Invited Speaker Text

A pricing model for the assessment of price of expensive orphan drugs

Mark Nuijten^a,b

^aA2M, Amsterdam, The Netherlands

^bITU, Istanbul, Turkey email address: [email protected]

Many pharmaceutical companies are now commercialising innovative expensive medicinal products for orphan diseases, with an incremental cost-effectiveness ratio (ICER), which will probably exceed the accepted threshold values for reimbursement, e.g. more than €100,000 per QALY. The goal of this presentation is to propose a new methodology to assess the price of expensive innovative orphan drugs from a broader perspective by applying concepts from business valuation.

The health authorities leave the responsibility for medical innovation to the market. Therefore medical innovation relies on the market mechanisms in the finance market of biotechnology including the incentives of the capital providers, who demand a required return of investment.

The prices for orphan drugs should be much higher than for other drugs, because the costs for development can only be recouped on fewer patients. The Discounted Cash Flow method can be used to validate the price of the new drug from an investor’s perspective. The Discounted Cash Flow method is based on the expected free cash flows and the required cost of capital. This approach may provide evidence, if the price of an orphan drug can be justified from an investor’s perspective, when the ICER exceeds the threshold.

Towards rational design of peptide antibiotics

Adam Mechler

La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia email address: [email protected]

The rise of multidrug-resistant strains of bacteria poses a challenge due to the lack of new drug targets and limited success of developing new antibiotics. The looming health crisis directs the attention to unorthodox solutions. Antimicrobial peptides (AMPs) have been considered, and discarded, for drug development before, largely due to limitations of standard medicinal chemistry methodology. AMPs provide innate immunity in most complex organisms, usually secreted at the site of injury and/or increased infection risk. AMPs evolved to evade most of the defenses bacteria can muster, and therefore they would provide excellent drugs. Unfortunately small and efficient amphibian/insect AMPs also kill mammalian cells, even though they are specific and selective in their host organism.

A novel approach to turn AMPs into antibiotics focuses on the mechanism of action, identifying the roles of specific moieties and developing design rules for artificial AMPs designed for human therapeutics. The best candidates for this approach are membrane disrupting AMPs that kill bacteria like “smart bullets”, opening holes through their plasma membranes. It will be demonstrated how to analyse the mechanism of a range of representative AMPs, and the identified design rules to date that will allow rational design of peptide antibiotics.

Advances in non-viral growth hormone gene therapy in dwarf “little” mice via muscular electroporation

Paolo Bartolini^a, Claudia R. Cecchi^b, Eliza Higuti^c, Gustavo P. Jesus^a, Alissandra M. Gomes^a, Enio a Zacarias^a, and Cibele N Peroni^a

^aBiotechnology Center, IPEN-CNEN, Cidade Universitária, São Paulo, SP, Brazil

^bTranslational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark

^cSociedade Beneficente Albert Einstein, Hospital Israelita Albert Einstein, São Paulo, SP, Brazil email address: [email protected]

Previous non-viral gene therapy studies occurred in two animal models of dwarfism: immunodeficient (lit/scid) and immunocompetent (lit/lit) dwarf mice. The first, based on genomic hGH DNA administration into muscle, has in general obtained better results, while the latter, an homologous model based on genomic mGH DNA, was less efficient, but more recommended for pre-clinical assays.

Our goal was to improve growth parameters aiming at a complete recovery of the lit/lit phenotype.

Electrotransfer conditions: three pulses of 375 V/cm of 25 ms and mGH-DNA administration into two sites of each tibialis cranialis muscle.

The highest GH circulatory levels ever reported for GH non-viral gene therapy were obtained: 14.7 ± 3.7 ng mGH/mL. In a 36-day bioassay these levels at the end of the experiment still were 8.5 ± 2.3 ng/mL, i.e. significantly higher than in normal mice. The catch-up growth reached 40.9% for body weight, 38.2% for body length, 80.0% for femur length. At the end of the experiment, the catch-up for mIGF-1 levels was still low, from the previous value of 3.9% to the actual 8.5%.

A complete phenotypic recovery was not obtained but we believe this should be possible starting with much younger animals and/or increasing the number of injection sites.

Climate change effects on the biodiversity of salt marsh habitats

Oscar Vicente^a and Monica Boscaiu^b

¹Institute for the Preservation and Improvement of Valencian Agrodiversity (COMAV), Universitat Politècnica de València, Valencia, Spain

²Mediterranean Agroforestry Institute (IAM), Universitat Politècnica de València, Valencia, Spain email address: [email protected]

There is a general awareness that climate change represents the most severe threat to agricultural production worldwide. Less attention is given to its adverse effects on wild plants and their natural habitats. Mediterranean salt marshes are extremely fragile ecosystems, inhabited by specialised halophytic species. These plants are adapted to high soil salinity – with different tolerance thresholds – and high temperatures and lack of rain characteristic of Mediterranean summers. More extended, frequent and intense drought periods and ‘heatwaves’ – which will also increase soil salinity – expected in a climate change scenario, will disturb salt marsh habitats, modifying the distribution of the species, even leading to the local extinction of some of them, either directly or due to competition with invasive species better adapted to the new environment. Narrow endemics, with few populations of a limited number of individuals, are the most vulnerable taxa. Further studies on the mechanisms of stress tolerance in halophytes are required for the successful design and implementation of salt marsh conservation programmes, based on the recovery of structural, abundant halophytic species, fundamental for the functioning of these ecosystems, and on the conservation, reinforcement and/or reintroduction of endemic, rare and threatened species of high ecological value.

Angiogenesis in the ovary – some molecular mechanisms of regulation

Bajram Berisha^a,b

^aAnimal Biotechnology, Faculty of Agriculture and Veterinary, University of Prishtina, Prishtina, Kosovo

^bAnimal Physiology and Immunology, Technical University Munich, Weihenstephan, Germany email address: [email protected]

The ovarian cycle in ruminants is characterized by repeated patterns of specific cellular proliferation/differentiation and transformation that accompanies follicular development, ovulation and corpus luteum (CL) formation. The folliculo-luteal transition is a complex process that requires the development of angiogenesis (the development of new capillaries from pre-existing ones) in early CL, which is closely regulated by a variety of endocrine and autocrine/paracrine factors. The dominant regulators of angiogenesis in CL are the vascular endothelial growth factors (VEGF), fibroblast growth factors (FGF), angiopoietins (ANPT), hypoxia-inducible factor (HIF) family members etc. The CL in this study was assigned to the following stages: days 1–2, 3–4, 5–7, 8–12 13–16 and >18 of the estrous cycle and months 1–2, 3–4, 6–7 and >8 of pregnancy. Expression of mRNA was measured by RT-qPCR, steroid hormone concentration by EIA and protein localization by immunohistochemistry. The highest expression for VEGF, FGF, ANPT and HIF family members was found in CL during the early luteal phase (period of angiogenesis) followed by a lower plateau afterwards and during luteal regression. These results lead to the assumption that the examined factors are involved in the molecular mechanisms regulating ovarian angiogenesis especially during CL formation, development and function.

Flexible approaches are required for the successful production of functionally active, structurally stable, pharmaceutically important recombinant proteins

Tarlan Mammedov

Department of Agricultural Biotechnology, Akdeniz University, Antalya, Turkey email address: [email protected]

Plant transient expression system has become one of the promising expression platforms for production a variety of vaccines, therapeutic proteins, enzymes and antibodies. However, for the expression of various proteins with different post-translational modification status, more flexible approaches are needed to preserve their native sequence and biological function. We developed robust strategies for the production of a number of "difficult-to-express" therapeutic proteins, vaccines in plants (Mamedov et al., PLOS ONE, 2017; PLOS ONE, 2019). Using these technologies affordable, safe, correctly folded and structurally stable, protein-based vaccines and therapeutic proteins were successfully produced in plants. Although there have been many decades of effort, malaria remains the leading cause of morbidity and mortality among the human population globally and no vaccine is currently available that provides a satisfactory level of protection. Using in vivo enzymatic deglycosylation strategy, the most advanced, a full-length Pfs48/45 based malaria antigen has been produced in plants, which may have the potential to save millions life (Mamedov et al., Scientific Reports, 2019). A technology has also been developed for processing of target proteins in vivo by co-expressing engineered human furin to produce safe, affordable and functional active therapeutic proteins (human FIX, etc.) in plants.

Calpains – calcium dependent cysteine proteases – their evolution and biomedical importance

Juraj Krajcovic and Dominika Veselenyiova

Department of Biology, Faculty of Natural Sciences, University of Ss. Cyril and Methodius in Trnava, Trnava, Slovakia email address: [email protected]

Calpains are cysteine proteases activated by calcium ions. This large protein family has been increasingly studied because of their involvement in human diseases, such as muscular dystrophy, type II diabetes, neurodegenerative and cardiovascular diseases and even cancer. Despite the attention that is payed to calpain research, their exact function and biology remains unclear. Although calpains are mostly studied in humans, they are well evolutionary conserved and have been identified in most eukaryotes and even some bacteria. In our study, we have focused on the identification of calpains primarily among unicellular eukaryotes from the protist taxon Euglenozoa. We have shown that the sequence similarity with human calpains is relatively high. We have also identified new calpains, specific only for Euglenozoa and we have further studied their structure, catalytic sites, subcellular localization and their evolution. Our findings show a surprisingly high number of calpains (≥20), especially in case of Leishmania and Trypanosoma species, compared to plants (1 calpain) and even humans (15 calpains).We believe that identification of new calpains and new calpain homologs in species that are less complex and easier to study can bring new information about their function and can be useful for their biomedical even biotechnological applications.

