13277 (MRSA-252), and S. aureus NCTC 13143, and clinically isolated Pakistani strain of S. aureus in an in vitro Microplate Alamar Blue Assay (MABA).

Keywords : Biotransformation; fungus; steroid

Epidémiologie et transmission du parasite entérique Blastocystis

KHALED Salma a,b, El SAFADI Dima a , VISCOGLIOSO Eric b, DABBOUSSI Fouad a

a Université Libanaise, Laboratoire Microbiologie Santé et Environnement (LMSE), Faculté de la Santé Publique, EDST, Tripoli, Liban b Université de Lille, Institut Pasteur de Lille, CIIL, UMR CNRS 8204, Inserm U1019, Lille, France

Blastocystis sp. est actuellement le protiste intestinal le plus fréquemment retrouvé dans les selles humaines et il est considéré comme un parasite émergent avec une distribution mondiale. A cause de son impact sur la santé publique, nous renforçons l'image de la prévalence et la distribution moléculaire des sous-types de Blastocystis sp. en Afrique en réalisant une enquête sur une large cohorte humaine au Sénégal. Des échantillons de selles de 620 écoliers asymptomatiques vivant dans 8 villages localisés dans deux régions (Saint-Louis et lac de Guiers) ont été testés pour la présence de Blastocystis sp. par PCR en temps réelle en utilisant des amorces ciblant le gène SSU rDNA. 78.7% avec 117 infections mixtes par deux ou trois sous-types différents. Parmi les 371 infections simples, ST2 représente la moitié des sous-types détectés (51.5%) suivis des ST1 (26.4%), ST3 (20.8%), ST10 (0.5%), ST14 (0.5%) et ST7 (0.3%). Les sources potentielles d’infection par Blastocystis sp. y compris la transmission interhumaine et transmission zoonotique sont également discutées. La prévalence de Blastocystis sp. dans notre population sénégalaise est élevée et assez proche de celle de 100% obtenue auparavant au Sénégal. Les sous-types détectés sont 1, 2, 3, avec une très faible proportion d’isolats des sous-types 7, 10 et 14 indiquant probablement des cas de transmission zoonotique. Cette enquête fournit aux autorités sanitaires locales des informations précieuses pour mettre en place des mesures urgentes de prévention et de contrôle.

Mots clés : Blastocystis, épidémiologie, sous-type, transmission zoonotique, Sénégal.

Breastfeeding practices among Lebanese and Syrian Refugee mothers in Akkar -North of Lebanon- and its role in cancer prevention

Sara Daher1, Moemen Baroudi1, Farah Naja2, Fouad Ziadeh1, Lara Nasreddine2

1Faculty of Health Sciences, Lebanese University 2FacultyAgricultural and Food Sciences, American University of Beirut

Breast cancer is a major cause of cancer deaths among women. Breastfeeding reduces women's risk of breast cancer. Women that have a history of breastfeeding have been shown to have reduced rates of breast cancer. Since exclusive breastfeeding has a stronger hormonal effect, it could theoretically result in a greater reduction in breast cancer risk than any breastfeeding mode. We conducted a cross-sectional study that examined the prevalence of breastfeeding and exclusive breastfeeding among a representative sample of 750 Lebanese and Syrian refugee mothers in Akkar –North of Lebanon. A multi-component questionnaire was used to assess socio-demographic data, breastfeeding practices, and cancer history among mothers.Preliminary analysis of 240 questionnaires showed that 92.6% of mothers reported having ever breastfed their child while only 28.1% and 19.2% reported exclusively breastfeeding for up to 4 and 6 months respectively. In addition, 54.2% of mothers reported breastfeeding up to 12 months, and only 16.2% up to 24 months of age. On the other hand, 11.8% of mothers had a family history of breast cancer, with only 0.5% of mothers have had breast cancer but healed. However, only 95.1% of mothers had ever had a mammography.Despite low prevalence of breast cancer in this sample, there appear to be a need to increase awareness of mothers to increase exclusive breastfeeding till 6 months of baby’s age and increase breastfeeding till 24 –or at least 12 months in light of its role in breast cancer prevention. Also, there appear to be a need to encourage mothers to participate in yearly mammography in order to ensure early detection and treatment of breast cancer.

Keywords: Breastfeeding; Breast cancer; Lebanon; Akkar; Mothers; infants.

