Programme and Abstract Book

EUROBIOTECH 6th Central European Congress of Life Sciences Eurobiotech

11-14 September 2017

PROGRAM and ABSTRACT BOOK

Organizers and Partners

Polish Federation of Biotechnology

Patronages

Honorary Patronage Division II: Biological and Agricutural Sciences, PAS

Honorary Patronage Jacek Majchrowski Mayor of the City of Kraków

Honorary Patronage Jacek Krupa Marshal of the Malopolska Region

Partners

Committee of Biotechnology, PAS

Congress financed by the WFOŚiGW funds in Kraków

Media Patronages

EUROBIOTECH 6th Central European Congress of Life Sciences Eurobiotech

11-14 September 2017

PROGRAMME and ABSTRACT BOOK

Editor: Targi w Krakowie Ltd. Galicyjska Str 9, 31-586, Kraków Cover Layout: Anna Grzelak DTP, Print: TECHNET

Contents Welcome Message __________________________________________________________________________________ 5 Programme Overview ________________________________________________________________________________ 6 General Information __________________________________________________________________________________ 8 Detailed Conference Programme _________________________________________________________________________ 13 Posters ___________________________________________________________________________________________ 28 Index of Authors Presenting Posters _____________________________________________________________________ 29 List of Posters Presentations ___________________________________________________________________________ 31 Abstracts _________________________________________________________________________________________ 41 Sponsors & Partners _________________________________________________________________________________ 228 Exhibitors _________________________________________________________________________________________ 221 List of Exhibitors ____________________________________________________________________________________ 236

4 Committees

Scientific Committee: Prof. Ada E. Yonath, Nobel laureate, Weizmann Institute of Science, Israel, honorary Chairlady Prof. Tomasz Twardowski, President of the Biotechnology Committee, Polish Academy of Sciences, Chair Prof. Andrzej Kononowicz, President of the Polish Federation of Biotechnology, Co-chair Prof. Kazimierz Strzałka, Jagiellonian University, Co-chair Prof. Piotr Laidler, Jagiellonian University Medical College, Co-chair Prof. Jan Barciszewski, Institute of Bioorganic Chemistry, Polish Academy of Sciences Prof. Grzegorz Węgrzyn, Gdańsk University Prof. Józef Dulak, Jagiellonian University Prof. Maria A. Ciemerych-Litwinienko, University of Warsaw Prof. Jan Chłopek, AGH University of Science and Technology Ms. Ewa Woch, President, Targi w Krakowie Ltd.

Organizing Committee: Ewa Woch, Targi w Krakowie Ltd., Chair Prof. Tomasz Twardowski, President of the Biotechnology Committee PAS, Co-chair Sara Lamik, Targi w Krakowie Ltd. Weronika Plata, Targi w Krakowie Ltd. Prof. Kazimierz Strzałka, Małopolska Centre of Biotechnology

Session Organizers: Prof. Jan Barciszewski, Institute of Bioorganic Chemistry, Polish Academy of Sciences (sessions no.: 1; 13; 21) Prof. Piotr Laidler, Jagiellonian University in Kraków, Medical College (session no. 1) Prof. Józef Dulak, Jagiellonian University in Kraków (sessions no.: 2&5; 17; 22) Prof. Maria Anna Ciemerych-Litwinienko, Warsaw University (session no. 2&5) Prof. Andrzej Kononowicz, University of Lodz (session no. 3) Prof. Rafał Barański, University of Agriculture in Kraków Prof. Jerzy Długoński, University of Lodz (sessions no.: 4; 12) Prof. Jan Szopa (session no. 6) Dr Magdalena Żuk (session no. 6) Prof. Małgorzata Łobocka, Warsaw University of Life Sciences (session no. 7) Dr Kenji Yamada, Jagiellonian University in Kraków (session no. 8) Prof. Kazimierz Strzałka, Jagiellonian University in Kraków (session no. 8) Prof. Jan Chłopek, AGH University of Science and Technology in Kraków (session no. 9) Prof. Grzegorz Węgrzyn, University of Gdańsk (session no. 10) Dr Aleksandra Małyska, European Technology Platform ‘Plants for Europe’, Brussels (session no. 11) Prof. Joanna Surmacz- Górska, Silesian University of Technology, Gliwice (session no. 14) Prof. Jonathan Gardiner Heddle, Jagiellonian University in Kraków (session no. 15&18) Prof. Ryszard Słomski, Poznań University of Life Science (session no. 16) Prof. Grzegorz Węgrzyn, University of Gdańsk (session no. 17) Prof. Wojciech Karłowski, Adam Mickiewicz University in Poznań (session no. 19) Prof. Jolanta Jura, Jagiellonian University in Kraków (session no. 20) Prof. Ewa Bartnik, Warsaw University (session no. 22)

6th Central European Congress of Life Sciences Eurobiotech 2017

Welcome Message

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Ladies and Gentelman, Welcome to Krakรณw!

Welcome to Eurobiotech 2017!

Holistic view of science is more demanding, but it can also be much more satisfying, than studying individual disciplines in isolation. The use of molecular biology in the past 50 years has led to applications that have dramatically changed our society: towards a broader and more integrated approach to science, technology or economy, it helps address particular problems like environmental issues, rural and industrial development, many diseases therapies. Biotechnology became even subject to social sciences, including art and potential dual use of biotechnology.

Many industrial sectors, such as automotive, construction, textiles, packaging, food and feed, etc. have undergone dynamic transformation due to the increasing use of biological materials and biological methods of production and processing [mostly enzymatic processes]. All these [and many more!] aspects of modern and innovative bio-sciences are included in our sessions.

Once again: WELLCOME to KRAKร W! We wish you very productive and pleasant participation in scientific sessions and... do not forget to visit the Main Market and to see the beauty of Krakรณw at night.

Tomasz Twardowski

On behalf of the Scientific and Organizing Committees

6th Central European Congress of Life Sciences Eurobiotech 2017

PROgRAMME OvERviEW Time

Session room: Bratysława

Session room: Wiedeń

Session room: Budapeszt

Monday, 11 September 8:00-20:00

Registration, bagde and bag collection

10:00-12:00 Satellite Event: Roundtable discussion: “Microbiome towards securing sustainable food system”

11:00-12:00 12:30-14:00 14:15-16:00

Opening Ceremony

16:10-17:55

Stem Cells (S2)

Nucleic acids tools for regulation of gene expression (S1)

Plant biotechnology (S3)

Stem Cells (S5)

Microbial elimination of deleterious compounds (S4)

The tools of epigenetics (S6)

17.55-18.25 18:25-19:55 20:00-22:00

Tuesday, 12 September 8:00-9:45

Plenary session

9:45-11:15 11:15-13:00

Bacteriophages in biotechnology (S7)

Molecular mechanisms of plant response to environmental stresses (S8)

13:00-14:00 14:00-15:30

Plenary session

15:40-17:25

Microbial cell factories (S10)

Plants for the european bioeconomy (S11)

17:35-19:20

Modern instrumental analysis in biotechnology and biomedicine (S12)

Biotechnology for beauty (S13)

Biomaterials engineering – new material technologies for tissue engineering (S9)

Wednesday, 13 September 9:15-11:00

Plenary session

11:00-11:30 11:30-13:15

From molecular machines to molecular medicine: advances in biotechnology (S15)

Microorganisms – from environment to biotechnology (S14)

Dual use of biotechnology (S16)

From molecular machines to molecular medicine: advances in biotechnology (S18)

Stem cells in diseases and therapy (S17)

Big data and biotechnology (S19)

13:15-15:30 15:30-17:15 20:00

Thursday, 14 September 9:00-10:30

Plenary session

10.40-12:25

Tools in the treatment of human disorders (S20)

12.25-13.00 13:00-14:15

Closing Ceremony

Art in biotechnology (S21)

Bioethics (S22)

Programme Overview

Session room: Praga

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Session room: Lwรณw

Dunaj Hall

Satellite Event: Student Session Satellite Event: How to make money on science?

Satellite Event: Student Session Coffee break

Welcome Reception

Coffee break and poster session

Lunch

Coffee break

Lunch & Poster session

Coffee break 6th Central European Congress of Life Sciences Eurobiotech 2017

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General Information

REGISTRATION Badges and registration

For security reasons, badges must be worn at all times inside the congress venue and at all events related to the Eurobiotech Congress (this includes lunches and evening social programme for delegates and accompanying persons).

Registration opening hours:

Monday 11th September 2017 8:00–20:00 Tuesday 12 th September 2017 7:30–19:30 Wednesday 13th September 2017 8:30–17:30 Thursday 14th September 2017 8:00–14:00 On-site registration is possible during registration desk opening hours.

VENUE INFORMATION

The Congress takes place at the International Convention and Exhibition Centre EXPO Krakow, Galicyjska 9, 31-586 Kraków, Poland.

Conference rooms are: Bratysława (Plenary Hall), Wiedeń and Budapeszt.

Wi-Fi internet access

Free Wi-Fi internet access is provided.

Wireless network: Eurobiotech Password: Eurbiotech2017Expo

ATM

There is an ATM situated in the lobby of EXPO Krakow

Catering

Coffee breaks Coffee will be served in the exhibition and poster area. Coffee is included in the registration fees for congress delegates. Coffee and tea is served all the time. Small snacks will be served during coffee breaks according to the Congress program, as listed below: Monday, 11th September: 17.55–18.25 Tuesday, 12 th September: 9:45–10:15 Wednesday, 13th September: 11:00–11:30 Thursday, 14th September: 12.25–13.00 Lunches Lunches will be served in the exhibition and poster area (Dunaj Hall) on Tuesday and Wednesday.

Tuesday, 12 th September: 13:00–14:00 Wednesday, 13 th September: 13:15 - 14:15 On Monday and Thursday lunches will not be provided. Participants can buy something to eat in the bistro.

Speaker preview room

A speaker preview room is located on the ground floor, Meeting Room SO7.

Cloakroom

EXPO Krakow provides a cloakroom that is manned like Registration Opening Hours. The Cloakroom is located on the ground floor, Meeting Room S06.

Smoking

No smoking is allowed at EXPO Krakow. Please go to the designated areas outside the building.

6th Central European Congress of Life Sciences Eurobiotech 2017

General Information

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2 3

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1

7 6 8

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1.

hala WISŁA / WISŁA hall

2.

hala DUNAJ / DUNAJ hall

3.

sala konferencyjna BRATYSŁAWA / BRATYSŁAWA conference room

4.

sala konferencyjna WIEDE¡ / WIEDE¡ conference room

5.

sala konferencyjna LWÓW / LWÓW conference room

6.

sala konferencyjna BUDAPESZT / BUDAPESZT conference room

7.

sala konferencyjna PRAGA / PRAGA conference room

8.

pokoje spotkaƒ / meeting rooms 6th Central European Congress of Life Sciences Eurobiotech 2017

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General Information

Transfers

Transfers are provided from the M1 Shopping Mall parking to the venue.

Estimated time of the journey: 10 minutes one way.

Shuttle bus will rotate according to the following schedule – every fifteen / twenty minutes.

11.09

12.09

13.09

14.09

9:00–14:30 20:00–22:30

7:30–9:30 19:15–20:15

9:00–10:30 17:00–18:00

8:30–10:00 13:30–15:30

Shell Gas Station Eurobiotech Congress Shuttle Bus Stop

M1 Shopping mall

M1 Shopping mall

EXPO Kraków

6th Central European Congress of Life Sciences Eurobiotech 2017

General Information

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SOCIAL PROGRAMME

Monday, 11th September 2017

WELCOME RECEPTION 20:00–22:00 Location: Exhibition (Dunaj Hall) Dress Code: Business Casual A welcome reception will take place in the exhibition hall at EXPO Krakow. This event is free to all delegates.

Wednesday, 13th September 2017

GET-TOGETHER PARTY Start at 20:00 Location: Kogel Mogel Restaurant Address: Sienna 12, 31-041 Kraków Dress Code: Business Casual No coaches will be provided. We kindly ask delegates to make their own way. The Restaurant is centrally located.

Attendance is by invitation or pre-purchased as part of the registration process. Please bring your ticket, located in your badge holder. The registration team will be able to confirm whether any places remain available for the Get-together Party.

Main Market Square

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Mariacki Church Kogel Mogel Restaurant

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GALA DINNER

GET-TOGETHER PARTY Kogel Mogel Restaurant; 12 Sienna Str., 31-041 Kraków, Start at 20:00

6th Central European Congress of Life Sciences Eurobiotech 2017

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General Information

KRAKOW LOCAL INFORMATION

Currency

The official currency in Poland is the Polish Zloty (PLN). €1 equals approximately 4 PLN.

Emergency services

For Police, dial 997.

For Fire, dial 998.

For Ambulance, dial 999.

Using mobile phones: remember to dial the local code before the number, for example: 12 - 997 to call the police in Krakow.

The European Emergency Number 112 can also be used.

Taxis

The best way to order a taxi is by telephone.

Calling ahead will get you a better fare. Please find taxi numbers below: MPT: 19633 iCAR +48 12 653 5555, +48 12 888 0000 Mega Taxi +48 12 400 00 00, +48 12 196 25

KRAKÓW

Krakow is the former capital of Poland, the residence of Polish kings and the seat of the oldest university in central Europe.

The city was fortunately not destroyed during its volatile history and today competes with some of the most beautiful European cities. The medieval old town, which is on the UNESCO List of World Heritage Sites, is the dynamic heart of Krakow, buzzing with students, residents and tourists from all over the world.

The key points of interest are the main Market Square (the largest medieval square in Europe), the Royal Castle Wawel, the Collegium Maius of Jagiellonian University, the Cloth Hall, the old Jewish district of Kazimierz and many more. Krakow is also a modern metropolis with 1 million inhabitants. It is a vibrant academic centre with more than 200,000 students studying in over 20 higher education institutions and universities, and also has a vigorous new technologies and life sciences centre. It is Poland’s second business centre after the capital Warsaw and home to many international corporations.

Tram and bus tickets While Krakow has no underground metro system it does have an integrated bus and tram system which runs from 05:00 - 23:00, with night trams and buses continuing less frequently in between. Check timetables and network maps online at mpk.krakow.pl To get to the Venue using the tram you can use lines: 1,14,22 and take stop: RONDO 308 DYWIZJONU For the bus you can get off at M1 NOWOHUCKA Tickets can be bought at newsagents or in ticket machines at bus and tram stops. Ticket machines can also be found at a number of bus and tram stops. Drivers can sell tickets as well, but note: they only have “multiple trip valid 60 min” tickets and reduced tickets available and you can only pay in cash.