Could the molecular evaluation of stress resistance of wheat cultivars bring a new era on crop production?

Ariola Bacu^a, Vjollca Ibro^b, Magdalena Nushi^a, and Kathelina Kristollari^a

^aDepartment of Biotechnology, University of Tirana, Albania

^bDepartment of Agricultural Sciences, Agricultural University of Tirana, Albania email address: [email protected]

Bread wheat is among crops in demand worldwide, yet, the continuous efforts to improve yield and quality have not resulted in the approval of GM wheat cultivars. It is the latest of cereals transformed genetically, because of the complicated hexaploid genome, and many technical difficulties linked to transformation efficiencies, shortage of suitable plant explants, etc. Presentation discusses research leading to the belief that further progress in breeding of this crop is strongly dependent upon knowledge of functional genomics, tools and techniques available for accessing gene functions in wheat, applications of wheat genetic manipulation to increase yield, nutritional qualities, resistance to biotic/abiotic stresses. The actual progress and problematic on the above are discussed, aiming to explain perspective possibilities that the production, approval and acceptance of GM wheat from the market become a reality.

Integrated Approach to the Development of Pharma and Medicinal Biotechnology – the Role of Computer Modelling

Stanislav Miertus^a and Vladimir Frecer^b

^aDepartment of Biotechnology, University of SS. Cyril and Methodius, Trnava, Slovakia

^bFaculty of Pharmacy, Comenius University, Bratislava, Slovakia email address: [email protected]

Modern research in pharma and medicinal biotechnology includes integrated approaches ranging from computer modeling, through different chemistry fields, up to molecular biology and biotechnology. Computational (bio)modeling includes standard methods of bioinformatics, but also computational methods such as molecular mechanics and dynamics and even QM/MM methods with inclusion of solvent effects. The lecture presents examples of applications of such integrated approaches including drug design, biosensors development, molecular genetics, and design of materials for regenerative medicine.

The integrated approach focused on drug design and is demonstrated for HIV-1 protease inhibitors (potential anti-AIDS drugs) inhibitors of neuraminidase (influenza A virus) and Mpro SARS CoV-2 inhibitors.

Examples of biosensor development are focused on electrochemical biosensors ranging from the screen-printing technology up to nano-biosensors applicable in biomedicine and biotechnology.

Selected examples of applications of computational modelling on elucidation of DNA point mutations causing hereditable rare diseases (such as retinal degeneration), are also illustrated.

Polymeric scaffolds for artificial human urethra were studied using molecular modelling of interactions of human integrin domain with polymeric model surfaces combined with experimental evaluation of adhesion and proliferation of epithelial cells on polymer films.

Acknowledgement: The support by Slovak RDA grants APVV-15-0111, APVV-17-0239, APVV-18-0420 and PP-COVID-20-0010 is acknowledged.

Role of the copper dyshomeostasis in the neurodegeneration process

Michele Maffia, Chiara Carmela Spinelli, Simona Di Giulio, Lidia De Riccardis, and Marco Greco

Lab of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy email address: [email protected]

Cu dyshomeostasis and oxidative stress play a pivotal role in several neuropathologies such as Parkinson disease (PD). Metal accumulation in the central nervous system could result in loss-of-function of proteins involved in copper metabolism and in a copper redox cycling that generates reactive oxygen species. Moreover, neurodegenerative disorders imply the presence of an excess of misfolded proteins known to lead to neuronal damage: in PD, copper accumulates in the brain, binding alpha-synuclein and initiating its aggregation. In this work, we assessed the correlation between neuronal differentiation and copper homeostasis regulation, in both physiological and pathological conditions. At this purpose, we used SHSY5Y neuroblastoma cell line, treated with retinoic acid and brain derived growth factor (BDNF) in order to induce neuronal differentiation, and rotenone, able to cause neuron degeneration. Upon Cu treatment, we analyzed transcriptional and metabolic levels of proteins directly or indirectly involved within copper homeostasis such as Cu transporters and chaperones, together with α-synuclein and the prion protein (PrP). Aberrant conformations of these soluble proteins facilitate their precipitation and the tendency to form insoluble and toxic deposits: identifying Cu dependent alterations in the pathways responsible for the protein-misfolding may potentially offer new opportunities for clinical intervention.

Biocatalyst immobilization for continuous processing in microflow

Polona Znidarsic Plazl

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia email address: [email protected]

Immobilization of biocatalysts enables establishment of continuous bioprocesses with a long-term use of the same biocatalyst, which contributes to lower production costs. Microflow reactors have recently been introduced as promising tools both for early stages of the continuous biocatalytic process development, as well as for the production using the concept of numbering-up for increasing production capacity.

This work presents development of efficient microflow reactors using immobilized whole cells and enzymes for selected biotransformations, namely fumarase-catalyzed synthesis of L-malic acid and ω-transaminase-catalyzed enantioselective synthesis of optically pure amines. Various immobilization techniques including entrapment in hydrogels, covalent surface immobilization and the use of specific tags for oriented immobilization will be presented. Benefits of using miniaturized devices for the selection of the most appropriate immobilization technique with the aim to establish efficient continuous biotransformations will be highlighted.

Lysosomal sphingolipid catabolism disorders: implications in Parkinson’s disease

Tommaso Beccari^a, Maria Rachele Ceccarini^a, Elisabetta Albi^a, Carmela Conte^a, Michela Codini^a, Federica Patria^a, Anargyros Moulas^b, Matteo Bertelli^c

^aUniversity of Perugia, Dept. Pharmaceutical Sciences, Perugia, Italy

^bUniversity of Thessaly, Larissa, Greece

^cMagi Euregio Bolzano, Italy email address: [email protected]

Lysosomal storage disorders are a group of 60 different genetic diseases and they are classified on the basis of the biochemical nature of the accumulating substrate. Sphingolipidoses are a class of lysosomal diseases characterized by the unmetabolized sphingolipids and the consequently reduction of ceramide pool in lysosomes. Sphingolipidosis include several disorderds as Sandhoff disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, Krabbe disease, Niemann Pick disease, Farber disease and GM2 gangliosidosis. In sphingolipidoses, lysosomal lipid storage occurs in both the central nervous system and visceral tissues and central nervous system pathology is a common hallmark for all of them. Parkinson’s disease, the most common neurodegenerative movement disorder, is characterized by the accumulation and aggregation of misfolded α-synuclein that seems associated to some lysosomal disorders, in particular Gaucher disease. The lysosomal sphingolipid catabolism disorders are involved in Parkinson’s disease as demonstrated by several recent findings. The understanding of the pathophysiology of the endosomal-lysosomal-autophagic system will become of fundamental importance to develop new therapeutic strategies not only for lysosomal storage disorders but also for Parkinson’s disease.

Bacterial production of polyhydroxyalkanoates by extremophiles and stress-adapted bacterial strains

Stanislav Obruca^a, Iva Pernicova^a, Ivana Novackova^a, Xenie Kourilova^a, Eva Slaninova^a, Zuzana Sedrlova^a, Petr Sedlacek^a, Filip Mravec^a, Ota Samek^b, Jana Nebesarova^c, and Vladislav Krzyzanek^b

^aFaculty of Chemistry, Brno University of Technology, Brno, Czech Republic

^bInstitute of Scientific Instruments, Czech Academy of Sciences v.v.i, Brno, Czech Republic

^cFaculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic email address: [email protected]

Polyhydroxyalkanoates (PHA) represent group of bacterial polyesters which can serve as bio-alternative to synthetic polymers. To reduce production cost of these materials, we investigated several extremophiles for their capacity of PHA production. Among halophiles, we have identified that some Halomonas are very potent PHA producers from various low-cost substrates. Nevertheless; apart from many benefits, utilization of halophiles brings also many drawbacks related to high concentration of salt in cultivation media. Therefore; we focused on screening of thermophiles capable of PHA biosynthesis. It should be stated that, as compare to halophiles, PHA biosynthesis is far less explored so far in thermophiles. We screened selected thermophilic strains available in public collections of microorganisms. Moreover; we also developed isolation protocol enabling us to isolate PHA accumulating thermophiles from various microbial consortia and we isolated and identified very promising PHA producers capable of production of valuable PHA materials with tailored mechanical and technological properties. We have also performed evolutionary engineering experiments aimed at adaption of selected of PHA producers to various stressors to (i) gain PHA overproducing strains (ii) investigate role of PHA in stress-adaptation process. To sum up, it can be stated that stress-adapted bacteria are auspicious candidates for industrial production of PHA.

New strategies for the control of infectious and parasitic diseases in blood donors

Fabrizio Bruschi^a and Luca Galli^b

^aDepartment of Translational Research, N.T.M.S., Università di Pisa, Pisa, Italy

^bDepartment of Clinical Pathology, Azienda USL Toscana Nord Ovest, Pisa, Italy email address: [email protected]

Around 70 infectious agents are possible threats for blood safety. The risk for blood recipients is increasing because of new emergent agents like West Nile, Zika and Chikungunya viruses, or parasites such as Plasmodium and Trypanosoma cruzi in non-endemic regions, for instance. Screening programmes of the donors are more and more implemented in several Countries, but these cannot prevent completely infections, especially when they are caused by new agents. Pathogen inactivation (PI) methods might overcome the limits of the screening and different technologies have been set up in the last years. This review aims to describe the most widely used methods focusing on their efficacy as well as on the preservation integrity of blood components.