Formulation of bio-insecticides: application to α-pinene and camphor

Zahraa Hammoud1,2, Souha Haydar1, Jouda Mediouni-Ben-Jemâa3, Abdelhamid Elaissari2, Hélène Greige-Gerges1

1Bioactive Molecules Research Laboratory, Doctoral School of Sciences and Technologies, Faculty of Sciences, Section II, Lebanese University, Lebanon 2Univ Lyon, University Claude Bernard Lyon-1, CNRS, LAGEP-UMR 5007, F-69622 Lyon, France 3Laboratory of Biotechnology applied to agriculture, University

of Carthage, Tunisia

The essential oils α-pinene and camphor are reported as bio-insecticides. However, their applications to the agriculture and food sectors are limited due to their volatility, low aqueous solubility, and sensitivity to light and oxygen. In this study, conventional liposomes and drug-in-cyclodextrin-in-liposomes (DCLs) were evaluated as encapsulating materials for these natural insecticides. Hydroxypropyl-β-cyclodextrin/drug (HP-β-CD/drug) inclusion complexes were prepared in aqueous solution, and the optimal HP-ß-CD concentration was determined. The conventional liposomes and DCLs were prepared by the ethanol-injection method using phospholipon 90H in combination with cholesterol. The size of vesicles, the phospholipid and cholesterol incorporation rates, the drug encapsulation efficiency, and the drug loading rate were determined. The addition of HP-β-CD enhanced the solubility of α-pinene and camphor; the optimal solubilization occurred at HP-β-CD concentration of 50 and 75 mM for camphor and α-pinene, respectively. On the other hand, for the various liposomal batches, two distinct populations (nanometric and micrometric vesicles) were obtained, and the incorporation of both drugs did not influence the size distribution of blank liposomes. Nevertheless, HP-β-CD addition promoted the formation of larger vesicles compared to the blank batches. In addition, HP-β-CD entrapment into vesicles decreased the phospholipids and cholesterol incorporation rates into liposomes. Compared to conventional liposomes, DCLs improved the loading rate of camphor; however, they did not ameliorate that of α-pinene.

Increase of para-coumaric acid solubility via dendrimer-guest complex formation while maintaining its antioxidant activity

Sanaa Daakoura, Gihane Nasra, Hélène Greige-Gergesa

Bioactive Molecules Research Laboratory, Faculty of Sciences II, Lebanese University, Lebanon

Para-coumaric acid (p-CA), a hydroxyl derivative of cinnamic acid, is an antioxidant known for its therapeutic properties. However, the poor water solubility of this drug limits its absorption and bioavailability. Dendrimers, a new class of polymers, possess great potential for drug solubility improvement by virtue of their unique properties. They are hyperbranched polymer with well-defined chemical structures and a high density of functionalities on the surface. In the present work, we aim to improve the solubility of p-CA in the presence of Poly-amidoamine generation 4 (PAMAM G4) dendrimer and to study the interaction between them. Therefore, p-CA was incubated with PAMAM in Tris-HCl buffer at pH 7. The complex was characterized by various techniques. The infrared spectra confirmed that the complexation relies on the interaction between the carboxyl group of p-CA and the terminal primary amine group of PAMAM. 13C NMR and 1H NMR studies showed downfield and highfield shifts of protons adjacent to amino groups and to carboxyl groups, respectively. This suggests that electrostatic interactions contribute to p-CA/PAMAM complex formation. RP-HPLC analysis showed that the solubility of p-CA have risen with dendrimer concentration and increased three folds in presence of 100 μM of PAMAM at pH 7. In addition, the in vitro release study performed by dialysis method showed that p-CA release from drug-dendrimer complex was slightly delayed compared to pure p-CA (58.83% compared to 71.72% respectively within 60 min). The antioxidant activity of p-CA was highly maintained in presence of PAMAM dendrimers as evaluated by the decrease of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical absorbance in the UV-Visible. Our data prove that PAMAM dendrimer increases p-CA solubility due to the formation of water-soluble complexes by favorable electrostatic interactions while maintaining p-CA anti-oxidant activity.

Keywords: para-coumaric acid; poly-amidoamine dendrimer; complex; solubility; antioxidant activity

Study of the effect of cyclodextrins (β-CD, HP-β-CD and RAMEB) and cyclodextrins /Thymol inclusion complexes on the stability of liposome membranes

Ghenwa Nasr1,2, Hélène Greige1, Sophie Fourmentin3, Abdelhamid Elaissari2, Nathalie Khreich1*

1Bioactive Molecules Research Laboratory, Faculty of Sciences, Lebanese University, Lebanon.2Laboratoire d’Automatique et de Génie des Procédés (LAGEP), Université Claude Bernard, Lyon 1,

France.3Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV, EA 4492), SFR Condorcet FR