Ticket prices for all day routes and night routes: any one journey up to 40 minutes for 1 person: 3,80 PLN (standard), 1,90 PLN (half-rate) short-term for 1 person: 24 h ticket: 15,00 PLN (standard), 7,50 PLN (half-rate) 48 h ticket: 24,00 PLN (standard), 12,00 PLN (half-rate) 72 h ticket: 36,00 PLN (standard), 18,00 PLN (half-rate) 7 days ticket: 48,00 PLN (standard), 24,00 PLN (half-rate)

6th Central European Congress of Life Sciences Eurobiotech 2017

DETAILED CONFERENCE PROGRAMME

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Detailed Conference Programme

Monday, 11.09.2017

10:00–12:15 Satellite Event: Student Session Location: LWÓW CONFERENCE ROOM Chair: Monika Grzebyk, Jagiellonian University in Kraków TIME

PRESENTATION

10:00

Opening of the Workshop, Monika Grzebyk

10:10

Jeff Cole, University of Birmingham How to make sure your paper will be rejected?

10:30

Agata Tyczewska, “Biotechnologia”, Poznań How to increase the chance that your paper will be accepted?

10:45

Piotr Kochan, “World Journal of Medical Images, Videos and Cases”, Kraków Hints for best titles and good abstracts

11:00

Questions to the panel

11:20

Jeff Cole, University of Birmingham How to present your work at a scientific meeting?

11:50

Questions and discussion

12:00

Maria Szrajber, German Academic Exchange Service (DAAD), Warsaw Scholarships for language courses, studying and academic research in Germany

12:15–14:15 Student Session 1: Health & Medical Microbiology Student’s presentations

16:05–17:50 Student Session 2: Molecular, Industrial & Green Biotechnology Student’s presentations

12:30–14:00 Satellite Event: How to make money on science?

Location: BUDAPESZT CONFERENCE ROOM Chairs: Dorota Hubka, Fundacja Małopolskie Centrum Transferu Technologii (Technology Transfer Center Foundation in Malopolska), Kraków & Kamil Kipiel, Centrum Transferu Technologii Medycznych, Kraków

11:00–12:00 Satellite Event: Roundtable discussion: “Microbiome towards securing sustainable food system” Location: PRAGA CONFERENCE ROOM coordinator: Aleksandra Małyska Plenary opening of the discussion: Marios Markakis; European Commission, Brussels KEYNOTE SPEAKER: Roland Kozłowski, Life Science Biznes Consulting (LSBC), Kraków Panel discussion “How to make money on science?”: • Aleksandra Moscicka-Studzińska, National Center for Research and Development, Warsaw • Maciej Wierzbicki, Project Director, Evestra Oncology, Łódź • Dorota Hubka-Wójcik, Małopolska Center for Technology Transfer, Krakow • Kamil Kipiel, Center for Technology Transfer of Medical Technology Park, Krakow

6th Central European Congress of Life Sciences Eurobiotech 2017

Detailed Conference Programme

Monday, 11.09.2017

14:00–16:00 Opening ceremony

Location: BRATYSŁAWA CONFERENCE ROOM Chair: Tomasz Twardowski, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań TIME

PRESENTATION

14:15 14:30

Introduction OPENING SPEAKER: Ada Yonath, Nobel Prize Winner, Weizmann Institute of Science, Rehevot A bright Future for Antibiotics? Medical and Environmental Consideration PLENARY SPEAKER: Nikolaus Rajewsky, Max Delbruck Center, Berlin Linear and circular RNAs Jeff Cole, University of Birmingham Invitation to the 18th European Congress on Biotechnology, Geneva 2018

15.15 16:00

Session 1: Nucleic acids tools for regulation of gene expression

Location: WIEDEŃ CONFERENCE ROOM Chairs: Jan Barciszewski, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań & Marta Dziedzicka-Wasylewska, ­Jagiellonian University in Kraków TIME

PRESENTATION

16:10

KEYNOTE SPEAKER: Anna Maria Kietrys, Department of Chemistry, The Stanford University RNA modifications and NGS: It’s not a bug, it’s a feature Katarzyna Rolle, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań Pioneering ribozyme-based strategy for mitochondria transformation and gene regulation Kinga Kocemba-Pilarczyk, Jagiellonian University in Kraków Transcriptional silencing of the CD146 expression in breast cancer cells by promoter Methylation Natalia Zofia Pstrąg, Adam Mickiewicz University in Poznań Landscape of RNA and protein processing of TMEMs in clear cell renal cell carcinoma Renata Grzela, University of Warsaw Hydrolytic activity of the hNudt16 towards oligonucleotides bearing different native cap structures Marcin Równicki, University of Warsaw & Centre of New Technologies Vitamin B12 as a carrier of peptide nucleic acid oligomers into bacterial cells

16:40 16:55 17:10 17:25 17:40

Session 2: Stem cells

Location: BRATYSŁAWA CONFERENCE ROOM Chairs: Rita Perlingeiro, University of Minnesota & Maria A. Ciemerych-Litwinienko, University of Warsaw TIME

PRESENTATION

16:10

KEYNOTE SPEAKER: Zipora Yablonka-Reuveni, University of Washington, Seattle, Washington Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency Józef Dulak, Jagiellonian University in Kraków New modifiers of Duchenne muscular dystrophy Paulina Kasprzycka, University of Warsaw TGFß1 pathways and MMPs activity in myoblasts differentiation Iwona Bronisz, Jagiellonian University in Kraków Role of Nrf2 in muscle satellite cells differentiation and progression of Duchenne muscular dystrophy Roger Lijnen, KU Leuven ADAMTS5 knockdown impairs murine adipogenesis

16:40 17.10 17.25 17.40

6th Central European Congress of Life Sciences Eurobiotech 2017

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Detailed Conference Programme

Monday, 11.09.2017

Session 3: Plant biotechnology

Location: BUDAPESZT CONFERENCE ROOM Chair: Rafał Barański, University of Agriculture in Kraków & Stanisław Flasiński, Monsanto Company, St. Louis TIME

PRESENTATION

16:10

KEYNOTE SPEAKER: Stanisław Flasiński, Monsanto Company, St. Louis Expanding biotechnology approaches to control insect pests

16:40

Aydin Akbudak, Akdeniz University, Turkey Reduction of Lignin Content in Rice Straw for Biofuel Production

16:55

Agnieszka Szopa, Jagiellonian University in Kraków, Collegium Medicum Improved production of dibenzocyclooctadiene lignans in elicited microshoot cultures of Schisandra chinensis

17:10

Demet Erdonmez, Aksaray University,Turkey A dye degradation study: Green synthesis of Iron Nanoparticles

17:25

Weronika Babińska, Intercollegiate Faculty of Biotechnology University of Gdańsk and Medical University of Gdańsk First report on the occurrence of Pectobacterium carotovorum subsp. brasilienses in Polish waterways

17:40

Maider Astorkia, NEIKER-Tecnalia – Basque Institute of Agricultural Research and Development, Arcaute, Álava Useful catalogue of Candidate Gene Alleles established in different Guineensis, Oleifera and Hybrid Populations

17:55–18.25 Coffee break Location: DUNAJ HALL

Session 4: Microbial elimination of deleterious compounds Location: WIEDEŃ CONFERENCE ROOM Chair: Jerzy Długoński, University of Lodz TIME

PRESENTATION

18:25

KEYNOTE SPEAKER: Jerzy Falandysz, Uniwersity of Gdańsk Emerging persistent halogenated organic pollutants“ “New Trends in Agriculture Biotechnology”

18:55

Anna Węgrzyn, Silesian University of Technology, Gliwice Diversity of endophytic bacteria from Miscanthus exposed to diclofenac and sulfamethoxazole mixed pollution.

A cultivation dependent vs a cultivation independent

19:10

Przemysław Bernat, University of Lodz Lipidomic adaptations in the filamentous fungus, auxin herbicides degrader

19:25

Łukasz Chrzanowski, Poznan University of Technology Environmental impact of herbicidal ionic liquids – from synthesis to advanced field studies

19:40

Amanda Pacholak, Poznan University of Technology The impact of xylose based surfactants on environmental microorganisms

6th Central European Congress of Life Sciences Eurobiotech 2017

Detailed Conference Programme

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Monday, 11.09.2017

Session 5: Stem cells

Location: BRATYSŁAWA CONFERENCE ROOM Chair: Józef Dulak, Jagiellonian University in Kraków & Zipora Yablonka –Reuveni, University of Washington, Seattle, Washington TIME

PRESENTATION

18.25

Maria A. Ciemerych-Litwinienko, University of Warsaw From pluripotency to myogenesis

18.55

KEYNOTE SPEAKER Rita Perlingeiro, University of Minnesota iPS cell-derived myogenic progenitors for the treatment of muscular dystrophies: how far are we?

19.25

Jarosław Lewandowski, Institute of Human Genetics, Polish Academy of Sciences, Poznań The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin

19.40

Mateusz Jeż, Jagiellonian University in Kraków Role of heme oxygenase-1 in differentiation of human induced pluripotent stem cells to cardiomyocytes

Session 6: The tools of epigenetics

Location: BUDAPESZT CONFERENCE ROOM Chair: Magdalena Żuk, University of Wrocław TIME

PRESENTATION

18:25

KEYNOTE SPEAKER: Jan Szopa, University of Wrocław Crops improvement by target genome editing

18:45

Marta Koblowska, University of Warsaw HD2C histone deacetylase binds to SWI/SNF chromatin remodeling complex and act together to regulate Arabidopsis heat stress response

19:10

Magdalena Działo, University of Wrocław Oligodeoxynucleotides can regulate CHS gene expression in flax by changing DNA methylation in a sequencespecific manner

19:25

Bartłomiej Kraziński, University of Warmia and Mazury in Olsztyn Significance of microRNA-199a-5p and microRNA-199b-5p and their target genes in renal cancer cells

19:40

Marlena Szalata, Poznań University of Life Sciences Methylation analysis of HLA-E transgene in the transgenic pigs

20:00–22:00 Welcome Reception in DUNAJ HALL

6th Central European Congress of Life Sciences Eurobiotech 2017

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Detailed Conference Programme

Tuesday, 12.09.2017

Plenary session 8:00–9:45

Location: BRATYSŁAWA CONFERENCE ROOM Chair: Grzegorz Węgrzyn, University of Gdańsk TIME

PRESENTATION

8:00

PLENARY SPEAKER: Mauro Giacca, International Centre for Genetic Engineering and Biotechnology, Triest Biotechnological Drugs for Cardiac Repair and Regeneration

8:45

PLENARY SPEAKER: Krzysztof Bankiewicz, University of California, San Francisco Advances in gene therapy of brain degenerative diseases

9.30

Gold Sponsor Speech: Polpharma Biologics, Adriana Kiędzierska-Mencfeld Single use or stainless steel technology in manufacturing of biosimilar monoclonal antibodies

9:45–11.15 Coffee break & poster session & How to make money on science? – poster session Location: Dunaj Hall

Session 7: Bacteriophages in biotechnology

Location: BRATYSŁAWA CONFERENCE ROOM Chair: Małgorzata Łobocka, Warsaw University of Life Sciences TIME

PRESENTATION

11:15

KEYNOTE SPEAKER: Jeff Cole, University of Birmingham Microbial response to environmental stress when starved of oxygen

11:45

Izabela Sabała, International Institute of Molecular and Cell Biology in Warsaw Antistaphylococcal enzybiotics – structural bases of enzyme engineering

12:00

Elżbieta Jagielska, International Institute of Molecular and Cell Biology in Warsaw Auresine – new weapon against Staphylococcus

12:15

Aleksandra Głowacka, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw Analysis of phage gene encoding a new candidate for an antistaphylococcal agent

12:30

Agnieszka Necel, University of Gdańsk Bacteriophage vb_Eco4M-7 as the Golden mean in fight against Enterohemorrhagic Escherichia coli(EHEC)

12:45

Agnieszka Bednarek, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw Production of phage lytic enzyme of high anti-enterococcal activity in Escherichia coli

6th Central European Congress of Life Sciences Eurobiotech 2017

Detailed Conference Programme

Tuesday, 12.09.2017

Session 8: Molecular mechanisms of plant response to environmental stresses Location: WIEDEŃ CONFERENCE ROOM Chairs: Kenji Yamada & Kazimierz Strzałka, Jagiellonian University in Kraków TIME

PRESENTATION

11:15

KEYNOTE SPEAKERS: Przemysław Malec & Katarzyna Turnau, Jagiellonian University in Kraków Metal homeostasis and tolerance in plants: cellular and symbiotic aspects

11:45

Kenji Yamada, Jagiellonian University in Kraków Endoplasmic reticulum (ER) bodies constitute a defense system in Brassicaceae

12:15

Shino Goto-Yamada, Jagiellonian University in Kraków Roles of LON protease and autophagy in the peroxisomal quality control

12:30

Anna Wyrzykowska, Adam Mickiewicz University, Poznań Genome-wide identification of genes involved in the potato response to drought indicates functional evolutionary conservation with Arabidopsis plants

12:45

Jakub Kuczyński, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań MicroRNAs in soybean chilling stress response

13:00–14:00 Lunch Location: DUNAJ HALL

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Detailed Conference Programme

Tuesday, 12.09.2017

Plenary session 14:00–15:30

Location: BRATYSŁAWA CONFERENCE ROOM Chair: Józef Dulak, Jagiellonian University in Kraków TIME

PRESENTATION

14:00

PLENARY SPEAKER: Stefan Malepszy & Leszek A. Łyżnik, Warsaw University of Life Sciences Synthetic biotechnology as applied for crop improvement and molecular farming

14:45

PLENARY SPEAKER: Abhay Pandit, National University of Galway Going back to Roots: Biological-basis for Designing Biomaterials for the Injured and Degenerated Host

Session 9: Biomaterials engineering – new material technologies for tissue engineering

Location: BUDAPESZT CONFERENCE ROOM Chairs: Jan Chłopek, AGH University of Science and Technology in Kraków & Abhay Pandit, National University of Galway TIME

PRESENTATION

15:40

Klaudia Trembecka-Wójciga; Institute of Metallurgy and Materials Science, Polish Academy of Sciences, Krakow Bioinspired thin films materials structured by laser interference lithography for direct blood contact

15:55

Patrycja Domalik-Pyzik, AGH University of Science and Technology in Kraków 3D printing of chitosan and graphene oxide hydrogels for biomedical applications

16:10

Agnieszka Leszczyńska, Cracow University of Technology Improving the thermal stability of nanocrystalline cellulose: The influence of hydrolysis and modification conditions

16:25

Kinga Pielichowska, AGH University of Science and Technology in Kraków Preparation and characterization of polyoxymethylene-based nanocomposites containing functionalized hydroxyapatite for biomedical applications

16:40

Florian Ryszka, Pharmaceutical Research and Production Plant „Biochefa”, Sosnowiec Dynamics of content changes selected hormones (rh-GH or rh-PRL) in kidney prefunds rinse with Biolasol

16:55

Questions and answers

6th Central European Congress of Life Sciences Eurobiotech 2017

Detailed Conference Programme

Tuesday, 12.09.2017

Session 10: Microbial cell factories

Location: BRATYSŁAWA CONFERENCE ROOM Chair: Grzegorz Węgrzyn, University of Gdańsk TIME

PRESENTATION

15:40

KEYNOTE SPEAKER: Joanna Karczewska-Golec, University of Gdańsk Bacteriophages – cell factories for metal and metal oxide nanoparticle synthesis

16:10

Wojciech Smułek, Poznan University of Technology The perspectives of biodegradation of chloroaromatic pollutants enhanced with natural surfactants

16:25

Marta Woźniak-Karczewska, Poznan University of Technology The influence of growth conditions on the rhamnolipids production by 20 different Pseudomons aeruginosa species

16:40

Karolina Jakubczyk, Opole University Effect of phthalocyanines on S. epidermidis cells. In vitro studies

16:55

Agata Zdarta, Poznan University of Technology How do bacteria adapt to long-term hydrocarbons exposure?