Decrease in serum cholesterol level influences breast cancer cell viability

Elisabetta Albi, Maria Rachele Ceccarini, Federica Patria, Carmela Conte, Giovanna Traina, Tommaso Beccari, and Michela Codini

Department of Pharmaceutical Sciences, University of Perugia, Italy email address: [email protected]

Patients with cancer, including breast cancer, often have abnormal levels of high-density lipoprotein (HDL) and low-density lipoprotein (LDL), and the cancer outcomes observed in dyslipidemic patients are poorer.

The study was aimed to evaluate the effect of hypocholesterolemia in three breast cancer cell lines: MCF7, less aggressive cancer cells that have receptors for estrogen and progesterone and therefore respond to hormone therapy; MDAMB453, cells with medium degree of aggressiveness (they are positive for the receptor for epithelial growth factor, HER2 +); MDAMB231 as cancer cell lines of high level of aggressiveness. Cells were grown with a medium containing depleted lipid serum. The condition of hypocholesterolemia, corresponding to 80 mg/dL in the blood, has been obtained by mixing 1:2 the normal medium and the lipid medium depleted. The results showed that the growth and cell viability is similar in MCF7 and MB453 and do not change during culture with lipid depleted serum and in hypocolestrolemia conditions. The MB231 cells have a lower vitality than other cell lines in culture with control serum and with depleted lipid serum and it increases in hypocholesterolemia conditions. In conclusion, the effect of hypocholesterolemia is dependent on the tumor phenotype.

Therapeutic evaluation of thymus serpyllum derived silver nanoparticles in STZ-induced diabetic BALB/c mice

Attya Bhatti

Department of Healthcare Biotechnology, Atta-ur Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan email address: [email protected]

Type 2 Diabetes Mellitus (T2DM) is one of the common metabolic disorder which is characterized by abnormal blood sugar level and current medications used for T2DM have shown slow mode of action and various side effects. Silver nanoparticles (AgNP) are gaining more importance in current era due to their effective antidiabetic properties along with antioxidant, antibacterial potential. The present study investigated the antidiabetic Potential of AgNP from Thymus serpyllum. Characterization of NP was done by using FTIR, XRD and SEM techniques. The α amylase and DPPH radical scavenging assay were performed respectively. Efficacy of AgNP on blood glucose levels FBG, IPGTT, ITT tests was employed on STZ induced BALB/c mice. To assess the morphological changes in the anatomy of liver, pancreas and kidney of BALB/c mice due to AgNP, histological analysis was done through H&E staining system. Finally, AMPK and IRS1 genes expression analysis was carried out via real time PCR. AgNP were found to be spherical in shape with an average size of 42 nm. Our study suggests that AgNP specifically 10 mg/kg causes significant increase in expression of AMPK & IRS1 which ultimately increase the glucose uptake in cells. Thus, Thymus serpyllum mediated AgNP possess the potential to cure T2DM.

Stress response in recombinant Escherichia coli expressing polyhydroxyalkanoic acid synthesis genes derived from Arctic and Antarctic bacteria

Jakub Grzesiak^a, Małgorzata Marta Rogala^a, Jan Gawor^a, Michał Styczyński^b, and Łukasz Dziewit^b

^aInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

^bFaculty of Biology, University of Warsaw, Warsaw, Poland email address: [email protected]

Polyhydroxyalkanoic acids (PHAs), the intracellular storage materials of a variety of Prokaryotes, are known for their role in carbon starvation survival. Recent findings point towards PHA involvement in stress resistance to several environmental factors. Although this phenomenon has been observed in natural PHA producer Cupriavidus necator, it was unknown if heterologous expression of PHA genes provides similar responses. Therefore, a number of PHA gene clusters from cold-loving bacteria have been inserted into Escherichia coli host. Transformants displaying PHA accumulation have been cultured and subjected to a series of stressors, including: high temperature, freeze-thaw cycles, low pH, oxidative and osmotic stress as well as UV irradiation. Preliminary analysis shows that granule presence conveyed greater resistance to temperature and pH shocks but made the transformants more sensitive to osmotic stress compared to the control strain. However, increased contribution of cells containing PHA of the PHA + transformants positively correlated with their survival rates for all tested stressors.

Time scale analysis and characteristic times in microscale-based chemical and biochemical processes

Igor Plazl^a and Goran N Jovanovic^b

^aFaculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia; Chair of Microprocess Engineering and Technology – COMPETE, University of Ljubljana, Ljubljana, Slovenia

^bSchool of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR, USA; PTT-Laboratory of America, Corvallis, OR, USA email address: [email protected]

Time-Scale Analysis and Characteristics Times are suggested as a novel, and useful tool in the analysis of the performance of microscale-based bioreactors with immobilized enzymes, and plant flow-sheet diagrams of chemical processes. Transport rates, reaction kinetics, and phase contacting can be easily represented by unique time constants, which facilitate understanding and representation of these processes via the ‘heat-map tableau’ of Characteristic Times. Characteristic Times are estimated from first principles and are controlled by a user to support a meaningful chemical process analysis and provide insight or suggestions for the successful design choices. Characteristic Times embed themselves easily into the Time-Scale Analysis (TSA) approach, which enables assessment of preliminary designs of microreactors and unit operations. We demonstrate the feasibility and usefulness of this novel tool by considering a microbial biochemical reaction process performed in classic bioreactor vs. performance in a microscale-based bioreactor design. We believe that this technical approach will confidently take place in the toolbox of practicing chemical reaction engineers.

Synthesis and investigations on new analogues of antimicrobial peptide (KLAKLAK)2

Sirine Jaber^a, Emilia Naydenova^a, Tsvetelina Angelova^a, Ivan Iliev^b, Inna Sulikovska^b, Nelly Georgieva^a, Zdravka Zaharieva^c, Ivo Grabchev^d, and Dancho Danalev^a

^aUniversity of Chemical Technology and Metallurgy, Sofia, Bulgaria

^bInstitute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria

^cTesting center Global Test Ltd, Sofia, Bulgaria

^dDepartment of Chemistry and Biochemistry, Physiology, and Pathophysiology, Sofia University "St. Kliment Ohridski", Sofia, Bulgaria email address: [email protected]

(KLAKLAK)2 is an antimicrobial peptides with antitumor properties (Deslouches and Peter, Oncotarget., 2017;8(28):46635–46651; Javadpour et al., Med Chem., 1996;39:3107–3113; Thundimadathil, J Amino Acids., 2012;2012:967347). Herein, we report the synthesis of (KLAKLAK)2 analogues containing unnatural amino acids, as well as conjugates of newly synthesized molecules with compounds with proven anticancer properties in order to investigate peptide moiety vectorization potential. In vitro 3T3 Neutral Red Uptake (NRU) Cytotoxicity assay indicated that the newly synthesized analogues are non-toxic. In most of the peptides tested it was determined IC50 > 2 mM. A low level of toxicity has also been observed in human non-tumorigenic cell line MCF-10 (IC50 > 1 mM). The same peptides were tested on a panel of human and mouse tumor cell lines (MCF-7, MDA-MB-231 and EAC) for antitumor activity. More of compounds show antitumor activity at μM range. Two of tested compounds show good selectivity index (SI > 2.5). In addition, the antimicrobial potential of newly synthesized analogues is also studied against model microorganism.

PHB from cyanobacteria – why phototrophic biotechnology is interesting for Europe

Ines Fritz, Katharina Meixner, and Christina Daffert

Department IFA-Tulln, University for Natural Resources and Life Sciences, Tulln, Austria email address: [email protected]

While there is no doubt about the usefulness of PHB as a plastic replacement, ethical issues arise with single-use products made from potential feed and food; while issues of raw material availability and quality arise from the utilisation of production residuals, such fat residues or slaughterhouse waste.

Many cyanobacteria are known to naturally produce and accumulate PHB. They grow photo-autotrophically, using light as energy source and binding CO₂ from the atmosphere or, preferably from a CO₂ rich off-gas, such as exhaust gas from incineration. Independence from the availability of an organic substrate is balanced by slower growth rates when compared with heterotrophic bacteria. This makes the volumetric efficiency of cyanobacterial PHB to the weak point of the whole production concept. But, a photobioreactor does not occupy agricultural land nor is it dependent on crop imports and it can be operated in all European climates over the whole year.

Strengths and weaknesses of PHB production with cyanobacteria need to be evaluated carefully and there are still many questions not answered on a scientific level. Phototrophic and heterotrophic biotechnological production of PHB are not in concurrence, they can be seen as complementary strategies – which is the conclusion from ongoing research.