CNRS 3417, ULCO, F-59140 Dunkerque, France

Cyclodextrins (CDs) are cone-shaped cyclic oligosaccharides. Thanks to their hydrophobic central cavity, CDs are able to form inclusion complexes with various lipophilic drugs [1]. CDs can extract lipid membrane components and affect the physicochemical properties of the membrane; their effect depends on the membrane composition, CDs type and concentration [2]. The aim of our study was to test the effect of free CDs and CDs/Thymol inclusion complexes on the permeability of liposomal membranes. Three CDs were included, the native β-CD, its randomly methylated derivative RAMEB and its hydroxy-propylated derivative HP-β-CD. Therefore, CD/Thymol inclusion complexes were prepared after determining the concentration of CDs ensuring maximal inclusion rate of thymol by HPLC. Liposomes encapsulating a fluorophore, sulforhodamine B (SRB), were formed by the reverse evaporation phase from a mixture of DPPC:Chol at a molar ratio of (1:1) and liposomal suspensions were treated with either free CDs, thymol or CD/Thymol inclusion complexes at CD:DPPC molar ratio of (100:1). Membrane permeability was assessed by following the release kinetics of SRB from liposomes at 37°C during 24h by fluorescence spectroscopy. Results showed that free β-CD (24.78%) and free RAMEB (25.60%) exerted a membrane permeabilizing effect whereas free HP-β-CD had no significant effect (5.48%). The complexation of β-CD and RAMEB with thymol reduced their permeabilizing effect since SRB release rates from CDs-treated liposomes were higher than those of CD/Thymol-treated liposomes at 24h (14.26% and 19.77% respectively). However, HP-β-CD/Thymol complexes induced a slightly increased effect (8.95%) compared to free HP-β-CD; yet, this effect was significantly lower than that of free β-CD and free RAMEB. Furthermore, the inclusion of thymol in CDs cavities seems to have a protective effect on the membrane since thymol inclusion markedly reduced the permeabilizing effect of free thymol (22.62%). These results participate in the comprehension of CDs as drug delivery systems.

Keywords: cyclodextrins, liposomes, inclusion complex, membrane permeability, fluorescence

Stability of Cucurbitacins E and B in Gastro-intestinal Fluid

Pamela Waked, Helene Greige-Gerges, Suzanne Abbas

Department of Chemistry and Bio-chemistry, Laboratory of Bioactive molecules, Faculty of Sciences, Lebanese University, Fanar, Lebanon.

Cucurbitacins are tetracyclic triterpene natural compound found in Cucurbitaceae, which their pharmacological properties have been studied for decades and Cucurbitacin B (Cuc B) is known to have potent biological activity. However, the pharmacokinetic profile of this compound is limited and the bioavailability and absorption of Cucurbitacin B is low. This study aimed to investigate the stability of Cucurbitacin B in gastro-intestinal (GI) tract. A High Performance Liquid Chromatography (HPLC) method was developed and used for quantitative analysis of Cucurbitacin B, I and D. A sample of cucurbitacin B is incubated with Stimulated Gastric (SGF) and Stimlated intestinal Fluid (SIF), centrifuged and aliquot of supernatant is analysed by HPLC. The stability of Cucurbitacin B was found to be low in SGF, with 17% remaining Cuc B after 4 days. While the stability of Cuc B in SIF was better, reaching a lowest percentage of cuc B of 79% in 4 days. In addition, Cucurbitacin D (CucD) molecule was formed as Cuc B was being hydrolysed with rate of formation being highest in the first few hours of incubation in SGF. However, minimum Cuc D was seen formed in SIF. Therefore, it was found that Cucurbitacin B is less stable in stomach than the intestines, potentially affecting its absorption. In addition, Cuc B was chemically hydrolysed and formed Cuc D molecule. The loss in Cuc B stability was observed as lower Cuc B concentration found at each progressive time point and increase in Cuc D formed. However, the rate of decrease in remaining Cuc B was higher than the rate of increase in Cuc D formation after 2 days of incubation, suggesting that Cuc B might be hydrolysed to different metabolite(s) or that chemical degradation of Cuc B is taking place. The rate of hydrolysis was found to be rapid in SGF and is dependent on pH of solutions (SGF and SIF).

Keyword: Cucurbitacin B; Cucurbitacin D; Stability; HPLC; Gastrointestinal tract; Simulated gastric fluid; Simulated intestinal fluid.

Anti-obesity Effect of Ethanolic Extract from Micromeria barbata in 3T3-L1 Adipocytes

NAFEH Bariaa1,2, ZREIKA Sami 3,4, ALWAN Thaer4 EL OM9AR Fawaz1, EID Assaad2#, KASSEM Jinane1,3.

1 Applied Biotechnology and Cell Culture Laboratory, Doctoral School for Sciences and Technology, Lebanese University, Tripoli, Lebanon.

2 American University of Beirut, faculty of medicine, Department of Anatomy, cell Biology, and Physiological Sciences, Beirut, Lebanon.