17:10

Aleksandra Dydecka, University of Gdańsk The role of open reading frame 63 (orf63) in the development of phage lambda and Shiga toxin-converting bacterophage Phi24B

Session 11: Plants for the European bioeconomy

Location: WIEDEŃ CONFERENCE ROOM Chair: Aleksandra Małyska, European Technology Platform ‘Plants for Europe’, Brussels TIME

PRESENTATION

15:40

KEYNOTE SPEAKER: Dr. Marios Markakis; European Commission, Brussels Microbiome in the context of FOOD2030 and Food systems

16:10

Camilla Liput, Bayer CropScience NV EU legal framework for plants developed through biotechnology: present and future

16:25

Monika Bojko, Jagiellonian University in Kraków Leaf vegetables as important source of precursor omega–3 long chain polyunsaturated fatty acids essential in the human diet

16:45

Andrzej Czaplicki, Plant Breeding and Acclimatization Institute – NRI, Radzików The use of biodiversity of species of the Poaceae family in wheat cultivar T. aestivum L development

17:00

Masatake Kanai, National Institute for Basic Biology, Okazaki Extension of oil biosynthesis during the mid-phase of seed maturation increases seed oil in Arabidopsis thaliana

17:15

Questions and answers

6th Central European Congress of Life Sciences Eurobiotech 2017

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Detailed Conference Programme

Tuesday, 12.09.2017

Session 12: Modern instrumental analysis in biotechnology and biomedicine Location: BRATYSŁAWA CONFERENCE ROOM Chair: Jerzy Długoński; University of Lodz TIME

PRESENTATION

17:35

KEYNOTE SPEAKER: Andreas Otto, Ernst-Moritz-Arndt-University Greifswald Improving workflows for MS- based proteomics by spectral libraries

18:05

Rafał Szewczyk, University of Lodz Childhood tuberculosis – in search of diagnostic biomarkers

18:20

Adrian Topolski, Nicolaus Copernicus University in Toruń Spectrophotometry in quantitative determination of platinum complexes on titania nanomaterials

18:35

Anna Jasińska, University of Lodz Production, characterisation and mass spectrometry identification of novel multicopper oxidases from Myrothecium roridum

18:50

Angelika Podbielska, National Research Institute of Animal Production, Kraków DNA fragment analysis for sex identification of bird using capillary electrophoresis on 3100xl Genetic Analyzer

19:05

Neslihan Tekin, Aksaray University Evaluation of the effects of S-allyl cysteine on autophagy and apoptosis in human leukemia cell line

Session 13: Biotechnology for beauty

Location: WIEDEŃ CONFERENCE ROOM Chairs: Jan Barciszewski & Dominika Malińska, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Poznań TIME

PRESENTATION

17:35

KEYNOTE SPEAKER: Suresh I.S. Rattan, Aarhus University The science of healthy old age

18:05

Łukasz Ławniczak, Poznan University of Technology Finding the relationship between the chemical structure and anti-microbial properties of rhamnose-based glycoconjugates

18:20

Elżbieta Sikora, Cracow University of Technology Nano-emulsions as vehicles for topical delivery of forskolin

18:35

Questions and answers

6th Central European Congress of Life Sciences Eurobiotech 2017

Detailed Conference Programme

Wednesday, 13.09.2017

Plenary session

Location: BRATYSŁAWA CONFERENCE ROOM Chair: Kazimierz Strzałka, Jagiellonian University in Kraków TIME

PRESENTATION

9.15

PLENARY SPEAKER: Jonathan Gardiner Heddle, Jagiellonian University in Kraków Caging Proteins: Building Artificial Supramolecular Complexes Using Biological Building Blocks

10:00

PLENARY SPEAKER: Robert Huber (Nobel Prize Winner), Max PIanck Institute, Berlin New Ways of Vision: Protein Structures in Translational Medicine and Business Development, my Experience

10:45

Gold Sponsor Speech: Sartorius Sartorius as Total Solution Provider

11:00–11:30 Coffee break Location: DUNAJ HALL

Session 14: Microorganisms – from environment to biotechnology Location: WIEDEŃ CONFERENCE ROOM Chair: Joanna Surmacz- Górska, Silesian University of Technology, Gliwice TIME

PRESENTATION

11:30

KEYNOTE SPEAKER: Peter Schröder, Helmholtz Zentrum München Plant microbe interaction and the role of endophytes in phytoremediation of pharmaceuticals

12:00

Sabina Elzbieta Zoledowska, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk Pangenome studies of plant pathogenic bacterium Pectobacterium parmentieri

12:15

Kubra Erkan Turkmen, Hacettepe University, Ancara Inhibitory effects of quercus pubescens willd. Galls on nosocomial Acinetobacter baumannii biofilm formation and quorum quenching of gram negative bacteria

12:30

Rodrigo Andler, Westfälische Wilhelms-Universität Münster Cleavage of poly(cis<-1,4-isoprene) rubber particles by cultures of Gordonia polyisoprenivorans

12:45

Bronze Sponsor Speech: Applikon Biotechnology: Cristina Bernal Martinez Screening of feeding strategies based on Trigger events in a 24-micro bioreactor platform

13:00

Stanislav Obruca, Brno University of Technology Capability of polyhydroxyalkanoates accumulation enhances stress resistance and cell robustness of bacteria

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Detailed Conference Programme

Wednesday, 13.09.2017

Session 15: From molecular machines to molecular medicine: advances in biotechnology Location: BRATYSŁAWA CONFERENCE ROOM Chair: Jonathan Gardiner Heddle, Jagiellonian University in Kraków TIME

PRESENTATION

11:30

KEYNOTE SPEAKER: Sebastian Glatt, Jagiellonian University in Kraków Lost in translation tRNA modifications protect the proteome

12:00

Rościsław Krutyhołowa, Jagiellonian University in Kraków Regulatory mechanisms of tRNA modification

12:15

Jakub Szymczyk, University of Wrocław Phage Display selection and characterization of scFv specifically recognizing extracellular domain of FGFR1

12:30

Soumyananda Chakraborti, Jagiellonian University in Kraków Unusual mechanical stability of an artificial model protein cage

12:45

Sliver Sponsor Speech: Jakub Nowak, Nanotemper Biomolecular Interactions analytics using microscale thermophoresis

13:00

Questions and answers

Session 16: Dual use of biotechnology

Location: BUDAPESZT CONFERENCE ROOM Chair: Ryszard Słomski, Poznań University of Life Science TIME

PRESENTATION

11:30

KEYNOTE SPEAKER: Ryszard Słomski, Poznań University of Life Science Dual use of scientific discoveries and activity of United Nations Biosecurity Working Group

12:00

Tomasz Zimny, Institute of Law Studies, Polish Academy of Sciences, Warsaw New plant breeding techniques from the point of view of their marketability and patentability in the European Union

12:15

Remigiusz Olędzki, Wrocław University of Economics Effects of microwave radiation on the bioactive properties in selected vegetable species

12:30

Magdalena Julita Hryhorowicz, Poznań University of Life Science Preparation of genetic constructs for modification of animal cells using CRISPR/Cas9 technology

12:45

Questions and answers

13:15–15:30 Lunch & Poster session Location: DUNAJ HALL

6th Central European Congress of Life Sciences Eurobiotech 2017

Detailed Conference Programme

Wednesday, 13.09.2017

Session 17: Stem cells in diseases and therapy

Location: WIEDEŃ CONFERENCE ROOM Chairs: Grzegorz Węgrzyn, University of Gdańsk & Józef Dulak, Jagiellonian University in Kraków TIME

PRESENTATION

15:30

KEYNOTE SPEAKER: Michael Rudnicki, Ottawa Hospital Research Institute Molecular regulation of muscle stem cell function

16:15

Agata Szade, Jagiellonian University in Kraków Cobalt protoporphyrin increases G-CSF and mobilizes granulocytes and hematopoietic stem cells to the peripheral blood in mice

16.45

Jacek Stępniewski, Jagiellonian University in Kraków Comparison of the transcriptome of human mesenchymal cells isolated from right ventricle and epicardial fat

17:00

Questions and answers

Session 18: From molecular machines to molecular medicine: advances in biotechnology Location: BRATYSŁAWA CONFERENCE ROOM Chair: Soumyananda Chakraborti, Jagiellonian University in Kraków TIME

PRESENTATION

15:30

KEYNOTE SPEAKER: Dmitry Ghilarov, Jagiellonian University in Kraków RiPPs are ripe for harvest: structural biology of antibiotic biosynthesis

16:00

Kinga Borzęcka-Solarz, Jagiellonian University in Kraków Impact of gold clusters on cell viability and motility

16:15

Martyna Plens-Gałąska, Adam Mickiewicz University in Poznań Targeted inhibition of STATs as a potential treatment strategy in cardiovascular diseases

16:30

Agnieszka Łoboda, Jagiellonian University in Kraków Small-molecule inhibitors of heme oxygenase-1 as possible anti-cancer drugs

16:45

Questions and answers

Session 19: Big data and biotechnology

Session sponsored by KNOW UAM/IChB PAN Location: BUDAPESZT CONFERENCE ROOM Chair: Wojciech Karłowski, Adam Mickiewicz University in Poznań TIME

PRESENTATION

15:30

KEYNOTE SPEAKER: Georg Haberer, Helmholtz Zentrum München Tackling Three at Once: The Bread Wheat Genome Project

16:00

Marek Żywicki, Adam Mickiewicz University in Poznań High throughput estimation of biologically relevant RNA secondary structures for bioenginering and drug design

16:30

Bronze Sponsor Speech: Applikon Biotechnology: Cristina Bernal Martinez Turning data into information

16:45

Katarzyna Kowalska, National Research Institute of Animal Production, Kraków Evaluation of chromosome microrearrangements of an intersex horse applying array CGH

17:00

Veronika Kukučková, Slovak University of Agriculture in Nitra Common breeding history of different cattle breeds based on high-throughput genotyping data 6th Central European Congress of Life Sciences Eurobiotech 2017

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Detailed Conference Programme

Thursday, 14.09.2017

Plenary session

Location: BRATYSŁAWA CONFERENCE ROOM Chair: Jolanta Jura, Jagiellonian University in Kraków TIME

PRESENTATION

9:00

PLENARY SPEAKER: Magdalena Żernicka-Goetz, University of Cambridge Building the mammalian embryo in vivo and in vitro

9:45

PLENARY SPEAKER: Prof. Thomas Leung; The Hong Kong Polytechnic University Can enzymes be used to treat difficult cancers?

Session 20: Tools in the treatment of human disorders

Session sponsored by KNOW UJ Organizer: Jolanta Jura, Jagiellonian University in Kraków Chairs: Justyna Drukała & Jan Potempa; Jagiellonian University in Kraków TIME

PRESENTATION

10:40

KEYNOTE SPEAKER: Fiona M. Wood, University of Western Australia Striving for excellence in Health care

11:10

Hans-Urlich Demuth, Fraunhofer Institute of Cell Therapy and Immunology, Leipzig Molecular medicine – developments from bench to bedside

11:40

Jan Potempa, Jagiellonian University in Kraków From the basic research to potential prophylaxis and/or treatment of periodontal disease – targeting bacterial glutaminian cyclase

11:55

Justyna Drukała, Jagiellonian University in Kraków In vitro cultured epidermal cells for skin regeneration – clinical applications

12:10

Larry Chow, The Hong Kong Polytechnic University Reversing cancer drug resistance using novel flavonoid dimers

Session 21: Art in biotechnology

Location: WIEDEŃ CONFERENCE ROOM Chair: Jan Barciszewski, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań TIME

PRESENTATION

10:40

KEYNOTE SPEAKER: Joanna Hoffmann, University of Arts in Poznan, Faculty of Art Education Art in the Society of Knowledge. Twisting Symmetries

11:10

Andrzej Długoński, Cardinal Stefan Wyszyński University in Warsaw The significance of interdisciplinary research in the field of landscape architecture and environmental biotechnology. A case study of the green space revitalization in the City of Lodz (Polnad)

11:25

Florian Ryszka, Pharmaceutical Research and Production Plant „Biochefa”, Sosnowiec Influence of selected factors for permeate and absorption of prolactin by small intestine of suckling piglets

11:40

Questions and answers

6th Central European Congress of Life Sciences Eurobiotech 2017

Detailed Conference Programme

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Thursday, 14.09.2017

Session 22: Bioethics

Location: BUDAPESZT CONFERENCE ROOM Organizers: Ewa Bartnik, University of Warsaw & Józef Dulak, Jagiellonian University in Kraków Chairs: Prof. Tomasz Twardowski; Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań & Prof. Jonathan Kimmelman, McGill University Montreal TIME

PRESENTATION

10.40

Tomasz Zimny, Institute of Law Studies, Polish Academy of Sciences, Warsaw Eye of beholder: risk perception and precautionary principle in decision making in biotechnology

11.10

KEYNOTE LECTURE: Józef Dulak, Jagiellonian University in Kraków Science and medicine in an era of populism: a case of stem cell “therapies”

11.40

Questions and answers

12:25–13:00 Coffee break Location: DUNAJ HALL

13:00 Closing Ceremony

Location: BRATYSŁAWA CONFERENCE ROOM Chair Tomasz Twardowski, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań TIME

PRESENTATION

13:00

PLENARY SPEAKER: Jonathan Kimmelman, McGill University Montreal Why so many novel treatments work great in animals but not patients, and what you can do about it?

13:45

Tomasz Twardowski Annoucement of the awards of the best poster prize winers [free registration for the ECB2018 in Geneva] and closing of the Congress

Accompanying exhibitions – all congress long

• Multimedia presentation, Joanna Hoffman (Placed at Metting room S01) • Oil painting Exhibition, Urszula Lisiecka (Dunaj Hall) • Pictures slide show, Robert Bolla, Tin Duck Consulting, Chesterfield – slides will be presented on the screens during breaks

6th Central European Congress of Life Sciences Eurobiotech 2017

28 Posters

POSTERS Useful information for authors presenting posters The posters will be placed in the Dunaj Hall, next to the congress accompanying exhibition.

The poster boards are numbered. Posters must be set up according to the numbers listed below.

Authors are kindly asked to be present by their posters and discuss presented content, according to the following schedule: • Authors of the posters with even numbers from 10:15–11:15 on Tuesday • Authors of the posters with odd numbers from 14:30–15:30 on Wednesday Dimension of the poster: 140 cm (height) x 90 cm (width).