Biotechnologies in the Czech Republic – current status, trends and challenges

Ivana Marova

Brno University of Technology, Faculty of Chemistry, Brno, Czech Republic email address: [email protected]

In the time of the continuing global economic competition, the Czech Republic tries to keep pace with the group of the countries with the most advanced economy and the highest living standard by the transition to the knowledge economics. In future, pillars of the modern knowledge Czech economy should include the molecular biology, biotechnology and additional associated branches. To establish a competitive technology-rich economy, CR must provide rapid and coordinated support for R&D&I at the front line, including biotechnology. The support of the EU enabled extensive innovative projects from several operational programs, e.g. OP Research and Development for Innovations, through which the construction of centers of the scientific excellence of the European significance was financed. Several of these centers focus on the selected fields of the molecular biology and biotechnology. Besides these top interdisciplinary projects, a number of smaller, on sector focused institutions implementing applied research were subsidized. Biotech industry is an essential source of rich economy growth, interconnects high-grade academic science and commercial R&D&I and generates high innovative potential. The true mission for the near future is to make strategic decision on the priority definition in research area as a single discipline without artificial division into basic and applied science.

Yeast isogenic system as a biotechnological tool for studying protein-DNA interactions

Vaclav Brazda^a, Otilia Porubiakova^a, Natalia Bohalova^b, and Jan Coufal^b

^aFaculty of Chemistry, Brno University of Technology, Brno, Czech Republic

^bThe Czech Academy of Sciences, Institute of Biophysics, Brno, Czech Republic email address: [email protected]

Protein-DNA interaction is the fundamental part of life. There are many techniques for detection and evaluation of the protein DNA binding from in vitro to in vivo assays. Here we presented application of yeast isogenic system for in vivo transactivation studies in chromosomal context. This variable system is applicable for analyses of influence of DNA structures to protein-DNA binding in vivo. We used a panel of S. cerevisiae haploid isogenic strains, except for the different p53 target site with different DNA structures located upstream of the luciferase reporter gene. The targeting of p53 target sequence of interest by the replacement of the ICORE cassette, using transfected single strand oligonucleotides, was performed following the Delitto Perfetto technique. yLFM isogenic derivative yeast strains constructed for this study were transformed with different plasmids for the expression of wild-type human p53 or p73 proteins. Our results show that transactivation in vivo correlated more with the relative propensity of a response element to form cruciforms than to its expected in vitro DNA binding affinity. Structural features of target sites are therefore an important determinant of its transactivation function.

About the mechanism keeping intracellular PHA granules amorphous and its biophysical consequences

Petr Sedlacek^a, Eva Slaninova^a, Vojtech Enev^a, Vladislav Krzyzanek^b, Ota Samek^b, and Stanislav Obruca^a

^aFaculty of Chemistry, Brno University of Technology, Brno, Czech Republic

^bInstitute of Scientific Instruments, Czech Academy of Sciences, Czech Republic email address: [email protected]

Polyhydroxyalkanoates (PHA) represent biomolecules of great importance both from the view of their biological role in microorganisms and of the wide range of their potential material applications. Recent studies have brought a novel perspective on the complex functionality of PHA in bacterial cells, comprising not only the traditionally considered energy storage role but also their previously unexpected involvement in stress survival and adaptation of their microbial producers. PHA is accumulated in the microbial cells in the form of numerous polymer granules. Already from the time of pioneer works focusing on the material and morphological properties of PHA in vivo, a debate has continued questioning the mechanisms that keep intracellular PHA I the thermodynamically unfavorable amorphous form. In our study, we have combined investigation of the stress response of PHA accumulating bacteria with simultaneous monitoring of changes in PHA crystallinity in the cells. Based on the results of this study we have proposed an updated view of the mechanism of plasticization of PHA in vivo which combines kinetic effects resulting from the restricted volume of the granules with plasticization effects of intra-granular water. The study has further revealed how particular stress factors induce inactivation of this plasticization mechanism in living cells.

Established and advanced approaches for recovery of microbial polyhydroxyalkanoate (PHA) biopolyesters from surrounding microbial biomass

Martin Koller^a,b

^aUniversity of Graz, Institute of Chemistry, NAWI Graz, Austria

^bAssociation for Resource Efficient and Sustainable Technologies, Graz, Austria email address: [email protected]

Downstream processing for recovery of polyhydroxyalkanoate (PHA) from biomass constitutes an integral part of the PHA production chain; this process is the major cost factor beside the feedstocks used for PHA production.

Besides economic aspects, PHA recovery techniques need to be sustainable by avoiding excessive use of (often precarious!) solvents, other harmful chemicals, and energy. Moreover, the applied PHA recovery method is decisive for the molecular mass and purity of the obtained product, and the achievable recovery yield. In addition to the applied method, also the PHA content in biomass is decisive for the feasibility of a selected technique. Further, not all investigated recovery techniques are applicable for all types of PHA and all PHA-producing microorganisms.

The presentation shines a light on benefits and shortcomings of established solvent-based, chemical, enzymatic, and mechanical methods for PHA recovery. Most of all, it presents novel recovery strategies, encompassing the use of “green” solvents, the use of classical “PHA anti-solvents”, ionic liquids, supercritical solvenst, hypotonic cell disintegration for release of PHA granules, and even digestion of non-PHA biomass by animals.

The different established and novel techniques are compared in terms of PHA recovery yield, product purity, scalability, and demand for chemicals, energy, and time.

Genomics of Solanaceae species: fundamental and applied aspects

Alexander Kilchevsky, Olga Babak, Natalia Nekrashevich, Tatyana Nikitinskaya, and Konstantsia Yatsevich

Laboratory of Ecological Genetics and Biotechnology, Institute of Genetics and Cytology NASB, Minsk, Belarus email address: [email protected]

Solanaceae crops are in demand as functional food products due to their content of substances with antioxidant, oncoprotective and immunomodulating properties. Based on DNA markers to the genes of various carotenoids (CRTISO, CYCB) and flavonoids (SlMYB12, Ant1), tomato forms with different combinations of their alleles to study carotenoid and flavonoid accumulation relationship have been developed. A decrease in the CRTISO-gene expression level, as well as prolycopene accumulation in the fruits of tomato forms with a t/Ant allele combination as compared to t/ant and a decrease in the CYCB-gene expression level, as well as lycopene accumulation with a combination of b/Y alleles in the genotype as compared to b/y have been revealed.

Based on the analysis of Antocyanin 1 tomato orthologous sequences in pepper and eggplant forms with wide intraspecies diversity by anthocyanin accumulation in fruits, the allelic polymorphisms of Myb113-like TF-genes of pepper and Myb1 of eggplant have been identified in the forms with impaired or no anthocyanin accumulation in fruits. Optimal SCAR and CAPS markers have been developed to identify the revealed polymorphisms of Myb-genes under study. Breeding forms with different gene combinations have been selected to study flavonoids’ accumulation peculiarities and develop valuable agricultural forms.

Identification of possibly pathogenic variants in candidate genes in lymphedema patients by NGS analysis

Sercan Kenanoglu^a,b, Dominika Vešelényiová^b,c, Sandro Michelini^d, Paolo Enrico Maltese^e, Stefano Paolacci^e, Juraj Krajcovic^c, Munis Dundar^a, Tommaso Beccari^f, and Matteo Bertelli^b

^aDepartment of Medical Genetics, Erciyes University, Kayseri, Turkey

^bMagi Euregio, Bolzano, Italy

^cDepartment of Biology, Faculty of Natural Sciences, University of Ss. Cyril and Methodius in Trnava, Trnava, Slovakia

^dDepartment of Vascular Rehabilitation, San Giovanni Battista Hospital, Rome, Italy

^eMagi’s Lab, Rovereto (TN), Italy

^fDepartment of Pharmaceutical Sciences, University of Perugia, Perugia, Italy email address: [email protected]

Lymphedema is a chronic disease caused by the accumulation of protein-rich interstitial fluid. Clinical symptoms involve swelling of the extremities and fibrosis. Multiple genes have been implied to be associated with the development of lymphedema and are used in genetic testing. We applied Next Generation Sequencing of 65 candidate genes in 235 samples negative for the diagnostic test. From this analysis we found putative pathogenic variants in 13 genes, including ACKR2, ARAP3, CDH5, CYP26B1, NOTCH1, NRP1, NRP2, PECAM1, PROX1, RORC, SEMA3A, SVEP1 and TIE1. We performed variants segregation analysis in the family members of lymphedema patients and lymphoscintigraphy in family members who were found to carry the same variant as the proband. To predict the effects of the identified variants on the resulting proteins, we also performed in silico 3 D modelling. Taken together, we suggest the role of new candidate genes in the development of lymphatic system malformations, which can result in lymphedema.

Development of a new test for the detection of autoimmune diseases of the retina

Kristjana Dhuli^a, Stefano Paolacci^b, Elena Manara^c, Maria Rachele Ceccarini^d, Tommaso Beccari^d, Paolo Enrico Maltese^b, and Matteo Bertelli^a,b,c

^aEBTNA-LAB, Rovereto (TN), Italy

^bMagi’s Lab, Rovereto (TN), Italy

^cMagi Euregio, Bolzano, Italy

^dDepartment of Pharmaceutical Sciences, University of Perugia, Perugia, Italy email address: [email protected]

Autoimmune retinopathy (AIR) is a rare disease in which the patient's immune system attacks proteins in the retina, leading to a sudden loss of eyesight. Included in this spectrum of diseases are cancer-associated retinopathy (CAR), melanoma-associated retinopathy (MAR) and non-paraneoplastic autoimmune retinopathy (npAIR).