3 Lebanese University, faculty of Sciences III, Department of Biology, Tripoli, Lebanon.4 Faculty of Sciences, Jinan University, Tripoli, Lebanon.

Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from natural products such as oriental medicine attack grow attention. Micromeria barbata, member of Lamiaceae family, highly rich in flavonoids and polyphenolic acids., reported to have in vitro anti-oxidant, anti-microbial, anti-bacterial and recently anti-tubercular activities. Despite the known role of these compounds in lipid metabolism, the role of Micromeria barbata in alleviating obesity have not been reported previously. The present work aimed to investigate the effect of ethanolic extract of Micromeria barbata on the inhibition of adipogenesis and lipogenesis in 3T3-L1 pre-adipocytes. The extract suppresses reactive oxygen species (ROS), the differentiation of pre-adipocytes and triglycerides accumulation using DCF-DA and Oil red Oil staining respectively. After confirmation that the extract is not cytotoxic to these cells up to 100 μg/ml by using (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenultetrazolium bromide) MTT and trpan blue assay, the effect of ethanolic extract on the expression of various adipogenesis related proteins was analyzed via western blot. We demonstrate that the treatment inhibited adipocyte differentiation dose dependently by suppressing adipogenic proteins including peroxisome proliferation activated receptor (PPAR γ), P-AKT (S473) and by stimulating the anti-adipogenic proteins such as P-AMPK. In addition, the extract treatment significantly increased glycerol level and adiponectin secretion in adipocytes matures using glycerol assay kit and ELISA respectively. Taken together, our findings indicate that Micromeria ethanolic extract is the most effective candidate for preventing obesity and related metabolic disorders.

Keywords: Micromeria barbata, ethanolic extract, 3T3-L1 cell, cytotoxicity, adipogenesis, lipolysis, adiponectin, western, obesity.

Antiproliferative and Antioxidant Activities of Sulfated Polysaccharides, Fucoxanthin and Crude Extracts of Macroalgae harvested from the Lebanese Coast against Colon Cancer

Batoul Al SHARIF1, Ziad RIZK2, Chantal GHANEM2, Mona DIAB1, Mona TANNOURY*1

1. Lebanese University, Faculty of Sciences; 2. Lebanese Agricultural Research Institute (LARI)

Although the wide diversity of algae, many intersect at the biological activities that their contents hold. The main concern to researchers is to exploit these potentials in updating treatments for cancers, like human colorectal carcinoma. For this, we investigated different extracts from two algae harvested from the Lebanese coast: Cystoseira compressa L. (brown alga) and Ulva rigida L. (green alga). In accordance, we performed the extraction to obtain crude extracts from each alga, fucoxanthin (carotenoid) from brown alga C. compressa and sulfated polysaccharides (fucoidan from brown alga C. compressa and ulvan from green alga U. rigida). Then, cell viability of colorectal cancer cells (HCT 116 cell line) was tested by MTT and Trypan Blue Assay after their treatment with algae extracts. Moreover, antioxidant activity was studied by DPPH assay.For the two algae, as the concentration of crude extract, fucoxanthin and fucoidan increases with an increase of time of treatment from 24 to 72 hours, the percentage cell viability of HCT 116 significantly decreases reflecting the efficiency of the extracts. At concentration 1000 µg/ml and time 72 hours, the percentages cell viability of HCT 116 treated by sulfated polysaccharides (42.17% for fucoidan’s C. compressa and 41.73% for Ulvan’s U. rigida), which are greaters than that treated by fucoxanthin’s C. compressa (34.52%) or by crude extracts (25.3% and 34.48% respectively for C. compressa and U. rigida). Similarely, all extracts showed free radical scavenging activities especially for C. compressa extracts.Our study showed that Cystoseira compressa L. and Ulva rigida L can be used as natural medicaments for anticancer (colon cancer HCT 116 cell line) and antioxidant. Further study is needed to identify the nature of their bioactive molecules in the crude extracts.

Keywords: Algae; Cystoseira compressa L.; Ulva rigida L.; Colon cancer; HCT 116 Cell line; MTT test; Trypan Blue test; Antioxidant; DPPH

Targeting HSP90 in neuroblastoma cancer and investigating cancer pathways

Hanna R1,2, Abou-Antoun T1, Khazen G3, and Abdallah J1

1 School of Pharmacy, Lebanese American University, Byblos, Lebanon2 Faculty of Sciences, Lebanese University, Fanar, Lebanon