Posters should be put on the boards on Monday (September 11th). You will be provided with necessary materials: scissors, tapes and clips.

Posters will be presented throughout the Congress, until Thursday September 14th.

Please remove posters from the boards on Thursday till 14:00.

After this time, posters left behind will be removed and destroyed by the organizers.

If you want to participate in the best poster award prize contest we kindly remind you to put ,,The best poster award” in the upper right ­corner of the poster. Winners will be chosen by panel of judges. Authors participating in the contest are asked to be present by their posters during first poster session, from 10:15–11:15 on Tuesday The winners shall be announced during Closing Ceremony of the Conference.

More information can be found in the ,,Terms and Conditions – Best Poster Award Eurobiotech” on the website: www.eurbiotech.krakow.pl

Posters Locations – Dunaj Hall

6th Central European Congress of Life Sciences Eurobiotech 2017

Index of Author Presenting Posters

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INDEX OF AUTHOR PRESENTING POSTERS Achinas Spyridon ............................................................. P13.1 Achinas Spyridon ............................................................ P13.2 Akbudak M. Aydin ............................................................... P3.1 Antończyk Aleksandra Monika .......................................... P20.1 Astorkia Maider ............................................................... P3.40 Augustyniak Adrian .......................................................... P14.1 Augustynowicz Joanna ...................................................... P3.2 Babińska Weronika ............................................................ P3.3 Baranski Rafal ................................................................... P3.4 Bednarek Agnieszka ........................................................... P7.1 Bereźnicka Anna ............................................................... P12.1 Bialik-Wąs Katarzyna .......................................................... P9.1 Bialik-Wąs Katarzyna ......................................................... P9.2 Bratuhina Antonina ........................................................... P14.2 Buklaha Anna .................................................................. P20.2 Chytilová Aneta ................................................................ P14.3 Cielecka Izabela ................................................................. P9.3 Ciesielska Anita .................................................................. P1.1 Ciroglu Nayat Narot .................................................... P15&18.1 Czarny Piotr ....................................................................... P9.4 Czechowska Joanna .......................................................... P9.5 Czubatka-Bieńkowska Anna ............................................ P20.3 Danalia-Guidea Silvana Mihaela ......................................... P3.5 Dolińska Barbara .............................................................. P21.1 Dolińska Barbara ............................................................... P9.6 Drabczyk Anna ................................................................. P14.4 Dreger Mariola ................................................................... P3.6 Drożdż Anna .................................................................... P20.4 Durbas Małgorzata Aleksandra .................................. P15&18.2 Durska Anna ....................................................................... P8.1 Dwużnik Dorota ............................................................... P20.5 Dyba Barbara .................................................................... P8.2 Dyba Barbara ................................................................... P16.1 Dziadek Kinga .................................................................... P3.7 Ekiert Halina ...................................................................... P3.8 Ekiert Halina ...................................................................... P3.9 Ferenc-Mrozek Aleksandra ................................................ P1.2 Fornal Agnieszka ................................................................ P1.3 Gadzała Małgorzata ......................................................... P20.6 Gajek Gabriela ................................................................. P20.7 Gąsior Tomasz ................................................................... P7.2 Goraj Weronika ................................................................. P14.5 Goraj Weronika ................................................................. P14.6 Góralczyk Aleksandra ...................................................... P12.2 Gracz Joanna .................................................................... P8.3 Gumna Julita .................................................................... P14.7 Hałat-Łaś Małgorzata ......................................................... P4.1 Hus Konrad Kamil ............................................................ P20.8 Ismael Bairam Solomon .................................................... P3.10 Jahundoviča Inese ............................................................ P3.11 Janek Tomasz ................................................................... P10.1 Janek Tomasz ................................................................... P12.3 Jarocki Piotr ....................................................................... P7.3 Jedynak Marta ........................................................... P15&18.3 Jedynak Pawel .................................................................. P14.8 Jodłowska Iga Karolina .............................................. P15&18.4 Jurkowska Halina Katarzyna ............................................ P20.9 Jurkowska Halina Katarzyna ........................................... P20.10 Kamińska Agnieszka ....................................................... P20.11 Kamińska Katarzyna ........................................................... P1.4 Kaszuba Kinga ........................................................... P15&18.5

Kaszuba Kinga ........................................................... P15&18.6 Kaszycki Paweł ................................................................ P14.9 Kempa Michał Piotr .......................................................... P3.12 Kietrys Anna Maria ............................................................. P1.5 Kirjusina Muza ................................................................... P9.8 Klimek-Chodacka Magdalena ............................................ P3.13 Kłosowski Grzegorz ......................................................... P16.2 Kłosowski Grzegorz ........................................................ P14.10 Kocik Justyna Natalia ..................................................... P20.12 Kostecka-Gugała Anna ..................................................... P12.4 Kot Anna .......................................................................... P3.14 Kozakowska Magdalena ................................................ P2&5.1 Krzyścin Piotr ................................................................... P12.5 Krzyścin Piotr ................................................................... P12.6 Kubiak Marta Anna ............................................................. P1.6 Kučera Dan ..................................................................... P14.11 Kucia Małgorzata Anna .................................................... P13.3 Kuczyński Jakub ............................................................... P8.4 Kudłacik-Kramarczyk Sonia ............................................ P14.12 Kukučková Veronika ......................................................... P19.1 Kwaśniak Konrad ................................................................ P1.7 Kwiecień Inga ................................................................... P3.15 Kwiecień Inga ................................................................... P3.16 Langner Ewa .............................................................. P15&18.7 Langwinski Wojciech Jerzy ............................................ P20.13 Lasek Robert .................................................................. P14.13 Lasoń Elwira ..................................................................... P13.4 Lemieszek Marta Kinga ............................................. P15&18.8 Lewandowski Jarosław ................................................. P2&5.3 Liszniańska Magdalena ..................................................... P3.17 Lomza Pola ....................................................................... P4.2 Łęgowska Ewelina Angelika ............................................... P9.7 Macieja Anna .................................................................. P20.14 Magner Dorota ................................................................... P1.8 Mahmoud Medhat Helmy ................................................. P19.2 Makowski Wojciech .......................................................... P3.18 Malinowska Magdalena Anna .......................................... P13.5 Marciniak Beata .............................................................. P20.15 Markowska-Zagrajek Agnieszka .......................................... P1.9 Marszałek Małgorzata ...................................................... P3.19 Maruszewska-Cheruiyot Marta ....................................... P20.16 Matczynska Ewa.................................................................P19.9 Matras Ewelina .................................................................. P4.3 Mazurkiewicz Natalia ................................................. P15&18.9 Messerschmidt Katrin ..................................................... P16.3 Michalak Angelika ............................................................ P3.20 Michalczyk Alicja Katarzyna ........................................... P14.14 Miedziak Beata ................................................................. P1.10 Mierziak-Derecka Justyna Katarzyna ............................... P3.21 Miksh Korneliusz ............................................................ P14.15 Mikulski Dawid ................................................................. P16.4 Mikulski Dawid ................................................................ P16.5 Miluchová Martina ............................................................ P16.6 Miszkiewicz Joanna .......................................................... P1.11 Mitka Ilona ........................................................................ P1.12 Mitka Ilona ........................................................................ P1.13 Morańska Emilia .............................................................. P3.22 Morańska Emilia .............................................................. P3.23 Moravčíková Nina ............................................................. P16.7 Motyka Agata ..................................................................... P4.4 Mucha Olga Jolanta .......................................................... P1.14

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Index of Author Presenting Posters

Müllerová Lucie .............................................................. P14.16 Nalejska Ewelina ....................................................... P15&18.10 Natonek-Wiśniewska Małgorzata ...................................... P12.7 Natonek-Wiśniewska Małgorzata ...................................... P12.8 Navratilova Alica ............................................................. P14.17 Necel Agnieszka ................................................................. P7.4 Niedźwiecki Mateusz ....................................................... P3.24 Nikcevic Gordana .......................................................... P2&5.2 Nowak Jakub ............................................................ P15&18.11 Nykiel - Szymańska Justyna .............................................. P4.5 Obruca Stanislav ............................................................ P14.18 Oczkowicz Maria .............................................................. P1.15 Oczkowicz Maria .............................................................. P1.16 Oleszkiewicz Tomasz ....................................................... P3.25 Orlikowska Teresa ........................................................... P3.26 Orlikowska Teresa ........................................................... P3.27 Osinka Anna ................................................................... P14.19 Pacholak Amanda ............................................................... P4.6 Paduszyński Piotr ........................................................... P20.17 Paraszkiewicz Katarzyna .................................................. P12.9 Parzymies Marzena ......................................................... P3.28 Pauliukavets Anastasiya ................................................. P20.18 Pawełkowicz Magdalena Ewa .......................................... P3.29 Pawełkowicz Magdalena Ewa .......................................... P3.30 Pelc Katarzyna ................................................................ P3.31 Pernicová Iva ................................................................. P14.20 Petryszak Przemyslaw .................................................... P14.21 Pieron Katarzyna ............................................................. P3.32 Pierzynowska Karolina ................................................... P20.19 Piestrzynska-Kajtoch Agata .............................................. P1.17 Piórkowska Katarzyna ...................................................... P1.18 Piórkowska Katarzyna ........................................................ P6.1 Pluta Klaudia Anna ............................................................ P9.9 Podbielska Angelika Paulina ............................................. P1.19 Podkalicka Paulina ........................................................ P20.20 Polak Justyna Maria ...................................................... P20.21 Radzik Paulina .................................................................. P9.10 Rolle Katarzyna ............................................................... P1.20 Ropka-Molik Katarzyna ................................................... P1.21 Ropka-Molik Katarzyna ................................................... P1.22 Sarnik Joanna ................................................................ P20.22 Sawicki Krzysztof Bogumił ............................................ P20.23 Sedlacek Petr ................................................................ P14.22 Sereikaite Jolanta .......................................................... P14.23 Sharafi Ali ........................................................................ P3.33 Siewiera Paulina Ksenia ...................................................... P4.7 Sikora Elżbieta ................................................................. P3.34 Skarzyńska Agnieszka ..................................................... P3.35 Skarzyńska Agnieszka ..................................................... P3.36 Skrzypek Klaudia ............................................................. P1.23 Slaninova Eva ................................................................. P14.24

Słaba Mirosława ................................................................ P4.8 Smołucha Grzegorz .......................................................... P1.24 Smołucha Grzegorz ......................................................... P1.25 Smułek Wojciech ............................................................... P4.9 Smułek Wojciech .............................................................. P4.10 Soboń Adrian .................................................................... P4.10 Soboń Adrian .................................................................... P4.11 Sobuś Anna ..................................................................... P1.26 Sobuś Anna ................................................................... P2&5.4 Sochalska Maja ............................................................. P20.24 Stefaniuk-Szmukier Monika Karolina ............................... P1.27 Supel Paulina ................................................................. P14.25 Sydow Mateusz ................................................................ P19.3 Szade Agata ..................................................................... P17.1 Szade Agata .................................................................. P20.25 Szalata Milena ................................................................. P3.37 Szczerba Hubert .............................................................. P3.38 Szulczewska Katarzyna Małgorzata ............................... P14.26 Szymczyk Jakub Paweł ............................................ P15&18.12 Śliwa Karolina Anna .......................................................... P13.6 Tavassoli Elham .................................................................. P7.5 Tomczyńska Małgorzata ........................................... P15&18.13 Topka Gracja ...................................................................... P7.6 Trakovická Anna ............................................................ P20.26 Trzebuniak Kamil Filip ...................................................... P3.39 Tyczewska Agata ............................................................... P8.5 Tyra Mirosław .................................................................. P1.28 Tyra Mirosław .................................................................. P1.29 Wartalski Kamil ............................................................. P2&5.5 Wartalski Kamil ........................................................ P15&18.14 Warzecha Joanna ............................................................ P1.30 Wasak Agata .................................................................... P19.4 Wasak Agata .................................................................... P19.5 Wiater Jerzy ..................................................................... P9.11 Wigner Paulina ................................................................. P9.12 Wiśniewska Ewa ............................................................... P9.13 Witarski Wojciech ............................................................. P1.31 Witarski Wojciech ............................................................ P1.32 Witman-Zając Sylwia ....................................................... P16.8 Wojtowicz Wojciech ....................................................... P12.10 Woźniak Ewa .................................................................... P19.6 Zawadzka Katarzyna ......................................................... P4.12 Zdarta Agata ..................................................................... P4.13 Ziółkowski Robert ........................................................... P12.11 Zuwala-Jagiełło Jolanta ................................................. P20.27 Zuwala-Jagiełło Jolanta ................................................. P20.28 Żbik Paweł ..................................................................... P14.27 Żukowska Monika .......................................................... P2&5.6 Żukowski Kacper .............................................................. P19.7 Żukowski Kacper .............................................................. P19.8 Żurek Grzegorz ................................................................. P11.1

6th Central European Congress of Life Sciences Eurobiotech 2017

List of Posters Presentations

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LIST OF POSTERS PRESENTATIONS

POSTER NUMBER

TITLE, AUTHORS

Session 1: Nucleic Acids Tools for Regulation of Gene Expression P1.1

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Trichophyton rubru m; A. Ciesielska, P. Stączek

P1.2

Effect of His-tag sequence location in Decapping Scavenger enzymes on their hydrolytic activity towards dinucleotide cap analogs; A. Ferenc-Mrozek, E. Bojarska, M. Łukaszewicz, E. Darżynkiewicz

P1.3

Development of microsatellites additional panel for horse parentage testing based on Polish population; A. Fornal, A. Radko, A. Piestrzyńska-Kajtoch

P1.4

Changes in transcriptome and epigenome in prostate malignant transformation model; K. Kamińska, J. Kowalewski, S. Huang, M. A. Lewandowska

P1.5

RNA modifications and NGS: It’s not a bug, it’s a feature; A. M. Kietrys, E. T. Kool

P1.6

Spatial chromosomal organization of ROD1 regulates gene expression in prostate cancer cell lines; M. Kubiak, S. Huang, M. A. Lewandowska

P1.7

Assessment expression of non-coding RNAs in patients with non-small cell lung cancer; K. Kwaśniak, J. CzarnikKwaśniak, K. Pogoda, M. Pyszniak, P. Rybojad, J. Tabarkiewicz

P1.8

Point mutations of APP gene (Amyloid beta precursor protein) as a target for SNP-selective RNA degradation by ribonuclease H using modified antisense oligonucleotides; D. Magner, R. Kierzek

P1.9

Bacterial growth inhibition by peptide nucleic acids complementary to ribosomal RNA; A. Markowska-Zagrajek, R. Grzela, J. Trylska

P1.10

Analysis of Nudt16 localization and colocalization using confocal microscopy; B. Miedziak, R. Grzela, E. Darżynkiewicz

P1.11

The cleavage of bacterial mRNA by a hammerhead ribozyme; J. Miszkiewicz, K. Maximova, M. Łukaszewicz, E. Darżynkiewicz, J. Trylska