Although the first case report is from 1976, the prevalence of AIR is currently unknown. Diagnosis of AIR can be difficult due to the overlap of symptoms with hereditary retinal degeneration, and to the lack of standard diagnostic consensus and standard diagnostic tests for the detection of circulating autoantibody against specific retinal antigens. With the aim to develop a new test for the detection of serum autoantibody against the main retinal target (i.e. alpha-enolase, recoverin and rhodopsin) we developed an indirect ELISA that we used to screen the serum of 30 AIR patients enrolled in Italy. The validation of this test for diagnostic purpose could lead to a better understanding of this spectrum of disease, increasing the information about AIR clinical course and prognosis, in addition could help in patient selection for available clinical trial and appropriate patient management.

MAGI an innovative SME that promotes scientific collaboration in genetic and rare disease

Matteo Bertelli^a,b,c, Jan Miertus^b,c,d, Stefano Paolacci^b, Paolo Enrico Maltese^b, Elena Manara^c, Stefano Cecchin^b, and Giuseppe Marceddu^c

^aEBTNA-LAB, Rovereto (TN), Italy

^bMagi’s Lab, Rovereto (TN), Italy

^cMagi Euregio, Bolzano, Italy

^dGenius n.o., Trnava, Slovakia email address: [email protected]

MAGI is a no profit reality that deals with research and diagnosis of genetic diseases and biotechnological research. It has been active since 2006 in the national and international context. In diagnostics, we have one of the largest platforms for the genetic analysis with ISO 15189 certification and about 2000 analyzed genes. Our diagnostic process includes the integrated application of Sanger sequencing, MLPA, real time PCR, and NGS (Illumina). We are now implementing the use of long read sequencing (Nanopore, PacBio). The MAGI staff is made up of geneticists, researchers, doctors and technicians and today has more than 35 employees. We aim to offer state-of-the-art diagnostic tests, constantly upgrading our NGS custom panels; trying to gain further insight into the disease-causing mechanism of lymphedema and defining the pathogenic role of new identified variants.

In the international context, the group has built a non-profit diagnostic center in Tirana and spin-off within the University of Krasnoyarsk. The number of publications increases exponentially in the last 3 years reaching the number of about 30 articles in 2019. Our collaborations involve Italian, European and American institutes and universities.

Mendelian anorexia: a preliminary genetic study

Paolo Enrico Maltese^a, Stefano Paolacci^a, Elena Manara^b, Tommaso Beccari^c, Maria Rachele Ceccarini^c, Laura Dalla Ragione^d, Matteo Bertelli^a,b,e

^aMagi's LAB, Rovereto (TN), Italy

^bMagi EUREGIO, Bolzano, Italy

^cDepartment of Pharmaceutical Sciences, University of Perugia, Perugia, Italy

^dCenter for the Treatment of Eating Disorders, Residenza Palazzo Francisci, Todi (PG), Italy

^eEBTNA-LAB, Rovereto (TN), Italy email address: [email protected]

Anorexia nervosa is a multifactorial eating disorder that manifests with self-starvation, body image disturbances, hyperactivity and intense fear of gaining weight. It has one of the highest mortality rates of any psychiatric disease.

The disease can be familiar showing strong heritability, however the genetic bases remain obscure. Only recently, research studies identified several genetic loci involved in molecular pathways that might lead to anorexia.

On the basis of an analysis of the literature, we developed a Next Generation Sequencing-based genetic test including all genes that can be putatively associated with the disease, such as genes involved in serotonin, opioid, dopamine and endocannabinoid pathways or genes related to appetite-regulating hormones. Our custom designed panel comprising a total of 156 genes was then used to perform targeted analysis of familiar cases (68 probands, 63 females and 5 males) of anorexia nervosa. Here we report the preliminary genetic results of our study.

Dopamine receptor gene variants in eating disorders susceptibility

Maria Rachele Ceccarini^a, Luana Fontana^b, Federica Filomena Patria^a, Simona Fittipaldi^b, Michela Codini^a, Elisabetta Albi^a, Laura Dalla Ragione^c, Tommaso Beccari^a, and Annalisa Botta^b

^aDepartment of Pharmaceutical Science, University of Perugia, Perugia, Italy

^bDepartment Biomedicine and Prevention, Tor Vergata University of Rome, Rome, Italy

^cUniversity of Campus Biomedico, Rome, Italy email address: [email protected]

Eating Disorders (EDs) refer to a group of complex psychiatric conditions in which disorderly food intake results in impaired psychological functioning or physical health. Dopamine (DA) is a neurotransmitter implicated in the feeding behavior and is therefore a strong candidate for EDs. In this study, we carried out a genetic association studies in a huge cohort of Italian patients with EDs consisting of individuals affected by AN (n = 323), BN (n = 118) and BE (n = 132) disorders. We primarily focused on six polymorphisms within the DRD2 and DRD4 genes that encode DA receptors. Our initial data confirm and extend previous genetic studies showing a strong association of EDs with the rs936461 SNP in the DRD4 gene. The prevalence of homozygous GG individuals is significantly higher in BN (44%) and BE (42%) patients compared with controls (n = 206) (25%). Moreover, a strong association has been demonstrated also for the DRD4 120-bp TR polymorphism, with a 23% frequency of Omo L/L BE patients compared to 61% of controls. These preliminary results straighten the potential role of dopamine receptor genes in the hereditability of EDs symptoms and traits.

Shapes of metabolism of lactic acid bacteria Lactobacillus amylovorus DSM 20531 T and Leuconostoc mesenteroides LMG 7954

Anita Slavica

Faculty of Food Technology and Biotechnology, Department of Biochemical Engineering, University of Zagreb, Zagreb, Croatia email address: [email protected]

Metabolic features of an obligately homofermentative amylolytic strain Lactobacillus amylovorus DSM 20531 T and an obligately heterofermentative strain Leuconostoc mesenteroides LMG 7954 have been explored in order to establish their biotechnological relevance. Hydrolysis of complex carbon and nitrogen sources and simultaneous utilization of hydrolytic products thereof in a fermentative production of D-/L-lactate by the strain Lactobacillus amylovorus DSM 20531 T were characterized in many details. Obtained metabolic features have been successfully embedded within an integrated bioprocess for (1) a direct production of the lactates from starch by entrapped L. amylovorus DSM 20531 T cells, and (2) a subsequent formation of lactic acids ethyl esters. Final products – ethyl lactates were identified by GS/MS method. Permeabilization of Leuconostoc mesenteroides LMG 7954 cells affected number of applicable nuances of metabolism of these apparently obligately heterofermentative strain. Growth of L. mesenteroides LMG 7954 was not considerably affected by use of two permeabilization appliances – ultrasound and a non-ionic surfactant Tween 80, as production of the lactates, ethanol and acetate was. Besides rerouting metabolism of carbohydrates in which pyruvate gives rise to not more than one end product, the permeabilization procedures showed potential of L. mesenteroides LMG 7954 strain for production of high added value biochemicals.

Blood compatibility studies of silk fibroin based polyelectrolyte complexes

Terin Adali^a,b and Moyo Mthabisi Talent George^a,b

^aDepartment of Biomedical Engineering, Faculty of Engineering, Near East University, , Istanbul, Turkey

^bTissue Engineering and Biomaterials Research Center, Center of Excellence, Near East University, Istanbul, Turkey email address: [email protected]

Polyelectrolyte multilayer (PEC) films of silk fibroin (SF) and sodium alginate (NAL) were prepared and loaded with anticoagulant agent, clopidogrel bisulphate (CB) aiming to design new hydrophilic PECs with controlled physicochemical properties and drug release behaviour that find application as components of blood contacting devices and transdermal drug delivery systems. The SF based PEC films were characterized by AFM, SEM, TGA, XRD and FTIR spectroscopy. The in vitro blood compatibility studies were analysed by blood coagulation factors prothrombin time (PT), activated partial thromboplastin time (APTT) and International Normalized Ratio (INR). The number of layers and the nature of the outer layer affect the physicochemical characteristics, blood compatibility properties, CB loading and release behaviour of the films. The three-layer film PEC-04, which is composed of NAL outer layer on SF – NAL double layer, is characterized by the lowest roughness (Rq = 16.1 nm) among other films. The “in vitro” coagulation and periphery smear tests analyses indicated that the three-layer film PEC-04 exhibited better hemocompatibility in comparison with pure SF, pure NAL and two-layer films.

These results proposed that, three-layer film PEC-04 has potential applications as coating biofilm for blood contacting devices and transdermal drug delivery systems.

Small bugs, big opportunities: turning wastes to valuable biomaterials

Iza Radecka^a, Hazel Gibson^a, Abhishek Gupta^b, Sam Swingler^a, Anabel Itohowo Ekere^a, Brian Johnston^a, Grazyna Adamus^c, and Marek Kowalczuk^c

^aWolverhampton School of Sciences, Faculty of Science & Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK

^bSchool of Pharmacy, Faculty of Science and Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK

^cCentre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland email address: [email protected]

Naturally-occurring polymers are produced by a range of prokaryotic and eukaryotic organisms. Microbial polymers have an enormous potential as they can be produced from renewable biogenic resources under well controlled conditions. Many of microbial polymers are biodegradable and biocompatible. Moreover, their expected impact is not simply limited to easing the environmental problems inevitably associated with their production, but also in reducing dependency on fossil-fuel based polymers. Over the past decades a large amount of biopolymers originating from various types of microorganisms have been reported. This presentation will discuss an integrated biotechnological/chemical process which allows transformation of agricultural and other biorefinery by-products into value-added biodegradable polymeric materials which may be used in the areas of agriculture, cosmetics, medicine, as well as in household products, coating systems and as additives to compostable plastic packages of extended shelf life.