3 School of Arts and Sciences, Lebanese American University, Byblos, Lebanon

Neuroblastoma is a pediatric solid tumor that commonly arises in early childhood. It originates from the neural crest elements, leading to an extra-cranial tumor in the adrenal glands and/or sympathetic ganglia. MYCN amplification has been observed in around 20% of cases and correlates with a highly malignant, treatment-evasive form of the tumor with very poor prognosis. The aim of our study is to determine if the chemical inhibition of the tumorigenic molecule HSP90, a chaperone that contributes to tumor growth, using the drug 17-AAG, would have a therapeutic effect on the malignant MYCN-amplified human IMR-32 cell line. Our data showed that targeting HSP90 led to anti-cancer activities, including the inhibition of cell proliferation, viability and migration, the induction of apoptosis and the crucial reduction of sphere-formation capacity. More interestingly, it caused differential expression of other tumorigenic proteins including p-L1CAM, prohibitin (PHB) and MYCN which were upregulated and L1CAM, FABP5, Oct4 and HMGA1 which were downregulated. In conclusion, our data indicate that HSP90 inhibition should be considered as a potential effective treatment in neuroblastoma cases. Finally, our findings point to the important networking between these malignant pathways that if targeted with combinatorial therapy may be important in eradicating the malignant, high-risk form of this deadly childhood cancer.

Medical Potential of Sulfated Polysaccharides, Fucoxanthin and Crude extracts of Macroalgae harvested from the Lebanese Coast against Lung Cancer

Sara SAAD1, Ziad RIZK2, Chantal GHANEM2, Mona DIAB1, Mona TANNOURY*1

1. Lebanese University, Faculty of Sciences; 2. Lebanese Agricultural Research Institute (LARI)

Despite the improvement of the treatment choice for lung cancer, mortality rates remains alarmingly high. The present chemotherapy regimens appear to have reached a therapeutic plateau leading to decline in patient’s condition after treatment. Consequently, there are investigations to identify novel chemotherapeutic agents with high cytotoxicity and minimum side effects. Seaweeds contain a wide spectrum of bioactive molecules. Pharmacologically, the interest in natural remedies has increased due to minimal side effects and high efficiency. Many studies showed that algae exhibit anticancer activity on different cell lines and antioxidant activities. Hence, in our study, we tested different extracts from two algae harvested on the Lebanese Coast (Berbara, Jbeil): a brown seaweed Padina pavonica L. and a green algae Ulva compressa L: sulfated polysaccharides (fucoidan from P. pavonica and ulvan from U. compressa), carotenoid (fucoxanthin of Padina pavonica) and crude extracts for the two algae (Padina and Ulva). The evaluation of the anticancer potential was done on adenocarcinoma lung cancer A549 cell line, treated by the extracts, using MTT assay and Trypan blue test. For the antioxidant activity, we used the DPPH test. Our results proved that the crude extract, fucoxanthin and fucoidan of P. pavonica L. have interesting values of IC50 56.87, 51.73 and 241.33µg/ml respectively. Moreover, the crude extract and ulvan of U. compressa L. showed interesting values, as well, of IC 50 68.95 and 274.72µg/ml, respectively. Although the antioxidant activity of both crude extracts of the brown alga P. pavonica and the green alga U. compressa showed the highest free radicals scavenging percentages, the fucoidan, fucoxanthin and ulvan had moderate antioxidant activity too.

Keywords: Padina pavonica L., Ulva compressa L., Lung Cancer - A 549 cell line; Antiproliferative activity; MTT Test; Trypan Blue Assay; Antioxidant DPPH test; Sulfated polysaccharides; Fucoxanthin; Crude extract.

Effect of Leucetamine B Analogues on Alzheimer Zebrafish Induced Model

Riad Nassour, Samar Bou Zeid1, Laurene Aoun1, Samar Eid1*, Fadia Najjar1*, Aline Hamade1*

Lebanese University, Faculty of sciences II, Laboratory of therapeutic innovation (L.I.T), Fanar, Lebanon

Alzheimer’s disease continues to be a global issue with around 37 million patients worldwide, and a rise in incidence taking place in recent years. Despite research efforts, a cure has yet to be discovered, and affected people only have access to symptomatic treatment that comes with multiple side effects and drug interactions. It has been established that Amyloid Beta plaques and phosphorylated Tau neurofibrillary tangles are involved in AD pathophysiology, with a large number of potential contributing factors. Research has revealed that disturbance in pre m-RNA alternative splicing mechanisms are at play in the pathogenesis of AD, linking it to specific groups of protein kinases such as DYRKs and CLKs. Thus, research into discovering modulators of these factors was encouraged. Consequently, the discovery of the modulatory effects of Leucetamine B and its analogues on the aforementioned kinases has promoted the synthesis of more analogues for fundamental research purposes and for drug discovery revolving around AD.With the research requirements comes the need to establish practical animal models of the disease, which explains the emergence of the zebrafish model of AD as a simple, cost-effective and efficient research model, as the neuroanatomical and neurochemical pathways of the zebrafish brain are very similar to those in humans. Moreover, given the role of aluminium neurotoxicity and its implication in cases of human AD, aluminuim salts have been deployed in AD induction in the zebrafish model. In this study, we aim to establish a quick and cost-effective model of Alzheimer disease in zebrafish using aluminium salts, more specifically, ones that we are chronically exposed to as humans in our daily life. In addition, we aim to test the effects of a novel Leucetamine B analogue on normal zebrafish and its effect on the zebrafish AD model as a pre-treatment drug, as well as its effect as a treatment after AD induction. Our hope is to establish the first step in studying SZ46, which will pave the way for further research into this substance, with the optimal target of advancing our knowledge about AD mechanisms, and finding better medications to improve AD patients’ quality of life.