P1.12

Effect of GPAT2 gene polymorphism on meat texture parameters in pigs; I. Mitka, K. Ropka-Molik, M. Tyra

P1.13

Effect of selected ACTN2 gene polymorphisms on meat quality in pigs; M. Tyra, K. Ropka-Molik, K. Piórkowska, I. Mitka

P1.14

Searching for the right tool for heme oxygenase-1 inhibition – the comparison of small-molecule inhibitors, shRNA and CRISPR/Cas9 system; O. Mucha, P. Podkalicka, M. Czarnek, A. Biela, M. Mieczkowski, S. Czauderna, N. Kachamakova-Trojanowska, J. Stepniewski, A. Jozkowicz, A. Loboda, J. Dulak

P1.15

Characteristic of miRNAome in three different tissues of pigs; M.Oczkowicz, K, Pawlina-Tyszko, M. Świątkiewicz, J. Warzecha

P1.16

qPCR validation of RNA-seq analysis of backfat transcriptome changes after different diets in pigs; M. Oczkowicz, M. Świątkiewicz, K. Pawlina-Tyszko, J. Warzecha

P1.17

PRNP gene polymorphism in red deer and roe deer; A. Piestrzynska-Kajtoch, A. Radko, A. Szumiec, M. Kościelny

P1.18

Gene expression changes in fast-growing chicken hypothalamus during ontogenesis; K. Piórkowska, K. Żukowski, K. Połtowicz, J. Nowak, K. Ropka-Molik, D.Wojtysiak, N. Derebecka, J. Wesoły

P1.19

Use of multiplex microsatellite panel for assessment of polymorphism domestic cats and free-living cats; A. Podbielska, A. Radko, W. Niżański, J. Kochan, A. Nowak, M. Bugno-Poniewierska

P1.20

Enantiomeric catalytic nucleic acids in downregulation of gene expression; A. Belter, E. Wyszko, K. Rolle, J. Barciszewski

P1.21

Exercise-related modification of the PLIN2 and FASN genes expression in Arabian horses skeletal muscle; K. Ropka-Molik, M. Stefaniuk-Szmukier, K. Żukowski, K. Piórkowska, M. Bugno-Poniewierska

P1.22

Whole transcriptome analysis of porcine muscle tissue in the terms of muscle growth and development; K. Ropka-Molik, K. Żukowski, K. Piórkowska, G. Żak, J. Dulska, N. Derebecka, J. Wesoły,

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List of Posters Presentations

POSTER NUMBER

TITLE, AUTHORS

P1.23

Genome editing in tumor and iPS cells for the study of SNAI1 role in human diseases; K. Skrzypek, A. Ulman, B. Badyra, M. Sulkowski, M. Majka

P1.24

Identification of mutations in BMPR1B, BMP-15 and GDF-9 genes in Cakiel podhalański and Wrzosówka sheep breeds; Grzegorz Smołucha, Agata Piestrzyńska-Kajtoch

P1.25

Polymorphism of the Growth Hormone (GH) and Myostatin (MSTN) genes in Kielecka and Landes geese; G. Smołucha, A. Kozubska-Sobocińska, A. Koseniuk, M. Lisowski, B. Grajewski

P1.26

Peripheral blood microRNA as potential biomarker of significant carotid vessels stenosis; M. P. Kawa, A. Sobuś, A. Kazimierczak, P. Rynio, A. Rybicka, D. Rogińska, K. Babiak, Z. Litwińska, K. Łuczkowska, A. Machalińska, P. Gutowski, B. Machaliński

P1.27

Preliminary study of training induced changes in transposable elements (TEs) expression in blood of Arabian horses; M. Stefaniuk-Szmukier, K. Ropka-Molik, K Żukowski, K Piórkowska, M. Bugno-Poniewierska

P1.28

ACTN2 polymorphisms in pigs and its effect on meat texture parameters; M. Tyra, K. Ropka-Molik, K. Piórkowska, I. Mitka, A. Bereta, M. Szyndler-Nędza

P1.29

Association of new PPARGC1 gene polymorphisms with meat texture parameters in pigs; M. Tyra, K. Ropka-Molik, K. Piórkowska, I. Mitka, M. Szyndler-Nędza, A. Bereta

P1.30

Genetic diversity among different geese breeds based on molecular markers; J. Warzecha, A. Fornal, D. Rubiś, M. Oczkowicz, M. Bugno-Poniewierska

P1.31

Premature centromere division (PCD) as a possible cause of abnormal X chromosome morphology in a hucul mare; W. Witarski, M. Bugno-Poniewierska, K. Kowalska

P1.32

The differences in CDC42BPA and TIMP3 gene expression in equine sarcoid and fibroblast in vitro cell cultures; W. Witarski, P. Podstawski, K. Ropka-Molik, M. Bugno-Poniewierska

Session 2: Stem cells P2&5.1

Lack of heme oxygenase-1 induces inflammatory reaction and proliferation of muscle satellite cells after cardiotoxininduced skeletal muscle injury; M. Kozakowska, K. Pietraszek-Gremplewicz, M. Ciesla, M. Seczynska, I. Bronisz, K. Bukowska-Strakova, A. Jozkowicz, J. Dulak

P2&5.2

The use of canine mesenchymal stem cells for the autologous treatment of osteoarthritis and for enhancing their therapeutic capacity; G. Nikcevic, S. Srzentic Drazilov, V. Spasovski, J. Mrkovacki, A. Fazlagic, S. Pavlovic

P2&5.3

The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin; J. Lewandowski, N. Rozwadowska, T. Kolanowski, A. Malcher, A. Zimna, A. Rugowska, W. Łabędź, Ł. Kubaszewski, J. Karczmarczyk, K. Chojnacka, K. Bednarek-Rajewska, P. Majewski, M. Kurpisz

P2&5.4

Biological activity of hematopoietic stem/progenitor cells collected from patients with abnormal secretion of glucocorticoids; M. P. Kawa, A. Sobuś, K. Łuczkowska, L. Osowicz-Korolonek, A. Cymbaluk-Płoska, E. Pius-Sadowska, D. Rogińska, Z. Litwińska, E. Paczkowska, E. Dąbkowska, A. Syrenicz, B. Machaliński

P2&5.5

Adult stem cells from the porcine ovaries as a source of potential neural cells – in vitro studies; K. Wartalski, G. Gorczyca, J. Wiater, M. Duda

P2&5.6

Does nuclear heme oxygenase-1 interact with G-quadruplexes and influence their stability? M. Zukowska, A. Czmoczek, A. Konturek, M. Cieśla, W. Nowak, K. Szade, J. Dulak, A. Jozkowicz

Session 3: Plant biotechnology P3.1

Effect of Gene Order in DNA Constructs on Gene Expression upon Integration into Plant Genome; M. A. Akbudak, V. Srivastava

P3.2

The use of Callitriche to phytoremediate chromium in bottom sediments of natural watercourses; J. Augustynowicz, A. Baran, E. Sitek, B. Ostachowicz, T. Bryniarski, M. Urbańska-Stopa

P3.3

First report on the occurrence of Pectobacterium carotovorum subsp. brasiliense in Polish waterways; W. Babinska, A. Motyka, S. Zołedowska, W. Sledz, E. Lojkowska

P3.4

Development of carotenoid gene knockouts in a model callus system using CRISPR/Cas9 vectors; T. Oleszkiewicz, M. Klimek-Chodacka, L. G. Lowder, Y. Qi, R. Baranski

6th Central European Congress of Life Sciences Eurobiotech 2017

List of Posters Presentations

POSTER NUMBER

TITLE, AUTHORS

P3.5

Antifungal activity of some steroidal glycoalkaloids extracted from species of the genus Solanum on Venturia inaequalisextension; S. Cristea, M. S. Manole, C. Zala, Ștefana Jurcoane, S. M. Dănăilă-Guidea, M. Florentina, B. Dumitriu, L. Olariu

P3.6

Effect of thidiazuron on shoot induction of fibre hemp (Cannabis sativa L.); T. Wróbel, M. Dreger, G. Mańkowska, M. Szalata, K. Wielgus, R. Słomski

P3.7

Antioxidant activity of fruit, petioles and leaves of sweet cherry (Prunus avium L.); K. Dziadek, A. Kopeć

P3.8

High production of phenolic acids in in vitro cultures of two aronia species after addition of phenylalanine as their biosynthetic precursor; A. Szopa, P. Kubica, A. Walkowicz-Bożek, H. Ekiert

P3.9

The influence of egzogenic cinnamic acid on the production of phenolic acids in in vitro cultures of two aronia species; A. Szopa, P. Kubica, A. Walkowicz-Bożek, H. Ekiert

P3.10

Use of nano-silver to optimize disinfection of Carex muskingumensis Schwein. rhizome explants in in vitro cultures; B. Ismael, D. Kozak, M. Parzymies, A. Świstowska

P3.11

Confocal laser scanning microscopy for smart and simple diagnostics of different in vitro callus cell types; I. Mickeviča, I. Rubeniņa, J. Kirilova, S. Osipovs, I. Kokina, S. Kecko, M. Kirjušina, I. Jahundoviča

P3.12

LTP2 protein detection in spring barley grains exposed to multiple abiotic stresses; M. Kempa, A. Kuczyńska, K. Mikołajczak, P. Ogrodowicz, M. Surma, T. Adamski, H. Ćwiek-Kupczyńska

P3.13

Agrobacterium tumefaciens-mediated genetic transformation of Nigella damascena L.; M. Klimek-Chodacka, T. Oleszkiewicz, R. Barański

P3.14

A multiplex PCR assay for simultaneous detection of the most common fungal wheat pathogens; A. Kot, H. Szczerba, A. Ostrowska, M. Nowak, M. Muszyńska, A. Kuzdraliński

P3.15

Accumulation of secondary metabolites in in vitro cultures of Scutellaria subvelutina Rech. f. – preliminary results; B. Kawka, I. Kwiecień, H. Ekiert

P3.16

Sulfurtransferases activity in cells of Brassica cretica ssp. botrytis cultured in vitro; I. Kwiecień

P3.17

Characteristic of the photosynthetic apparatus of Rumex tianschanicus x Rumex patientia, a high biomass yielding plant; M. Liszniańska, H. Ślesak, K. Tokarz

P3.18

Acclimatization strategy of Dionaea muscipula J. Ellis to light stress in in vitro – physiological study; W. Makowski, B. Piwowarczyk, R. Banasiuk, A. Królicka, M. Hanula, K. Tokarz

P3.19

Plant transgenesis- new source of biofuels; M. Marszałek, J. Zeyland, A. Nowak, R. Słomski, D. Lipiński

P3.20

Rhizobium rhizogenes transformation of monocotyledons Iris pseudacorus plants; A. Michalak, A. Królicka

P3.21

Effect of beta-ketothiolase gene expression modification on benzenoid synthesis pathway in flax; J. Mierziak, D. Nitarska, K. Kostyn, A. Boba, J. Szopa-Skórkowski, A. Kulma

P3.22

Assessment of the genetic relationships among raspberry cultivars using molecular markers; M. Simlat, A. Ptak, E. Morańska, A. Kula, A. Orzeł

P3.23

Oxidative stress in Leucojum aestivum L. in vitro cultures; E. Morańska, M. Simlat, E. Skrzypek, M. Warchoł, A. Ptak

P3.24

Roundup utilization by chemical methods; M. H. Kudzin, R. Żyłła, Z. Mrozińska, M. Niedzwiecki, P. Urbaniak, J. Sójka-Ledakowicz

P3.25

The impact of exogenous plant growth regulators on carotenoid composition and carotenoid pathway gene expression in carrot cells in vitro; T. Oleszkiewicz, M. Klimek-Chodacka, A. Kostyn, A. Boba, J. Szopa, R. Barański

P3.26

Elimination of contaminating bacteria from plant tissue culture; T. Orlikowska, T. Malinowski, L. Ogórek

P3.27

Piriformospora indica as growth stimulator and bioprotector of rhododendron plants for Phytophthora cinnamomi; A. Trzewik, L. Ogórek, T. Orlikowska, E. Klocke

P3.28

Use of tissue culture for ex situ conservation of the endangered plant species -Salix lapponu; M. Parzymies, M. Pogorzelec, A. Świstowska

P3.29

Comparative genomics of homozygous B10 cucumber line individuals shows dynamics within functional loci; P. Osipowski, M. Wojcieszek, M. Pawełkowicz, A. Skarzyńska, Z. Przybecki, W. Pląder

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List of Posters Presentations

POSTER NUMBER

TITLE, AUTHORS

P3.30

Genomic comparison of cucumber growth type mutants; M. Wojcieszek, M. Pawełkowicz, Z. Przybecki, W. Pląder

P3.31

The relationship between the silencing of the GMPase gene and the sugar composition of flax; K. Pelc, M. Preisner, A. Metryka, J. Szopa, A. Kulma

P3.32

Silver thiosulfate enhance accumulation of phenolics in hairy roots of Lactuca virosa; K. Pieron, J. Malarz

P3.33

In vitro regeneration and wogonin production in genetically transformed culture of Scutellaria bornmuelleriana; Z. Gharari, K. Bagheri, A. Sharafi, H. Danafar

P3.34

The effect of Methylomethionine Sulfonate (Vitamins U) on physicochemical changes of rapeseed oil under accelerated oxidation conditions; M. Kucia, E. Kondratowicz-Pietruszka, presenter: E. Sikora

P3.35

Different approaches of NGS reads correction for structural variant calling of cucumber somaclonal lines; A. Skarzyńska, P. Osipowski, M. Pawełkowicz, W. Pląder

P3.36

Is transgenesis significantly altering the cucumber genome? A. Skarzyńska, M. Pawełkowicz, M. Szwacka, W. Pląder

P3.37

Determination of cannabinoids content in water extracts prepared from fibre type hemp (Cannabis sativa L.); M. Szalata, K. Wielgus

P3.38

Novel primer sets for identification of Tapesia yallundae, T. acuformis and Rhizoctonia sp. based on PCR; H. Szczerba, A. Kot, A. Ostrowska, M. Nowak, M. Muszyńska, A. Kuzdraliński

P3.39

Insights into unequal distribution of norflurazon through the wheat seedlings; K. F. Trzebuniak, B. Myśliwa-Kurdziel

P3.40

Useful catalogue of Candidate Gene Alleles established in different Guineensis, Oleifera and Hybrid Populations; M. ­Astorkia, B. Santika, M. Hernández, A. Herrán, L. Barandalla, F. Wendra S., N. Quezada, E. Pratiwi, F. Orellana, O. Leon, F. Vera, S. Morales, K. Ponce, S. Zulhermana, D. Asmono, E. Ritter

Session 4: Microbial elimination of deleterious compounds P4.1

Bioremediation of model post-fermentation sludge liquors with bacteria and yeasts; M. Hałat-Łaś, P. Kaszycki, P. Malec P., S. Borowski, J. Burczyk

P4.2

Enzyme activity in petroleum contaminated soil as an indicator of physiology state of indigenous microorganisms in the context of biodegradation; P. Lomza, Ł. Drewniak