This presentation will describe the biosynthesis and production of biodegradable microbial polymers, including polyhydroxyalkanoates (PHA), bacterial cellulose (BC) and poly-gamma glutamic acid (PgGA), their most important industrial applications and ecological advantages.

Study of metabolic adaptation of red yeasts to waste animal fat substrate

Martin Szotkowski^a, Jiří Holub^a, Samuel Šimansky^a, Dana Byrtusová^a, Volha Shapaval^b, and Ivana Márová^a

^aBrno University of Technology, Brno, Czech Republic

^bNorwegian University of Life Sciences, As, Norway email address: [email protected]

Carotenogenic yeasts are oleaginous microorganisms capable of utilizing various food and agricultural industry waste substrates. In this work, four red yeast strains (Cystofilobasidium, Rhodotorula and Sporobolomyces sp.) were cultivated in media containing crude, emulsified, and enzymatically hydrolyzed animal waste fat, compared to glycerol and glucose, as carbon sources. During experiment production of total biomass, carotenoids, ergosterol, ubiquinone (HPLC-PDA), lipids (GC-FID), lipase and biosurfactants in yeast cells was studied in various conditions depending on media composition, carbon source and carbon/nitrogen ratio. Results showed that all studied strains are able to utilize solid and processed fat. Yeasts cultivated on hydrolyzed/emulsified fat in media with low C/N ratio = 13 produced more biomass, compared to the control media. Lipid and lipidic metabolites production whereas enhanced several times on fat. The production of total lipids increased in all strains to C/N = 50. Oppositely, the production of carotenoids, ubiquinone and ergosterol dramatically decreased with increased C/N in all strains. Compounds accumulated in red yeasts have a great application potential and can be produced efficiently during the valorization of animal waste fat under the biorefinery concept. Simultaneous induction of lipase and biosurfactants was observed on the crude fat substrate.

Studying the co-production of lipids and β-glucans in carotenogenic Basidiomycetes and Ascomycetes yeasts

Dana Byrtusova^a, Volha Shapaval^b, Achim Kohler^b, Andrea Nemcova^a, and Ivana Marova^a

^aFaculty of Chemistry, Brno University of Technology, Brno, Czech Republic

^bFaculty of Science and Technology, Norwegian University of Life Sciences, As, Norway email address: [email protected]

Beta-glucans represent a polymer, consisting of D-glucose monomers linked through β-glycosidic bonds. These polysaccharides can be found in cell walls of many organisms – high fungi (mushrooms), yeast, bacteria, plants, and algae, where they are responsible for the mechanical and structural properties. Due to the anti-inflammatory, antitumor, immunostimulatory and antimicrobial activities, they belong to biological response modifiers (BRMs) having beneficial effects on human and animal health. Currently, the commercial sources of β-glucans are higher fungi, while yeast sources are limited to only baker’s yeast Saccharomyces cerevisiae's cell wall. Other yeast sources, as Ascomycetes yeast of the Metschnikowia genus and carotenogenic yeasts of the phylum Basidiomycetes, represent biotechnologically unexplored potential sources of β-glucans. These yeasts are able of the co-production of β-glucans and lipids at certain conditions. This could significantly improve the economical sustainability of the both microbial lipids and β-glucans production. This study shows screening of fifteen Ascomycetes and Basidiomycetes yeast strains for the simultaneous production of lipids and β-glucans and influence of C/N ratio and osmotic stress the biosynthesis of both.

Genetic markers from urine as a non-invasive prostate cancer screening – challenges in translation from lab to clinic

Lejla Pojskic^a, Jasmin Ramic^a, Benjamin Kulovac^b, Naida Lojo Kadric^a, Maida Hadzic^a, Djenana Eminagic^c, Kasim Bajrovic^a, and Naris Pojskic^a

^aLaboratory for Human Genetics, Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Sarajevo, Bosnia And Herzegovina

^bClinic of Urology, University Clinical Centre Sarajevo, University of Sarajevo, Sarajevo, Bosnia And Herzegovina

^cClinic of Oncology, University Clinical Centre Sarajevo, University of Sarajevo, Sarajevo, Bosnia And Herzegovina email address: [email protected]

Standard diagnostic procedures for prostate carcinoma are time consuming, invasive and tiresome for most of the patients. In more than 50% of cases is unnecessary, as diagnosis is a benign prostatic hyperplasia.

In a series of experiments, we studied conditions for detection and measurement of the relative level of transcriptional level of the KLK3, PCA3 and TEMPRSS: ERG genes from the urine samples of patients with prostatic diseases and healthy controls. Urine was sample of choice because the sample is taken in non-invasive manner, and in order to potentially make better selection to biopsy. One of the selected genes (KLK3) differed significantly subject cohorts of various pathological conditions of the prostate.

This gene was evaluated further for its potential applications in the improvement of diagnostic procedures. However, additional validation should be performed in a larger cohort for having this marker qualified as a reliable tool for screening and non-invasive detection of prostate cancer from urine samples. We will present the methodology of our research and validation we used and also recommendations for rational and cost-efficient method verification. This model could be also employed in validation strategies for other non-invasive approaches that would utilize total gene information from body fluids.

Plant protein hydrolysates as animal cell culture media supplement

Igor Slivac, Marijan Logarusic, Visnja Gaurina Srcek, Kristina Radosevic, and Andreja Lebos Pavunc

Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia email address: [email protected]

Animal cells are widely used expression system applied in biotech production of therapeutics and bio-similars. In this regard, cell growth microenvironment plays an important role and it is largely defined by composition of growth media. Content and origin of proteins/peptides have a key role in preparation of growth media. Fetal bovine serum is still used as a major protein source, despite its composition inconsistency and safety related risks. To overcome these problems chemically-defined media have been developed, but their application often results in reduced cell growth and protein production when compared to serum-containing media. Due to the complexity of nutritional requirements of animal cells, it is time-consuming to formulate the chemically defined media. Therefore, media supplemented with protein hydrolysates, which contain oligopeptides, amino acids, iron salts, lipids, vitamins, and other low molar mass substances in trace elements, is a useful alternative. Among the most promising substances for the hydrolysate production are protein rich crops like soy or chickpeas, as well byproducts of corn processing (wheat and rice) or cooking oil industry (rape-seeds, hemp-seeds and flax-seed). This diversity of hydrolysate source requires thorough investigation when defining the kind and quantity of hydrolysates to be used

Flow Field-flow fractionation coupled to light scattering for the characterization of Nano-Drug-Delivery systems

Christoph Johann

Wyatt Technology, Santa Barbara, CA, USA email address: [email protected]

Nano Drug-Delivery systems are complex nano suspensions, consisting of the API, which can be proteins, oligo nucleotides or small molecules and the carrier, which are nanoparticles with sizes between 10 and 1000 nm. Asymmetrical Flow Field-flow fractionation (AF4) coupled to a multi-angle light scattering (MALS) and dynamic light scattering detectors is becoming recognized as a useful tool to determine the size distribution of the nano particles and quantify the conjugated and free drug in one analysis run (Caputo et al., J Control Release., 2019;299:31–43; Mol Pharm. 2019;16(2):756–767). AF4 is a separation without a stationary phase, using a thin ribbon-like channel. Based on diffusion, smaller sample components migrate into flow streams having higher velocity compared to bigger ones which results in increasing retention time with size. AF4 has high resolution, separates from a few nanometers to a few micrometers and is gentle and compatible with biological samples. Applications for various drug delivery systems will be discussed.

Current status of personalised medicine in drug development and clinical practice

Nikolai Zhelev^a, Christene Leiper^b, Vesselin Georgiev^a, Adebola O Oluwaseun^a, Lewis Reynolds^a, Larisa N Sivitskaya^c, Natalia Akentieva^d, Victor V Revin^e, Tatyana Vlaykova^f, Vili Stoyanova^g, and Marianna Murdjeva^g

^aUniversity of Dundee, Dundee, UK

^bOnorach Ltd, Dundee, UK

^cInstitute of Genetics and Cytology, Minsk, Belarus

^dIPCP, RAS, Moscow, Russia

^eMordovia State University, Saransk, Russia

^fTrakia University, Stara Zagora, Bulgaria

^gMedical University of Plovdiv, Plovdiv, Bulgaria email address: [email protected]

Personalised medicine, the use of marker-assisted diagnosis and targeted therapies derived from an individual’s molecular profile, have a substantial impact on the way drugs are developed and prescribed in clinical practice. We review the use of Next Generation Sequencing (NGS), biomarkers, biosensors, stem cell technologies and systems pathology in drug development and clinical practice with examples from our work in the field of cancer and cardiovascular diseases. Results from the development and validation of live-cell biosensors are presented. Recent advances in stem cell biology and 3 D cultures, representing a substantial advancement in structural and functional complexity over traditional in-vitro cell culture models are illustrated with examples. Advantages and challenges of implementing clinical NGS testing in a clinical molecular diagnostic laboratory are discussed. Novel approaches for more efficient management of clinical trials worldwide such as patFINDER are presented. We also summarise and critically review some of the ongoing challenges for realising the promise of personalised medicine

Applications in wearable smart systems and future challenges

Osman Erogul

Department of Biomedical Engineering, TOBB University of Economics and Technology, Ankara, Turkey email address: [email protected]

Technology and health are nested with each other, also developments in technology help people to access their health information with the use of data received from wearable sensors. People can monitor their mental or physical health, which may help for precaution or seeking help in case of an emergency like bleeding wounds. In occupations who are under high stress and physical load factors, such as athletes, construction workers, emergency room staff, can observe their status in real time. Wearable sensor technologies are also used to monitoring sleep parameters. Wearable smart watch that is known as actigraph can monitor body movements. In the light of recent developments, a wearable ring may include sensors for sensing motion signals in response to body motion, monitoring vital signals (e.g. heart rate, respiration, arousal) and a GPS signal to determine the location of the person. Wireless transmission and developments in manufacturing conductive paths in textile clothes significantly reduce cable mess. In this study, current applications in wearable smart systems and future challenges are discussed in details.