Allergies, genetic polymorphisms of Th2 interleukins and childhood acute lymphoid leukaemia: the ESTELLE study

CHANDAB Ghinaj¹ ², AJROUCHE Roula¹*, CLAVEL Jacqueline², BONAVENTURE Audrey²

¹ Faculty of Pharmacy, Lebanese University, Hadath, Lebanon

² INSERM, Paris-Descartes University, Sorbonne-Paris-Cité University, CRESS U1153, EPICEA-Epidémiologie des cancers de l’enfant et de l’adolescent, Villejuif, France

A negative association between a history of allergy and childhood acute leukemia (cAL) has been reported in previous studies, but it remains debated. This work aimed to investigate the association between the risk of cAL and medical history of allergy accounting for genetic polymorphisms of the Th2 pathway cytokines (IL4, IL10, IL13 and IL4-receptor (IL4R)) suspected to modify this association. Analyses were based on the French population-based case-control study ESTELLE (2010-2011). The complete sample included 635 cases and 1,421 controls frequency-matched on age and gender. Child’s medical history was collected through maternal standardized telephone interview. Biological samples were collected and genotyped data was available for 487 cases and 706 controls of European origin. Odds ratios (OR) were estimated using unconditional regression models adjusted for potential confounders. In the complete sample, significant inverse associations were observed between cAL and reported history of allergies possibly IgE-mediated (OR=0.78 [0.63-0.97]) and history of allergic rhinitis (OR=0.65 [0.44-0.96]) and. Furthermore, gene-environment interaction analyses showed an interaction between IL4R-rs1801275 polymorphism and the history of allergy (IOR=0.54; p interaction=0.02), especially asthma (IOR=0.27; pinteraction=0.01), allergic rhinitis (IOR=0.35; pinteraction=0.03) and eczema (IOR= 0.50; pinteraction=0.06). We also observed a borderline interaction in children with a history of atopic dermatitis and carriers of a variant haplotype of IL10 gene. No interaction was observed with the candidate polymorphisms in IL13. This study provides arguments in favor of the presence of an inverse association between cAL and history of allergy, which may be more marked in children with certain genetic polymorphisms.

Study on the Combined Effects of Curcumin and Ionizing Radiation on Bladder Cancer Cell Lines

Anthony Waked †, Joyce Azzi §, Aline Hamade †, Fadia Najjar †, Larry Bodgi §, Mira Maalouf †

† Department of Chemistry and Bio-chemistry, Laboratoire d’Innovation Thérapeutique, Faculty of Sciences, Lebanese University, Fanar, Lebanon § Department of Radiation Oncology, American

University of Beirut Medical Center, Beirut, Lebanon

Bladder cancer (BC) is the sixth most commonly occurring cancer in men, with 550,000 new cases in 2018. Lebanon is the country with the highest rate of BC worldwide. The disease’s treatment is increasingly shifting toward strategies that aim to preserve the bladder, usually involving radiotherapy (RT) supported by radiosensitizing chemotherapy. This thesis aimed to study the radiosensitizing effect of curcumin on two bladder cancer cell lines, UM-UC5 and UM-UC6 by characterizing the effects of combining X-ray radiation and curcumin on the viability, reproductive capacity, migration ability, and stemness potency of those cells. The impact of curcumin on radiation-induced DNA damage recognition and repair was also explored. Cell survival curves revealed UM-UC6 cells to be more radiosensitive than UM-UC5. Viability and colony-forming assays showed that combining ionizing radiation (IR) with curcumin resulted in a significant decrease in cell viability and reproductive capacity of both cell lines, an effect stronger than that observed with IR or curcumin alone. An immunofluorescence assay showed that curcumin did not affect the kinetics of pATM and γH2AX proteins, but resulted in increased DNA double strand breaks found 24 hours after IR in UM-UC6 cells. This effect was not observed in UM-UC5 cells, confirming their radioresistance. Additionally, a wound healing assay performed on UM-UC6 cells and a sphere-formation assay on UM-UC5 cells revealed that the combination treatment significantly reduced cell migration and the stemness potency of cells, respectively, more than curcumin or IR alone did. In conclusion, these results present curcumin as a promising natural radiosensitizing agent to be used as an adjuvant to BC radiotherapy, encouraging further research in this area.