P4.3

Surface charge dependent fungistatic proprieties of silver nanoparticles; E. Matras, M. Oćwieja, A. Gorczyca

P4.4

Rapid eradication of plant pathogenic bacteria by direct current atmospheric pressure glow discharge generated in contact with a flowing liquid cathode; A. Motyka, A. Dzimitrowicz, P. Jamroz, E. Lojkowska, P. Pohl, W. Sledz

P4.5

Elimination of 2,4-dichlorophenoxyacetic acid by the microscopic filamentous fungus Umbelopsis isabellina; J. Nykiel – Szymańska, P. Siewiera, J. Datskowa, P. Bernat

P4.6

The effect of saponins on biodegradation of clotrimazole by activated sludge; W. Smułek, A. Zdarta, E. Kaczorek

P4.7

Efficient dibutyltin (DBT) elimination by the microscopic fungus Metarhizium robertsii under conditions of intensive aeration and ascorbic acid supplementation; P. Siewiera, J. Nykiel-Szymańska, P. Bernat

P4.8

Chromium (VI) toxicity to tolerant fungal strains under sulfates starvation; M. Słaba, J. Nykiel-Szymańska, A. Zawierucha, S. Różalska

P4.9

The enhanced biodegradation of brominated diphenyl ether by environmental bacteria; W. Smułek, A. Zdarta, E. Kaczorek

P4.10

Cytosolic fraction proteins of Metarhizium robertsii IM 2358 involved in the degradation of 4-n-nonylphenol; A. Litwin, A. Soboń, S. Różalska

P4.11

Impact of tributyltin (TBT) on Cunninghamella echinulata IM 2611 proteome; A. Soboń, R. Szewczyk, J. Długoński

P4.12

Fungal metabolism of carbazole, quinoline and their derivatives with pharmacological activity; K. Zawadzka, A. Felczak, M. Nowak, K. Lisowska

P4.13

Hydrocarbons sorption on natural and synthetic materials; A. Zdarta, W. Smułek, E. Kaczorek

Session 6: The tools of epigenetics P6.1

Deep sequencing of QTL-rich region on porcine chromosome; K. Piórkowska, K. Żukowski, M. Tyra, K. Ropka-Molik

6th Central European Congress of Life Sciences Eurobiotech 2017

List of Posters Presentations

POSTER NUMBER

TITLE, AUTHORS

Session 7: Bacteriophages in Biotechnology P7.1

Production of phage lytic enzyme of high anti-enterococcal activity in Escherichia coli; Agnieszka Gozdek, A. Bednarek, J. Kosakowski, A. Różycka, E. Sadowy, B. Weber-Dąbrowska, A. Górski, M. Łobocka

P7.2

Novel Shiga toxin-converting bacteriophages – a comparative analysis of genomic sequences; T. Gąsior, K. Licznerska, A. Dydecka, S. Bloch, G. Topka, B. Nejman-Faleńczyk, G. Węgrzyn, A. Węgrzyn

P7.3

Characterization and stability analysis of prophage ΦBH1 identified in the genome of probiotic Lactobacillus rhamnosus Pen; P. Jarocki, M. Podleśny, O. Kholiavskyi, M. Dworniczak, E. Komoń-Janczara, Z. Targoński

P7.4

Bacteriophage vb_Eco4M-7 as the Golden mean in fight against Enterohemorrhagic Escherichia coli (EHEC); A. Necel, G. Topka, A. Dydecka, S. Bloch, B. Nejman-Faleńczyk, A. Jurczak-Kurek, T. Gąsior, K. Kwaśnicka-Kosznik, Ł. Nowakowski, Ł. Grabowski, A. Węgrzyn, G. Węgrzyn

P7.5

Identification of Fully Human Fab Fragment Antibody Targeting Protease Activated Receptor 1; E. Tavassoli, F, Rahimi Jamnani, H. Jahandar

P7.6

Characterization of selected bacteriophages isolated from urban sewages and their potential biotechnological importance; G. Topka, S. Bloch, B. Nejman-Faleńczyk, A. Jurczak-Kurek, A. Necel,Aleksandra Dydecka, T. Gąsior, G. Węgrzyn, A. Węgrzyn

Session 8: Molecular mechanisms of plant response to environmental stresses P8.1

Identification of Pisum sativum L. genes differentially expressed in resistant reaction to Didymella pinodes in – preliminary results; A. Durska, M. Gawłowska, W. Święcicki, L. Boros

P8.2

Structural and biochemical response of native model membranes to ozone stress – protective effects of gallic acid; B. Dyba, E. Rudolphi- Skórska, A. Sieprawska, J. Biesaga- Kościelniak, A. Kornaś, M. Filek

P8.3

Gene onthology analysis of miRNA targets involved in soybean chilling stress response; J. Gracz, W. Karłowski, J. Kuczyński, A. Tyczewska

P8.4

MicroRNAs in soybean chilling stress response; J. Kuczyński, J. Gracz, A. Tyczewska

P8.5

Proteome changes under herbicidal stress in maize; A. Tyczewska, I. Bielińska, J. Gracz, M. Sikora, T. Twardowski

Session 9: Biomaterials Engineering – New Material Technologies for Tissue Engineering P9.1

Selection of reaction conditions of polymeric carriers based on polyhydroxyalkanoates (PHAs); K. Bialik-Wąs, D. Malina, K. Pluta, A. Kulawik-Pióro, M. Zielina

P9.2

Studies on preparation of hydrogel materials containing extract from Echinacea purpurea; K. Bialik-Wąs

P9.3

Bacterial cellulose composites with CMC and HEC as potential dressings for chronic wounds; I. Cielecka, P. Rytczak, E. Gendaszewska-Darmach, S. Bielecki

P9.4

Association between single-nucleotide polymorphisms of genes responsible for repair, replication and degradation of mitochondrial DNA, and depressive disorder; P. Czarny, P. Wigner, J. Szemraj, T. Śliwiński

P9.5

Antibacterial hybrid material for bone regeneration; J. Czechowska, A. Zima, D. Siek, A. Ślosarczyk

P9.6

Permeate level of calcium to small intestine according to composition their formulation; B. Dolińska, M. Jelińska, A. Ostróżka-Cieślik, F. Ryszka

P9.7

Study of an active substance release from hydrogel polymer nanocomposites; E. Łęgowska, M. Grzywińska, M. Miotke, J. Strankowska, M. Strankowski, Ł. Piszczyk, J. Kwela

P9.8

Diagnostic of parasites using novel luminescent dyes and confocal laser scanning microscopy; M. Kirjušina, A. Pučkins, I. Mickeviča, J. Kirilova, S. Osipovs, I. Rubeniņa, I. Jahundoviča

P9.9

Bioactivity tests of calcium phosphates with variant molar ratios of main components; K. Pluta, A. Sobczak-Kupiec, O. Półtorak, D. Malina, B. Tyliszczak

P9.10

The influence of cellulose nanocrystals (CNC) on the structure and dynamic mechanical properties of biopolyamide 10.10; P. Radzik, A. Leszczyńska, A. Niedziela, K. Leszko, K. Pielichowski

P9.11

Analysis of downregulation of α-gal epitope expression in the skin and liver of transgenic pigs as a model for xenotransplantation; J. Wiater, Z. Smorąg, R. Słomski, J. Karasiński, M. Romek

P9.12

Association between single nucleotide polymorphisms of TPH1 and TPH2 genes and depressive disorders; P. Wigner, P. Czarny, T. Sliwinski 6th Central European Congress of Life Sciences Eurobiotech 2017

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List of Posters Presentations

POSTER NUMBER

TITLE, AUTHORS

P9.13

Growth of adipose Mesenchymal stromal cells on different scaffolds – polymers, bacterial cellulose and acellular swine pericardium; E. Wiśniewska, K. Kulik, A. Langrzyk, M. Szustak, S. Bielecki, M. Sobota, J. Włodarczyk, J. Kasperczyk, P. Gach, K. Jendryczko, M. Faber, P. Wilczek, J. Dulak

Session 10: Microbial Cell Factories P10.1

NADH-dependent erythrose reductase from Yarrowia lipolytica: production and biochemical characterization of the enzyme; T. Janek, A. Dobrowolski, A. Biegalska, A. M. Mirończuk

Session 11: Plants for the European Bioeconomy P11.1

Factors affecting seed yield in selected Festuca species; G. Żurek, K. Prokopiuk, D. Martyniak, E. Paszkowski, U. Woźna – Pawlak, M. Jurkowski

Session 12: Modern instrumental analysis in biotechnology and biomedicine P12.1

Expression of human α1,4-galactosyltransferase in CHO Lec-2 cell line; A. Bereźnicka, M. Wasik, R. Kaczmarek, K. Mikołajczyk, M. Duk, M. Czerwiński

P12.2

Extracellular proteome of Myrothecium roridum – a valuable tool for dyes degradation; A. Góralczyk, A. Jasińska, A. Soboń, J. Długoński

P12.3

Metal-amphisin conjugates: bioactivity of metal-containing compounds; T. Janek, L. R. Rodrigues, Ż. Czyżnikowska

P12.4

Low-frequency EPR spectrometry as a tool to monitor chromate bioremediation in living systems; A. Kostecka-Gugała, A. Dubicka-Lisowska, H. Gabryś, T. Walczak, M. Chochlińska, P. Kaszycki

P12.5

Determination of a universal animal DNA marker; M. Natonek-Wiśniewska, P. Krzyścin

P12.6

Evaluation of the suitability of mitochondrial DNA for the species identification of animal origin traces; M. NatonekWiśniewska, P. Krzyścin

P12.7

Use of cytochrome b polymorphism for species identification of spots of unknown origin; M. Natonek-Wiśniewska, A. Radko, D. Rubiś, P. Krzyścin, A. Podbielska

P12.8

Quantitative and qualitative determination of ducks and geese DNA from different matrices; M. Natonek-Wiśniewska, P. Krzyścin

P12.9

Bacilli biosurfactants production in mixed (bacterial-fungal) cultures determined by mass spectrometry; A. Kuśmierska, P. Bernat, J. Długoński, K. Paraszkiewicz

P12.10

Monitoring over time cell culturing medium metabolites changes in breast cancer cell line; W. Wojtowicz, A. Ząbek, P. Młynarz

P12.11

Electrochemical stem-loop biosensor for fast and sensitive detection of Bacillus anthracis DNA; R. Ziółkowski, K. Zacharczuk, A. A. Zasada, E. Malinowska

Session 13: Biotechnology for beauty P13.1

Catechins isolation from green tea: Effect of extraction time and temperature; S. Achinas, V. Achinas

P13.2

Phenolic compounds extraction for Olea europaea leaves using water-based solvent; Spyridon Achinas, Vasileios Achinas

P13.3

The effect of cosmetic base on the release of Methylmethionine Sulfonate; M. Kucia, A. Leśniak, E. Sikora

P13.4

NLC as a potential carrier system for transdermal delivery of forskolin; E. Lasoń, E. Sikora, M. Miastkowska, E. Escribano, M. Jose Garcia-Celma, C. Solans, M. Llinas, J. Ogonowski

P13.5

Safety evaluation and total antioxidant activity of the new semisynthetic lupeol derivatives; M. Malinowska, M. Pasikowska, J. Ogonowski, E. Sikora, I. Eris

P13.6

Ultrasonic assistance micelle-mediated extraction of Calendula Anthodiu; K. Śliwa, P. Śliwa, E. Sikora, J. Ogonowski, P. Nowicka, J. Oszmiański

Session 14: Microorganisms – from environment to biotechnology P14.1

Nanomaterials as stimulating agents for bacterial metabolism; A. Augustyniak, K. Cendrowski, M. Barylak, E. Mijowska, P. Nawrotek

P14.2

The influence of low-intensity extremely high-frequency electromagnetic radiation on variability and biosynthetic activity of streptomycetes; A. Bratuhina 6th Central European Congress of Life Sciences Eurobiotech 2017

List of Posters Presentations

37

POSTER NUMBER

TITLE, AUTHORS

P14.3

Production and characterization of extracellular polysaccharide of extremely halophilic Archaea Haloferax mediterranei; Aneta Chytilová, Kateřina Drábková, Paulína Strečanská, Vojtěch Enev, Dan Kučera, Pavla Benešová, Stanislav Obruča

P14.4

Physicochemical properties and cytotoxicity of hydrogels based on Beetosan® containing sage, bee pollen and caffeine; A. Drabczyk, S. Kudłacik – Kramarczyk, B. Grabowska, M. Kędzierska, B. Tyliszczak

P14.5

Methanotrophs community – from environmental sample to enrichment culture; W. Goraj, A. Pytlak, A. Szafranek-Nakonieczna, Z. Stępniewska

P14.6

Production of ectoine and hydroksyectoine by methanotrophs isolated from Wieliczka Salt Mine; W. Goraj, Z. Stępniewska

P14.7

Structural determinants of Ty1 genomic RNA dimerization: the role of Gag and p22 restriction factor; J. Gumna, K. J. Purzycka, A. Saha, D. Garfinkel, K. Pachulska-Wieczorek

P14.8

Two sides of the green coin – lipid profiling of microalgal consortium selected for treatment of ammonium-rich wastewater; P. Jedynak, P. Petryszak, K. Mungunkhuyag, J. Burczyk, M. Kędra, P. Kaszycki, P. Malec

P14.9

Response of environmental microalgae to anthropogenic pollutants: ammonia and chromate. Implications for bioremediation; P. Kaszycki, K. Regdos, Z. Gajewski, C. Gutiérrez González

P14.10

Optimization of native S. cerevisiae yeast submerged fermentation (SmF) conditions for phytase production; G. Kłosowski, D. Mikulski, O. Jankowiak

P14.11

Strategies to improve the yield of PHAs produced from wood material; D. Kučera, P. Benešová, M. Kovářová, S. Obruča

P14.12

Beetosan – proecological chitosan – preparation, characterization, and in vitro cytotoxicity; S. Kudłacik-Kramarczyk, A. Drabczyk, M. Krzan, E. Olejnik, B. Tyliszczak

P14.13

Unleashing the biotechnological potential of the cold-adapted Psychrobacter spp.; R. Lasek, M. Krolikowski, A. Sówka, D. Bartosik

P14.14

Waste and agricultural products as sources of carbon and nitrogen in D-lactic acid biosynthesis; A. Michalczyk, A. Cieniecka-Rosłonkiewicz, A. Bialek, S. Garbaczewska

P14.15

The genotypic structure changeability and biodiversity of bacterial community in vertical flow constructed wetlands during sulfamethoxazole-rich wastewater treatment; A. Ziembińska-Buczyńska, K. Ślipko, M. Nowrotek, K. Miksch

P14.16

Bacterial autofluoresce – A useful tool in biotechnological processes; L. Müllerová, F. Mravec, K. Bílková, S. Obruča

P14.17

The effect of nickel and aluminum exposition on cell division under Tor1 signaling deficiency in Schizosaccharomyces pombe; M. Pozgajova, A. Navratilova, A. Trakovicka