Application of mathematical modelling in biorefinery processes design: Production of nanocellulose

Tonci Rezic^a, Martina Andlar^a, and Roland Ludwig^b

^aDepartment of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia

^bDepartment of Food Sciences and Technology, University of Natural Resources and Life Sciences, Vienna, Austria email address: [email protected]

A biorefinery is a facility that integrates biomass conversion processes and equipment to produce bio-products from biomass. Biorefineries will have to employ the best possible processes (for biomass cultivation, harvesting, storage and transport, pre-treatment, fermentation and bio-chemical conversion) to ensure efficient bio-based production. Nevertheless, numerous technical, strategic and commercial challenges have to be overcome to reinforce importance of bio-based production. Mathematical models can be used to simulate all aspects of bio-based production systems including the process design, production economics, supply logistics and environmental impacts. Those models can provide powerful tools to optimize biorefinery processes and evaluate its technical feasibility as well as economics and environmental impacts. In the presentation, examples of different mathematical models will be discussed, from the physical base model including kinetic, transport and diffusion equations to the statistical base model and data analyses. In the end, life cycle assessment analysis of the nanocellulose production will be provided as the example of modelling application in better understanding of the bio-based production environmental impact.

Processing and applications of polyhydroxyalkanoates

Adriana Kovalcik

Department of Food Chemistry and Biotechnology, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic email address: [email protected]

The biodegradable properties of polyhydroxyalkanoates (PHA) have been identified as a promising solution to decrease packaging waste from cities, environment and landfill. Moreover, PHAs are biocompatible and meet several criteria as biomaterials for the utilisation in pharmaceutical, cosmetic and medical applications. They also may contribute to the circular economy if their biosynthesis is planned, including the use of local food and agricultural intermediates and wastes. However, PHAs may not necessarily provide all required physical properties for polymer processing and thermo-mechanical behaviour of final products. Taking a different chemical nature and wide range of physical properties approach, this paper addresses the issue for the diversity of PHAs, their thermal, viscoelastic and mechanical properties. Depending on the chemical structure and physical properties, various polymer processing methods will be suggested and discussed, such as solution casting and melt processing. This work provides an insight into PHÁs hydrolytic degradation, biodegradation in model fluids and compost. The resulting picture suggests that PHAs display environmental benefits if they are synthesised within the circulation economy schema. This work was funded through the internal Brno University of Technology project FCH-S-20-3613 and by the Ministry of Education, Youth and Sport of Czech Republic (project 7AMB19AT).

Fabrication and characterization of biodegradable nanofibers and nanoparticles with antioxidant and antimicrobial effect

Petra Matouskova, Renata Pavelkova, Lucia Dzuricka, Julie Hoova, and Ivana Marova

Department of Food Science and Biotechnology, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic email address: [email protected]

This work was focused on preparation and characterization of PHB nanoparticles and nanofibers containing some biologically active components, mainly lipid extracts of selected plant species. Prepared and functionalized PHB nanomaterials were tested for their potential antimicrobial and antioxidant effects. For potential medical application cytotoxicity of selected particles and fibers was tested by MTT assay using human keratinocytes (HaCat).

PHB nanofibers were prepared via different techniques but mainly electrospinning and forcespinning technique were used. The nanofibers morphology was confirmed by SEM. FLIM and FTIR-ATR methods were used for detailed fiber characterization. PHB nanoparticles were prepared using sonication techniques and the encapsulation efficiency as well as size and colloid stability of prepared nanoparticles were measured. Determination of antioxidant activity and content of active component in the nanostructure was performed by spectrophotometry. The results showed high antioxidant effect of tested PHB nanomaterials, ability to release active substances under different conditions as well as good antimicrobial activity against a wide range of microorganisms. Prepared functionalized PHB nanomaterials also exhibited no cytotoxicity effect and thus they can be used for skin application. Prepared PHB nanomaterials with antimicrobial and antioxidant effects are promising for local treatment applications in wound dressing and other clinical and cosmetic applications.

Applications of biotechnologies in sex-sorted sperm in farm animals

Dana Tapaloaga^a, Mara Georgescu^a, Anca Alexandra Popescu^b, Carmen Daniela Petcu^a, Vlad Cristian Dobrea^b, Makki Khalaf Hussein Al Dulaimi^c, Lucian Ionel Ilie^a, Paul Rodian Tapaloaga^a, and Dragos Georgescu^b

^aDepartment of Animal Production and Public Health, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania

^bUniversity of Medicine and Pharmacy Carol Davila Bucharest, Romania

^cAl-Furat Al-Awasat Technical University, Iraq email address: [email protected]

In the animal industry, changing the sex ratio of offspring can increase genetic progress, productivity and animal welfare. Numerous techniques requiring to alter the sex ratio have been proposed in mammals, but none of these were able to produce statistically significant separation of fertile X and/or Y sperm populations or were not repeatable even sex-sorted semen is in high demand. While the current technique based on modified flow cytometry separates X and Y chromosome-bearing sperm with high accuracy is limited because each sperm cell has to be characterized and sorted individually, its commercial utilization differs significantly among species. New qualitative methods, in combination with nanoparticles and microfluidic systems are genetic techniques which provide a simplified system to generate offspring, not only of the required sex, but also incorporating other important genetic characteristics. Gene editing has become a fast-growing research area since the development of sequence-specific nucleases and four different editing processes: meganuclease, zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats entered the animal experimental reproduction. Sex-sorted sperm in farm animals will play a major role in providing food, with sustainable protection of the environment, to meet the needs of a growing world population.

Obtaining functional and structural biocomposite materials of new generation based on microbial polysaccharides

Victor Revin

National Research Mordovia State University, Saransk, Mordovia Republic email address: [email protected]

Currently, the number of publications and technological developments on the creation of new materials based on microbial polysaccharides has increased. We have obtained highly productive strains that form up to 7 g/l of bacterial cellulose biopolymer. Technologies of biocomposite materials, aerogels have been developed on the basis of BC. It is shown that the strength properties of aerogels depend on the density of the material (it is 12 times greater than the density of air) and the uniformity of the porous structure of aerogels (the average pore size is from 80 to 150 microns). The resulting material has an extremely low thermal conductivity-0.027 W/(m K), comparable to air and superior to mineral wool and expanded polystyrene. Its sound absorption is higher than that of materials on the market. The second polysaccharide under study is Levan. Based on it, technologies for biocomposite materials, including pressed materials from wood waste, have been developed. The obtained samples are not inferior in their parameters to traditional ones based on toxic phenol-formaldehyde resins. The tensile strength under static bending of the best Levan-based biocomposites was about 24–29 MPa, the density was 1170–1250 kg/m3, and the water absorption was about 7%.

Classification of Colon polyps by comparing deep learning and machine learning methods

Refika Sultan Doğan^a and Bülent Yılmaz^b

^aBioengineering Department, School of Life and Natural Sciences, Abdullah Gul University, Kayseri, Turkey

^bElectrical Electronics Engineering Department, School of Engineering, Abdullah Gul University, Kayseri, Turkey email address: [email protected]

Colorectal cancer usually starts with the development of certain tissues (polyps) in the large intestines. Polyps have hyperplastic, adenoma and serrated types. Here, we investigated an automatic image analysis method that can classify different types of polyps on colonoscopy images as (1) resection or non-resection, and (2) adenoma, serrated, or hyperplastic to guide gastroenterologists. In machine learning (ML) part we extracted histogram of gradient features and used random forest classifier. In deep learning (DL) part we constructed a convolutional neural network (CNN) architecture. In the training and test phases 40 adenoma, 21 hyperplastic, and 15 serrated polyp videos (75,678 frames) obtained using white light imaging (WLI, 36,285 frames) and narrow-band imaging (NBI, 39,393 frames) were used. This dataset includes the decisions of 7 doctors whose accuracies were 80% for two and 61% for three categories on average. Our results showed that using ML we achieved better accuracies than all doctors, in both NBI (acc. 87%) and WLI (acc. 94%) modalities for the binary classification. For the three-category classification our performances were similar to the doctors. DL-based classifications resulted in lower accuracies than ML-based approach. DL and ML tools are feasible to predict histopathological character of colon polyps to guide polypectomy.