Keywords: Bladder cancer; Radiotherapy; Curcumin; DNA damage; Radiosensitivity; Cytotoxicity

Synthesis and evaluation of the anti-proliferative activity of Vanillin/hyaluronic acid conjugates

Christer Abou Anny, Mira Maalouf*, and Maher Abla*

Laboratoire de l’Innovation Thérapeutique (LIT), Lebanese University, Faculty of sciences II, Fanar, Jdeidet el Matn, Lebanon

Vanillin, like other phenolic compounds, is known for its therapeutic activities, for instance anti-cancer and anti-oxidant. However, once in the body, several obstacles prevent them from carrying out their therapeutic activity. Among these obstacles, one can cite: a low solubility in an aqueous medium, and a lack of affinity to the target cells. In order to limit these obstacles; we proceeded with the grafting of vanillin moieties on the hyaluronic acid (HA) forming HA-Va conjugates. HA is a biocompatible, biodegradable and hydrophilic biopolymer. It is widely used as tumor-targeted drug delivery system because of its high interactions with CD44 receptors that are overexpressed on tumor cells. The conjugates were prepared following Steglich esterification then purified by dialysis. Once lyophilized, HA-Va structures were confirmed by FTIR and NMR. In addition, EDX spectroscopy confirmed the absence of any metal contamination. The amount of vanillin grafted on the HA was estimated by UV spectrophotometry. The size and shape of the HA-Va conjugates were determined by granulometry and SEM and were correlated to the amount of vanillin attached to the biopolymer. These experiments showed that increasing the amount of vanillin; (i) improved the aqueous solubility of HA-Va; (ii) increased the size of their assemblies in water; (iii) and converted the shape of these latter from globular to lamellar. The evaluation of the cytotoxic effects of HA-Va conjugates on MCF-7 cancer cells showed very promising results compared to those obtained with vanillin. In conclusion, the grafting of phenolic compounds such as vanillin on HA promotes their anti-proliferative effect and eliminates the known pitfalls of their use mentioned above.

Anti-tumor effect of mir002, a potential inhibitor of DNA polymerase alpha, in Primary Effusion Lymphoma cells

Rady Chaaban1, Louna Karam1, Soumaiah Abou Staitieh1, Rana Lebdy1,2, Cyril Ribeyre2, Claudio Pisano3, Nadine Darwiche4, Raghida Abou Merhi1

1: Laboratoire de Surveillance Génomique et Biothérapie (GSBT), Université Libanaise, Faculté des Sciences I, Hadath 2: Institut de Génétique Humaine, Montpellier 3: Biogem Research Institute, Ariano

Irpino, Italy 4: Department of Biochemistry and Molecular Genetics, American University of Beirut, Beirut

Primary Effusion Lymphoma (PEL) is a rare and aggressive large B cell neoplasm presenting as serious lymphomatous effusions and known to be associated Human Herpes Virus 8 (HHV-8) infection. The use of retinoids as anti-tumor agents is being widely studied ever since natural retinoids such as ATRA were FDA approved for the treatment of solid malignancies. In PEL, the use of the synthetic retinoid ST1926, showed a potential anti-tumor and therapeutic activity, yet associated with several drawbacks. For that, a novel synthetic analog belonging to the class of Retinoid Related Molecules (RRMs), was suggested and called “mir002”.In this study, we report for the first time, the anti-tumor effect of mir002 on PEL cells and ascites that were retrieved from NOD/SCID mice representing PEL as a model. Mir002, at sub-micromolar concentrations, displayed potent and irreversible antiproliferative effects on PEL cells, and in BC1 and BC3 ascites. Furthermore, treatment with mir002 resulted in cell cycle arrest, early DNA damage assessed by the phosphorylation of ATM and ATR pathways and their downstream regulators in BC1 and BC3 ascites, which were prompted to programmed cell death observed via PARP cleavage, p53 and p21 upregulation and in the modification of expression of other proteins involved in the apoptotic pathway such as Bax and Bcl2. Moreover, we showed that mir002 induced remarkable stall in the replication fork progression assessed by DNA fibers technique and resulted in a downregulation of the POLA1, which is responsible of the initiation of replication. Our ongoing work shows the localization of specific proteins and helicases involved in fork progression, post treatment with mir002. Finally, we suggested that mir002 stimulated HHV-8 specific latent-lytic switch after modulation of the early lytic RTA transcript. Taken together, our results suggest that mir002 may be considered as a potential synthetic retinoid compound implicated in the treatment of PEL.