P14.18

Role of polyhydroxyalkanoates in biofilm formation of Burholderia cepacia and Burholderia sacchari; S. Obruča, M. Rucká, K. Mrázová, E. Slaninová, O. Samek, J. Krouska, V. Krzyzanek

P14.19

Newly identified ciliary proteins as a potential novel markers in PCD diagnosis; P. Urbanska, E. Waclawek, R. Bazan, M. Poprzeczko, M. Niziolek, H. Farahat, A. Osinka, E. Joachimiak, H. Fabczak, D. Wloga

P14.20

Interconnection of microbial degradation of chicken feather and polyhydroxyalkanoates production employing selected Pseudomonas strains; I. Pernicová, Z. Šuráňová, P. Innemanova, S. Obruča

P14.21

Analysis of cellular fatty acids of selected microalgal species; P. Petryszak, P. Jedynak, K. Mungunkhuyag, J. Burczyk, P. Malec, P. Kaszycki

P14.22

In-situ monitoring of a stress-induced crystallization of bacterial polyhydroxyalkanoates; P. Sedláček, S. Obruča, E. Slaninová, F. Mravec, O. Samek, V. Krzyžánek

P14.23

Nisin-loaded magnetic nanoparticles; R. Gruskiene, T. Krivorotova, R. Staneviciene, D. Ratautas, E. Serviene, J. Sereikaite

P14.24

Accumulation of PHA granules protects bacterial cells from adverse effect of UV-irradiation; E. Slaninová, P. Sedláček, F. Mravec, O. Hesko, L. Müllerová, S. Obruca

P14.25

Biotransformation of aliphatic and aromatic hydrocarbons with Rhodococcus sp. CUP11 isolated from brown coal deposit; P. Supel, P. Petryszak, P. Kapusta, J. Brzeszcz, P. Kaszycki

P14.26

Psychrophilic laccase Kabatiella bupleuri – a novel enzyme useful in biotechnology; K. M. Szulczewska, A. Białkowska, M. Turkiewicz 6th Central European Congress of Life Sciences Eurobiotech 2017

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List of Posters Presentations

POSTER NUMBER

TITLE, AUTHORS

P14.27

Myxoxanthophylls – carotenoid glycosides from cyanobacteria: isolation and intial characterization; P. Żbik, P. Kaszycki, P. Malec

Sessions 15&18: From Molecular Machines to Molecular Medicine: Advances in Biotechnology P15&18.1

Effects of Rotenone in Neuroblastoma SK-N-AS Cells and its Neutralization, N. N. Ciroglu, P. Obakan Yerlikaya, A. Sendemir Urkmez, A. Coker Gurkan, E. D. Arisan and M. E. Genc

P15&18.2

GD2 ganglioside-binding antibody and specific aurora A kinase inhibitor induce autophagy in IMR-32 neuroblastoma cells; M. Durbas, P. Pabisz, K. Wawak, E. Boratyn, I. Nowak, I. Horwacik, H. Rokita

P15&18.3

Application of Adenovirus dodecahedron delivery vector as a new approach in overcoming drug resistance; M. Jedynak, P. Dolega, M. Podsiadla-Bialoskorska, J. Chroboczek, E. Szolajska

P15&18.4

MONOAMINE OXIDASE FROM GEOMYCES SP. P3 AS A TOOL IN SYNTHESIS OF CHIRAL AMINE BUILDING BLOCKS OF PHARMACEUTICAL DRUGS; I. Jodłowska, K. Szmejda, T. Florczak

P15&18.5

Study on the effect of dental material Optibond on sulfurtransferases expression in human fibroblast cell line Hs27; A. Bentke, K. Kaszuba, A. Krawczyk, M. Wróbel

P15&18.6

The expression of antioxidative enzymes and lipids peroxidation in the heart of frogs exposed to heavy metal ions; M. Kaczor-Kamińska, P. Sura, K. Kaszuba, M. Wróbel

P15&18.7

Quinaldic acid alters cell cycle progression of colorectal adenocarcinoma cells in vitro; E. Langner, P. Pożarowski, G. Rajtar

P15&18.8

Neuroprotective properties of young barley water extract. In vitro study in different models of neurodegeneration; M. Kinga Lemieszek, W. Rzeski

P15&18.9

Preparation of the genetic construct for expression of human CD47 gene in the swine Rosa26 locus using the Cas9 nuclease for xenotransplantation purposes; N. Mazurkiewicz, M. Hryhorowicz, A. Nowak, J. Zeyland, D. Lipiński, R. Słomski

P15&18.10

Is liquid biopsy a good molecular monitoring tool? Detection of L858R and T790M EGFR mutations in circulating tumor DNA derived from NSCLC patient treated with gefitinib (case study); E. Nalejska, Ł. Żołna, A. Chrząstek, B. Żurawski, J. Kowalewski, M. A. Lewandowska

P15&18.11

High-resolution protein stability analytics using nanodsf; J. Nowak

P15&18.12

Phage Display selection and characterization of scFv specifically recognizing extracellular domain of FGFR1; J. Szymczyk, J. Chudzian, Ł. Opaliński, M. Szczepara, M. Zakrzewska, J. Otlewski

P15&18.13

Cross-talk between platelets and lymphocytes in autoimmune thyroid disease; M. Tomczyńska, J. Saluk-Bijak

P15&18.14

Antiandrogens induce atresia and disturb maturation of porcine preantral ovarian follicles cultured in vitro; G. Gorczyca, K. Wartalski, Z. Tabarowski, M. Duda

Session 16: Dual use of biotechnology P16.1

The protective effect of selenium on human cells treated with zearalenone; A. Barbasz, B. Kreczmer, B. Dyba, M. Filek

P16.2

Influence of the application of variable raw material composition on the process of HG alcoholic fermentation conducted under bioreactor conditions; G. Kłosowski, D. Mikulski

P16.3

Orthogonal, light-inducible protein expression platform in yeast Sacchararomyces cerevisiae; K. Messerschmidt, F. Machens, L.a Hochrein, G. Naseri

P16.4

Effects of various parameters of acidic pretreatment on the enzymatic hydrolysis efficiency of distillery stillage of different origin; D. Mikulski, G. Kłosowski

P16.5

Optimization of the enzymatic hydrolysis process of distillery stillage of various origin subjected to the procedure of acidic pretreatment; D. Mikulski, G. Kłosowski

P16.6

Association of kappa –casein gene polymorphism with milk yield, fat and protein in Holstein cattle; M. Miluchová, M. Gábor, J. Candrák, A. Trakovická, K. Candráková

P16.7

Effective population size and genetic drift in warmblood horses based on microsatellite DNA markers; N. Moravčíková, R. Kasarda, O. Kadlečík

P16.8

Integrated system of sewage treatment, biogas production and methane enrichment of biogas unit; A. G. Chmielewski, J. Palige, O. Roubinek, S. Witman-Zając, J. Usidus, A. Kryłowicz 6th Central European Congress of Life Sciences Eurobiotech 2017

List of Posters Presentations

POSTER NUMBER

39

TITLE, AUTHORS

Session 17: Stem cells in diseases and therapy P17.1

Cobalt protoporphyrin increases G-CSF and mobilizes granulocytes and hematopoietic stem cells to the peripheral blood in mice, A. Szade, K. Szade, W. N. Nowak, K. Bukowska-Strakova, M. Cieśla, N. Kachamakova-Trojanowska, J. Dulak, A. Józkowicz

Session 19: Big Data and Biotechnology P19.1

Common breeding history of different cattle breeds based on high-throughput genotyping data; V. Kukučková, R. Kasarda, N. Moravčíková, I. Curik, M. Simčič, G. Mészáros

P19.2

Analysis of genetic basis of natural herbicide resistance in maize by high-throughput sequencing technology; M. Mahmoud, M. Żywicki, W. M.Karłowski

P19.3

Can ecotoxicity tests performed in artificial soils may be used for prediction of metals toxicity in natural soils?

M. Sydow, Ł. Chrzanowski, N. Cedergreen, M. Owsianiak

P19.4

Analysis of the influence the rotating magnetic field (RMF) on the horseradish peroxidase catalytic properties; A. Wasak, R. Drozd, R. Rakoczy

P19.5

Modification of catalytic properties of laccase with use the rotating magnetic field; A. Wasak, R. Drozd, R. Rakoczy

P19.6

Greater Poland Voivodeship as the bioenergy producer; E. Woźniak, E. Kalbarczyk

P19.7

Network analysis of Arabian horses family ties; K. Żukowski, K. Pietrzyk, M. Stefaniuk-Szmukier, A. Sosińska, K. Piórkowska, K. Ropka-Molik, M. Bugno-Poniewierska

P19.8

The utilization of multi-dimensional scaling for colour coat database misclassification in Holstein cattle; K. Żukowski, J. Dulska, A. Gurgul, K. Piórkowska, K. Ropka-Molik

P19.9

POLGENOM – Polish genomic reference database – as a result of whole genome sequencing of successfully aging long-lived Polish Caucasians; E. Matczynska, P. Lyszkiewicz, M. Krawczyk, D. Kowalczyk, R. Szymańczak, M. Mossakowska, M. Puzianowska-Kuznicka, M.Zagulski, A.Boguszewska-Chachulska

Session 20: Tools in the treatment of human disorders P20.1

Targeted inhibition of IRF1 as a potential treatment strategy in cardiovascular disease; A. Antonczyk, M. Szelag, J. Wesoly, H.A. Bluyssen

P20.2

Modulation amino acid pool of blood plasma adding pyridoxine phosphate in composition of amino acids; A. Buklaha, A. Pauliukavets, V. Sheibak

P20.3

Mechanisms of cellular uptake of S-glycosylated thiosemicarbazides; A. Czubatka-Bienkowska, J. Sarnik, A. Macieja, Z. J. Witczak, T. Poplawski

P20.4

Microvesicles stimulates endothelial cell migration in hyperglycemic conditions; A. Drozdz, R. Jach, H. Huras, E. Stepien

P20.5

The role of red fox as a zoonotic reservoir for Alaria alata flukes; D. Dwużnik, D. Kakietek, M. Religa, E. Mierzejewska, A. Bajer

P20.6

De novo method of protein folding simulations; M. Gadzała, D. Dułak, B. Kalinowska, I. Roterman-Konieczna

P20.7

Antioxidant properties of hyaluronic acid in the human cancer cells in vitro; G. Gajek, R. Kotek

P20.8

Preliminary analysis of Naja ashei venom proteome in terms of its pharmacological potential; K. K. Hus, J. Buczkowicz, A. Łyskowski, V. Petrilla, M. Petrillová, J. Legáth, A. Bocian

P20.9

The expression and activity of hydrogen sulphide generating enzymes in INFγ- and LPS-stimulated murine macrophage J774A.1 cells; M. Wróbel, J. Marcinkiewicz, H. Jurkowska, P. Bronowicka-Adamska, K. Kaszuba, D. Szlęzak, P. Skalska

P20.10

The inhibition of cancer cell cycle and induction of apoptosis by 4-hydroxybenzyl isothiocyanate, a natural H2S-donor; H. Jurkowska, D. Szlęzak, M. Wróbel, E. Jasek-Gajda

P20.11

Spectroscopic and proteomic analysis of urinary extracellular vesicles-potential biomarkers of renal failure; A. Kamińska, M. Platt, J. Kasprzyk, A. Drożdż, K. Dziedzic Kocurek, B. R. Jany, W. Piekoszewski, M. Kuźniewski, F. Krok, E. Ł. Stępień

P20.12

Human osteosarcoma U-2 OS cells with acquired resistance to RG7388, a small-molecule inhibitor of p53-MDM2 interaction; J. Kocik, J. Polak, T. A. Holak, Ł. Skalniak 6th Central European Congress of Life Sciences Eurobiotech 2017

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List of Posters Presentations

POSTER NUMBER

TITLE, AUTHORS

P20.13

Association between polymorphisms in TIMELESS and asthma risk in the population of Polish children; W. Langwinski, B. Narozna, P. Sobkowiak, A. Breborowicz, A. Szczepankiewicz

P20.14

Comparison of two HRR (homologous recombination repair) inhibitors as a potential therapeutic agents against cisplatin-sensitive and cisplatin-resistant human ovarian carcinoma cells; A. Macieja, J. Sarnik, A. Czubatka-Bieńkowska, T. Popławski

P20.15

Antiproliferative capacity of Sea buckthorn (Hippophae rhamnoides L.) extracts in selected human cell lines; B. Marciniak, R. Kontek

P20.16

Dendritic cells activation with parasite components; M. Maruszewska-Cheruiyot, K. Donskow-Łysoniewska, K. Krawczak, M. Doligalska

P20.17

New betulin derivatives as candidates for anticancer agents; P. Paduszyński, K. Jelonek, M. Jaworska-Kik, A. Orchel, E. Chrobak, E. Bębenek, S. Boryczka, E. Chodurek, J. Kasperczyk

P20.18

Dietari supplement on the basis of amino acids and zinc; A. Pauliukavets, V. Sheibak, A. Buklaha

P20.19

Improvement in behavior and biochemical parameters in the streptozotocin-induced rat model of Alzheimer disease treated with genistein; K. Pierzynowska, M. Podlacha, D. Myślińska, I. Majkutewicz, J. Mantej, G. Węgrzyn

P20.20

Lack of miR-378 mitigates dystrophic phenotype in mdx mice – transcriptome analysis; P. Podkalicka, O. Mucha, K. Pietraszek-Gremplewicz, M. Kozakowska, I. Bronisz, M. Mikula, A. Jozkowicz, A. Loboda, J. Dulak

P20.21

The analysis of cell cycle arrest induction by RG7388 using the selected cell line models; J. Polak, J. Kocik, T. A Holak, Ł. Skalniak

P20.22

Inhibitory effect of Functional carb-pharmacophores with sulfur atom on proliferation of cancer cells; J. Sarnik, A. Czubatka-Bieńkowska, A. Macieja, Z. J Witczak, T. Popławski

P20.23

Influence of chlorpyrifos on the level of expression of vitamin D3 receptor in skin cells; K. Sawicki, A. Czerwonka, M. Kruszewski, L. Kapka-Skrzypczak

P20.24

Deragulated apoptosis of neutrophils as a potential mechanism underlying the pathobiology of periodontitis; M. Sochalska, M. Stańczyk, M. Użarowska & J. Potempa

P20.25

Comparative analysis of gene expression of human capillary (CAP) and high endothelial cells (HEC); A. Szade, K. Brulois, M. Lee, M. Rahman, E. Butcher

P20.26

Association study of polymorphisms in genes involved in lipid metabolism; A. Trakovická, N. Moravčíková, K. Candráková, M. Miluchová, M. Gábor, R. Kasarda

P20.27

Endocan: an early marker of bacterial infection in decompensated cirrhosis; J. Zuwala-Jagiello, J. Górka-Dynysiewicz, E. Grzebyk, E. Murawska-Ciałowicz, M. Pazgan-Simon

P20.28

New model using pentraxin 3 to predict liver fibrosis in patients with chronic hepatitis C; J. Górka-Dynysiewicz, M. Pazgan-Simon, J. Zuwala-Jagiello

Session 21: Art in Biotechnology P21.1

Directions for optimizing organ preservation solution; A. Ostróżka-Cieślik, B. Dolińska, F. Ryszka

6th Central European Congress of Life Sciences Eurobiotech 2017

ABSTRACTS

42 Abstracts

Plenary Lectures PL1

PL2

A bright Future for Antibiotics? Medical and Environmental Consideration

Linear and circular RNAs

Ada Yonath

Nikolaus Rajewsky

Department of Structural Biology, Weizmann Institute, Rehovot 76100, Israel

Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology

Resistance to antibiotics and the spread of antibiotics’ metabolites are severe problem in contemporary medicine.