Role of voltage dependent potassium (Kv) currents in human granulosa cell physiology

Bernard Fioretti^a, Lorenzo Monarca^a, Francesco Ragonese^b, Antonella De Luca^a, Chiara Pennetta^a, Federica Patria^a, Loretta Mancinelli^a, Rossana Giulietta Iannitti^c, and Lucio Leonardi^c

^aDepartment of Chemistry, Biology and Biotechnologies, University of Perugia, Perugia, Italy

^bDepartment of Experimental Medicine, Perugia Medical School, University of Perugia, Perugia, Italy

^cS&R Farmaceutici S.p.A Bastia Umbra, Perugia, Italy email address: [email protected]

Resveratrol is a nutraceutical molecule with several therapeutic effects. It has been shown to mimic effects of caloric restriction, exert anti-inflammatory and anti-oxidative effects, and affect the mitochondrial functionality but the involvements of ion channels activity for these effects are not completely defined. Recently, we described the effects of resveratrol (3 μM, 48 h) on promoting mitochondria biogenesis in human granulosa cells (h-GCs) and a depolarization of resting membrane potential by inhibiting voltage dependent potassium (Kv) currents. Now, we studied the pharmacological and molecular profile of Kv currents in h-GCs. Kv current modifies the biophysical properties and expression during time of culture h-GCs indicating an involvement of a cellular differentiation program. Kv currents are sensitive in various extents to PAP-1 (5-(4-phenoxybutoxy)psoralen) and RT PCR analysis demonstrated the presence of several member of kv1 sub family strictly associated to a differentiation program. Furthermore, we evaluated the functional expression of voltage dependent calcium currents (Cav) by using calcium imaging and patch clamp techniques. Our data suggests that potassium currents setting the resting membrane potential and intracellular calcium levels in h-GCs can be involved in differentiation, mitochondrial physiology and hormon regulation on the physiology of the female reproductive system.

Rethinking wastewater risks and monitoring in light of the COVID-19 pandemic

Ariel Kushmaro

Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben Gurion University, Beer Sheva, Israel email address: [email protected]

SARS-CoV-2 is an RNA virus, which had an acute and dramatic impact on health care systems, economies, and societies. Testing and tracing efforts are being employed in many countries in attempts to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common, and that the virus RNA can be detected in wastewater. This indicates that wastewater monitoring may provide a potentially efficient tool for epidemiological surveillance of SARS-CoV-2 in large populations at relevant scales. In particular, this provides important means of (i) estimating the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements, (ii), managing the early-warning system quantitatively and efficiently, and (iii) verifying disease elimination. Here we report different virus concentration methods or filtration techniques as well as different RNA extraction methodologies, providing important insights regarding detection of SARS-CoV-2 RNA in sewage. Virus RNA particles were detected in wastewater in several geographic locations in Israel. In addition, correlation of virus RNA concentration to morbidity was detected in Bnei-Barak city during April 2020. This study presents a proof of concept for the use of direct raw sewage associated virus data, during the pandemic in the country as a potential epidemiological tool.

Epidemiological model for the inhomogeneous spatial spreading of COVID-19 and other diseases

Rony Granek^a and Yoav Tsori^b

^aThe Avram and Stella Goldstein-Gorren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, , Israel

^bDepartment of Chemical Engineering, Ben-Gurion University of the Negev, Israel email address: [email protected]

We suggest a mathematical framework for the in-homogeneous spreading of an infectious disease in human population, with particular attention to COVID-19. Common epidemiological models, e.g. the well-known susceptible-exposed-infectious-recovered (SEIR) model, implicitly assume uniform (random) encounters between the infectious and susceptible sub-populations, resulting in homogeneous spatial distributions. However, in human population, especially under different levels of mobility restrictions, this assumption is likely to fail. We develop a novel “reaction-diffusion”' spatial model that includes five different sub-populations, in which the infectious sub-population is split into pre-symptomatic and symptomatic. Our model accounts for spreading of infectious population domains from initial epicenters. Importantly, we also account for the variable geographic density of the population, and show how weakly infected regions surrounding a densely populated area can cause rapid migration of the infection towards the populated area. Predicted infection “heat-maps” show remarkable similarity to publicly available heat-maps, e.g. from South Carolina. We further demonstrate how localized lockdown/quarantine conditions can slow down the spreading of disease from epicenters. Application of our model in different countries can provide a useful predictive tool for the authorities, in particular, for planning strong lockdown measures in localized areas – such as those underway in a few countries.

Bringing proof of value to the lateral flow immunoassay using proprietary innovations

Robert S. Marks

Department of Biotechnology Engineering, Ben Gurion University of the Negev, Israel email address: [email protected]

Diagnostics is an important component of healthcare, finding roots before antiquity, both East and West, and enabling the physician to direct the appropriate treatment. Some diseases are easily diagnosed by the eye (paracoccidioidomycosis, …), but most are not. One direction taken for the future in point-of-care diagnostics, such as the familiar and convenient pregnancy test based on lateral flow immunoassay technology and of course the glucose monitor, both being used at home. There are limitations in both, however, we modified the configuration of both so as to create useful proof of value. We will discuss, for thyroid cancer, creating quantitation ability using our proprietary electrolateral flow immunosensor, enabling multiplex testing with our vertical lateral immunoassay, providing self-calibration enabling cell phone reading with our capture flow immunoassay. All these bring us closer to convenient cheap home diagnostics.

Targeted modification of gut microbiota by individualized probiotic supplementation

Petr Rysávka^a, Martina Kohutkova Lánova^a, Radek Netusil^a, Eva Frysová^a, Marketa Hanackova^a, Jana Cmielova^a, Kamil Sťastny^b, Ivana Marova^c, and Petr Rysávka^d

^aMedi Pharma Vision s.r.o., Brno, Czech Republic

^bBrno University of Technology, Faculty of Chemistry, Brno, Czech Republic

^cVeterinary research institute v.v.i., Brno, Czech Republic

^dMedi Pharma Vision s.r.o., Brno, Czech Republic, Veterinary research institute v.v.i., Brno, Czech Republic email address: [email protected]

Study compares stool samples of 48 volunteers before and after individualised probiotic treatment. Stool sample bacterial composition of individual patients was characterised by 16 s rRNA sequencing. On the basis of this characteristic, the probiotic composition (species, number of species and CFU) was designed and prepared. After 3 months of probiotic supplementation, a control sample was analysed. Data confirm a significant increase of specific bacterial groups (lactobacilli, bifidobacteria, actinobacteria) as well as total number of species. Results clearly show that individualised probiotic treatment leads to enhancement of gut microbiome by beneficial bacterial groups and thus positive modulation of gut microbiota and host health.

Micro and nanoscale drug delivery systems: innovations and translational challenges

Shyam Mohapatra

Center Research & Education in Nano-bioengineering, Department of Internal Medicine, Morsani College of Medicine, and USF Health Taneja College of Pharmacy, Tampa, FL, USA email address: [email protected]

Nanoscale Drug Delivery Systems (NDDS) has rapidly become a revolutionary force that is driving innovation in the medical field. NDDS is generating solutions to many conventional challenges through the development of enhanced delivery systems for therapeutics, and theranostics. Thus, many novel nanocarriers that each express specifically designed physiochemical properties that optimize their desired pharmacokinetic profile. NCs are also being integrated into nanoscale platforms that further enhance their delivery by controlling and prolonging their release profile. Additionally, stimuli-responsive theranostic nanocarriers are quickly developing as vectors for specific targeting of diseased cells and tissues. Overall, BNT is an interdisciplinary approach towards health care, and has the potential to significantly improve the quality of life for humanity by significantly decreasing the treatment burden for patients, and by providing non-invasive therapeutics that confer enhanced therapeutic efficiency and safety. This presentation will review progress made in this field and challenges that remain using two examples: one focused on the NDDS for the respiratory syncytial virus infection causing lung disease and another NDDS relating to the delivery of drugs to the brain affected by traumatic brain injury.

Application of artificial intelligence in biomedicine

Ugur Sezerman^a and Munis Dundar^b

^aDepartment of Biostatistics and Bioinformatics, Acıbadem Mehmet Ali Aydınlar University, Istanbul, Turkey

^bDepartment of Medical Genetics, Medical Faculty, Erciyes University, Kayseri, Turkey email address: [email protected]

There are different mechanisms at play for complex diseases understanding key mechanisms that are affected to study overall disease aetiology is crucial to determine novel therapy targets. Complex disease aetiology can be studied in detail by integration of multi-omics data each presenting different dimension in the disease development mechanism. Throughout the great achievements in the next generation sequencing and related omics technologies, vast amounts of data have been produced and became accessible to the public. Integration of these multi-omics data enabled us to study disease causing mechanisms for complex diseases. To study individual mechanisms at play for disease aetiology one has to integrate different omics data in a pathway related manner to reveal altered pathways at a personalized level. The most important need here is to develop artificial intelligence (AI) algorithms that are in capable of analysing that big data, which will eventually be one of the most impactful applications employed in personalized medicine field. Especially in case of rare diseases, numerous variants are being reported in the literature. Filtering different types of variants, extracting the ones associated with the individual symptoms, and personalized diagnosis followed by determination of individualized treatment strategies can be presented as one of the biggest promising application area of AI-directed multi-omics strategies. Another important field of this application will be in cancer and other complex diseases like neurodegenerative diseases. There are numerous data produced from different sources including whole genome, transcriptome, proteome, and microbiome. Integration of all these data through AI leads identification of driver mechanisms behind the development and/or progression of the diseases, and enables development of new and more accurate diagnosis and treatment strategies. In this talk, we will go over briefly types of omics data and information they provide then briefly summarize AI methods employed to analyse 'omics data' to determine different mechanisms at play in complex disease development and identify novel therapeutic candidates. We will give examples from cancer neurological diseases and rare diseases.