Key words: PEL, Mir002, DNA damage, Apoptosis, Replication fork, POLA1.

Impact of Liver X Receptors knock out on myelin sheath physical and structural characteristics

T. El Jalkh1,2, P. Khoueiry1,3, A.Eid4, G. Azzi1, F. Harb1, S. Bernard2.

1. Department of Life and Earth Sciences, Faculty of Sciences, Lebanese University, Fanar, Lebanon.

2. Faculty of Basic and Biomedical Sciences, Paris Descartes University, INSERM UMR-S 1124, 45 rue des Saints-Pères, 75006 Paris, France.

3. Master Behavioral and Cognitive Neurosciences, Faculty of Sciences, Lebanese University, Fanar, Lebanon.

4. Department of Anatomy, Cell Biology and Physiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.

Recent work has proven Liver X Receptor (LXR) to be implicated in the myelination of the nervous system (Krishnan Sundaram et al., 2019). LXR double knock out lead to deregulation of crucial proteins expression of the myelin sheath membrane both in the peripheral and central nervous system, along with less nerve myelination (Makoukji et al., 2011; Meffre et al., 2015). Therefore, the team of C. Massaad in Paris Descartes University developed a specific LXR KO animal model. This model has portrayed locomotion difficulty as early as 8 weeks. In the present study, we investigated the impact of LXR knock out on peripheral nerve structural and physical integrity using multidisciplinary approaches. The apparent diameter and elastic Young’s modulus of teased fibers from sciatic nerves were measured by Atomic Force Microscopy. Electron microscopy was also used to obtain high definition images of the fibers. Myelination was evaluated with immunofluorescence staining of myelin Protein 0 (P0), the paranodal regions with CaspR and the neurofilaments with SMI 132. Our present data suggest that the knockout of LXR leads to the alteration of the peripheral nerve structural properties. LXR KO sciatic nerve fibers have smaller apparent diameters, less myelin, but there was no significant difference in Young’s modulus of Elasticity. Many hypotheses could be postulated in order to understand these results. Lipid profiling as well as cellular and molecular investigation could bring more clarity to the implication of LXR in peripheral nervous system myelination.

Molecular mechanism of cell death induced by aqueous extract of Althaea officinalisL. flowers in lung cancer A549 cells

Zainab Abdelghani1, Lara Daou1, Nancy Hourani2, Marguerite Mrad2, Ghassan Dbaibo2, 3,

Rouba Hage-Sleiman1

1Department of Biology, Faculty of Sciences, Lebanese University, Beirut, Lebanon, 2Department of Pediatrics and Adolescent Medicine, Department of Biochemistry and Molecular Genetics, Faculty of

Medicine, American University of Beirut, Lebanon, 3Center for Infectious Diseases Research, Faculty of Medicine, AmericanUniversity of Beirut, Lebanon

Althaea officinalis L. is a plant native to Lebanon belonging to the family Malvaceae. It has beendescribed in traditional medicine due to its healing properties. Most of the studies have been conducted on its mucilage-rich roots and little is known about its flowers consumed as hot water infusion in Lebanon. In this study, Althaea officinalis flowers collected from Damour were tested on human lung cancer A549 cells. Results showed a cytotoxic activity of the water extract (IC50 3.3 mg/ml) by MTT assay, accompanied by a time- and dose-dependent apoptosis illustrated by an increase in the percentage of early and late apoptotic A549 cells starting 6 mg/ml of the extract by Annexin / PI staining. Western blot results showed a significant increase in the expression levels of p53, Bax and Noxa in response to the water extract. When the expression level of p53 was reduced in A549 cells upon shRNA p53 transient transfections, the cell death response to A. officinalis flowers was also reduced, which reveals a p53-dependent apoptosis. The sphingolipid ceramide was generated in treated A549 cells via de novo and sphingomyelinase pathways. However, the induction of cell death was found to occur independently from ceramide. Upon the fractionation of the water extract, only the chloroform fraction showed a prominent cytotoxic effect (IC50 464.7 μg/ml). Apoptosis was induced as 46.875, 93.75, 187.5, 375 and 750 μg/ml of the chloroform fraction significantly distributed the A549 cells between early and late apoptosis in a dose- and time-dependent manner. These results highlight the anti-cancer effects of the crude extract and chloroform fraction, which could be attributed to several compounds such as phenolic acids, flavonoids and coumarins. Further experiments must be conducted to identify the active molecules present in the A. officinalis flowers that are responsible for the induction of the p53-dependent apoptosis.

Keywords: Althaea officinalis, apoptosis, p53, ceramide, cancer