Structures of complexes of eubacterial ribosomes with antibiotics paralyzing them, illuminated common pathways in the modes of antibiotics inhibitions, synergism, differentiation and resistance. Additionally, recent structures of ribosome from a multi-resistant pathogenic bacterium identified features that can account for species-specific diversity in infectious-diseases susceptibility. Careful analysis and comparisons to ribosomes from benign bacteria indicated novel structural features on the ribosome periphery, which are crucial for protein biosynthesis but do not belong to the primary ribosomal active site. Thus, opening new paths for the design of novel, next generation antibiotics, which are environmental-friendly degradable compounds, as well as species-specific, hence should minimize resistance alongside preserving the microbiome.

I will present projects where we employ massively parallel, high-throughput single-cell RNA sequencing. I will present (a) the perhaps first “complete” cell/lineage atlas of a complex animal (b) (collaboration with Zinzen lab) a “virtual embryo” where we have sequenced each cell of the drosophila embryo at stage 6 and reconstructed gene expression for 8,000 genes per cell in 3-D, (c) data about single-cell sequencing of an animal germline which reveal spatial organization of functional classes of RNA

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 43

PL3

PL4

Biotechnological Drugs for Cardiac Repair and Regeneration

Gene Delivery to the Brain; translational journey from bench to bedside

Mauro Giacca

Bankiewicz KS, Samaranch L, San Sebastian W, Naidoo J, Bringas J, Forsayeth J

International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

Interventional Neurology Center, Department of Neurological Surgery, University of California San Francisco, 1855 Folsom Street, MCB Rm 226, San Francisco, 94114, CA.

Degenerative disorders, including dementia and other neurodegenerative conditions, retinal degeneration, presbycusis and heart failure, are imposing an impressive toll on society in terms of morbidity, mortality and economic burden. Thus, there is an impelling need to develop novel therapeutic strategies aimed at preventing tissue degeneration and inducing regeneration. In particular, the number of patients diagnosed with heart failure is over 17 million every year worldwide, and rising. In contrast to other species that regenerate the heart during the adult life, in mammals damage to the myocardium is mended by a scarring mechanism. However, multiple evidence now indicates that a limited capacity of myocardial renewal also exists in adult individuals, and we are therefore actively searching for factors able to foster this regenerative capacity.

Using viral vectors based on the Adeno-Associated Virus (AAV), which transduce the heart at very high efficiency, we have undertaken an exhaustive approach to identify factors promoting cardiac repair, selected from an AAV library corresponding to the mouse secretome (1200+ secreted proteins). The identified proteins can be obtained as recombinant factors and administered systemically to prevent cardiac damage.

A second approach entails high throughput screening of microRNAs promoting cardiomyocyte proliferation. Starting from a whole genome human microRNA library, we have identified a few microRNAs endowed with the capacity of promoting expansion of cardiomyocytes in cell culture, inducing massive cardiac hyperplasia in the neonatal heart and improving cardiac function after myocardial infarction. These microRNA function by directly activating the proliferative potential of differentiated cardiomyocytes, thus bypassing the requirement of stem cell expansion and differentiation.

Protecting the heart from degeneration and inducing its regeneration by innovative biotech drugs might represent a new paradigm that could also extend to degenerative conditions in other organs.

Gene transfer technology can correct genetic mutations in the brain. Neuro gene delivery via direct intrapranchymal injections of adeno-associated viral (AAV) vectors is a locally administered treatment that requires accurate delivery to maximize safety and efficacy. The large volume and convoluted architecture of the human brain is a considerable barrier to translating small animal findings into efficacious clinical procedures. Too little target coverage and the treatment risks being ineffective. Conversely, excessive distribution or off-target gene delivery increases the possibility for unexpected adverse effects. Optimal viral vector delivery into the brain is challenging and brain distribution of viral vectors is uncertain. To address this issue we developed viral vector delivery system that permits direct MRI monitoring of vector distribution within the brain in real-time. This significant advance allows for the first time to adjust parameters of vector infusion while delivering gene therapy, giving surgeon full control over gene transfer technology.

To allow for precise intracerebral delivery of biologics for therapy of neurological disease we developed a skull-mounted aiming devices and integrated software platform for interventional MRI guided gene delivery to the brain. In our ongoing gene therapy clinical trials in brain disorders we adapted this device for real-time convection enhanced delivery (RCD) of therapeutics via a custom designed infusion cannula. Based on real-time MRI data, this system allows selection of brain targets, provides instructions for cannula insertion along a chosen trajectory, and permits visual monitoring of infusions.

Subsequent to our discovery that AAV2 vectors undergo anterograde transport along thalamocortical projections resulting in transduction of cortical neurons, we analyzed properties of several AAV serotypes and evaluated their potential for correcting genetic deficit in the brain via axonal transport. Combination of RCD and axonal transport may allow for predictable gene transfer over large cortical and sub-cortical regions of a human brain.

Our advanced gene delivery system is currently tested for delivery of therapeutic genes in Parkinson’s (PD), Huntington’s (HD) and Nieman-Pick, AADC deficiency in children and brain tumors. Data will be provided to demonstrate promises and challenges in successful clinical translation of gene transfer technology for CNS disorders based on our ongoing clinical investigations of gene therapy for several brain disorders.

6th Central European Congress of Life Sciences Eurobiotech 2017

44 Abstracts

PL5

PL6

Synthetic biotechnology as applied for crop improvement and molecular farming

Going back to Roots: Biological-basis for Designing Biomaterials for the Injured and Degenerated Host

Stefan Malepszy and Leszek A. Łyżnik

Abhay Pandit

Faculty of Horticulture, Biotechnology and Landscape Architecture, Warsaw University of Life Sciences

Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, Ireland, Abhay.pandit@nuigalway.ie

Synthetic biology makes it possible to produce user-defined biological organisms/systems. Whether used for medical applications, production of biological compounds of industrial, therapeutic, or pharmaceutical value (molecular farming), or for making animals and plants performing better in the ever-changing environmental conditions, the synthetic biology concepts have already proven their usefulness. The CRISPR genome editing system advances practical applications of synthetic biology even further.

New venues for biological studies have been opened up, thus employing new designs of experimental algorithms and enforcing re-evaluation of the established definitions of genetic manipulation products. An effort to apply new synthetic biotechnology principles in the context of plant breeding and crop improvement research programs will be discussed.

Biomaterials are no longer considered innate structures and using functionalisation strategies to modulate a desired response whether it is a host or implant is currently an important focus in current research paradigms. Fundamentally, a thorough understanding the host response will enable us to design proper functionalisation strategies.

The input from the host response need to be weighed in depending on the host disease condition. In addition biomaterials themselves provide immense therapeutic benefits which needs to be accounted for when using functionalisaton strategies. Using strategies such as enzymatic and hyperbranched linking systems, we have been able to link biomolecules to different structural moieties.

The programmed assembly of biomolecules into higher-order self-organized systems is central to innumerable biological processes and development of the next generation of functionalized scaffolds. Our recent design efforts have utilized a developmental biology approach toward both understanding and engineering supramolecular protein assemblies. Structural moieties have taken a variety of different forms such as nanofibers and nanoparticulate.

This approach has resulted in functionalisation of micro and nanoparticles with biomolecules that include designed peptide motifs, growth factors and a multitude of gene vector systems. In addition, nature itself has abundant structural complexity that can be harnessed for targeted clinical applications. This talk will elucidate some of these ongoing strategies in our laboratory.

Acknowledgements This publication has also emanated from research conducted with the financial support of Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Fund under Grant Number 13/RC/2073.

References Samal, J., Hoban, D.B., Naughton, C., Concannon, R., Dowd, E. and Pandit, A. ‘Fibrin-based Microsphere Reservoirs for Delivery of Neurotrophic Factors to the Brain.’ Nanomedicine. 2015: 10(5): 765–783.

Thomas, D., Fontana, G., Chen, X., Sanz-Nougés, C., Zeugolis, D., Dockery, P., O’Brien, T. and Pandit, A. A. Shape-controlled Tuneable Microgel Platform to Modulate Angiogenic Paracrine Responses in Stem Cells.’ Biomaterials. 2014: 35(31): 8757–66.

Lang, Y., del Monte, F., Collins, L., Rodriguez, B.J., Thompson, K., Dockery, P., Finn, D.P. and Pandit, A. ‘Functionalization of the Living Diatom Thalassiosira Weissflogii with Thiol Moieties.’ Nature Communications. 2013: 4: 3683 Hoban, D., Newland, B., Moloney, T., Howard, L., Pandit, A. and Dowd, E. ‘The Reduction in Immunogenicity of Neurotrophin Overexpressing Stem Cells after Intra-Striatal Transplantation by Encapsulation in an In Situ Gelling Collagen Hydrogel.’ Biomaterials. 2013: 2013: 34(37): 9420–9429.

Browne, S., Monaghan, M.G., Brauchle, E., Berrio, D.C., Chantepie, S., Papy-Garcia, D., Schenke-Layland, K. and Pandit, A. ‘Modulation of Inflammation and Angiogenesis and Changes in ECM GAG-activity Via Dual Delivery of Nucleic Acids.’

Biomaterials. 2015: 69: 133–147.

Dash, B.C., Thomas, D., Monaghan, M., Carroll, O., Chen, X., Woodhouse, K., O’Brien, T. and Pandit, A. ‘An Injectable Elastin-based Gene Delivery Platform for Dose-dependent Modulation of Angiogenesis and Inflammation for Critical Limb Ischemia.’ Biomaterials. 2015: 65:126–139.

Tapeinos, C. and Pandit, A. ‘Physical, Chemical and Biological Structures based on ROS-sensitive Moieties that are able to respond to Oxidative Microenvironments.’

Advanced Materials. 2016: 28(27): 5553–5585

6th Central European Congress of Life Sciences Eurobiotech 2017

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PL7

PL8

Caging Proteins: Building Artificial Supramolecular Complexes Using Biological Building Blocks

New Ways of Vision: Protein Structures in Translational Medicine and Business Development, my Experience

Jonathan G. Heddle

Robert Huber

Bionanoscience and Biochemistry Laboratory, Malopolska Centre of Biotechnology, Jagiellonian University 30-387, Krakow, Poland, jonathan.heddle@uj.edu.pl

Max-Planck-Institut fuer Biochemie D-82152 Martinsried, Germany; Technische Universität München, D-85747 Garching, Germany; Universität Duisburg-Essen, D-45117 Essen, Germany

Synthetic structural biology – the design and production of artificial molecular complexes using biological molecules is coming of age. DNA nanotechnology has led the way but proteins whose in vivo functionality make them a desirable target, have proved more difficult to produce.

It is still the case that large assemblies of proteins are currently not feasible for ab initio design. However, by using existing known protein structures as building blocks, which are further modified to provide connections to other building blocks, large protein complexes can be designed.

In recent years this has allowed novel protein cages to be produced via a number of techniques. In our own work we have used the toroidal TRAP protein to build large (20 nm) cages1,2. Interestingly, cage assembly is promoted by gold nanoparticles from a starting protein with 11-fold rotational symmetry. The resulting cages have a number of unusual features including high stability and the ability to be assembled/disassembled as well as a unique geometry as demonstrated by our recently solved high-resolution structure.

As a student in the early nineteen sixties, I had the privilege to attend winter seminars organized by my mentor, W. Hoppe, and by M. Perutz, which took place in a small guesthouse in the Bavarian-Austrian Alps. The entire community of a handful of protein crystallographers assembled in a room which served as living and dining room and as auditorium for the lectures.

Today structural biologists organize large congresses with thousands of attendants and there exist many hundreds of laboratories specialized in this field. It appears to dominate biology and biochemistry very visibly if we count covers in scientific journals displaying macromolecular structures.

Structural biology was successful, because it was recognized that understanding biological phenomena at the molecular and atomic level requires seeing those molecules.

Structural biology revealed the structure of genes and their basic mechanism of regulation, the mechanism of enzymes’ function, the structural basis of immune diversity, the mechanisms of energy production in cells by photosynthesis and its conversion into energy- rich chemical compounds and organic material, the mechanism that makes muscle work, the architecture of viruses and multi-enzyme complexes, and many more.

New methods have an essential impact on the development of structural biology. Methods seem to become available in cadence with the growing complexity of the problems and newly discovered methods bring biological problems within reach for researchers, a co-evolutionary process of the development of methods and answerable problems, not only in the field of x-ray diffraction, but also in optical microscopy, nuclear magnetic resonance, and electron microscopy.

An important additional incentive for structural biology came from its potential application for drug design and development using knowledge of drug receptor structures at the atomic level combined with theoretical approaches of ligand binding. The commercial interest in application spurred this direction of research enormously.

My lecture will focus on protein crystallography and start out with the major factors contributing to its development. Few examples shall illustrate how structure information contributes to our understanding of the physical and chemical basis of biological phenomena and may lead to medical application.

I then will let you share my experience with the foundation and development of two biotech companies with different business models, but both based on basic academic research in structural biology: Proteros (www.Proteros.com) offers enabling technology services for Pharma- and Crop science companies imbedding all steps of the workflow molecular and struc-

References 1 Malay, A. D. et al. Gold Nanoparticle-Induced Formation of Artificial Protein Capsids. Nano Lett. 12, 2056–2059, (2012).

2 Imamura, M. et al. Probing structural dynamics of an artificial protein cage using high-speed atomic force microscopy. Nano Lett. 15, 1331–1335, (2015).

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46 Abstracts

tural biology can provide and commands and uses its platform for the generation of leads from identified targets to in vivo Proof of Concept (PoC).

Suppremol (www.Suppremol.com) specializes in the development of novel immune-regulatory therapeutics for the treatment of autoimmune diseases on the basis of a recombinant, soluble, non-glycosylated version of the human Fcg receptor IIB and of receptor binding antibodies.

Suppremol was recently acquired by Baxter International Inc. (NYSE:BAX) offering an ideal setting for its therapeutic projects.

PL9 Building the mammalian embryo in vivo and in vitro Magdalena Żernicka-Goetz University of Cambridge Mammalian embryogenesis requires intricate interactions between embryonic and extra-embryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. In both mouse and human embryos the first most dramatic series of morphogenetic transformations in embryo architecture and potency is initiated during embryo implantation into the body of the mother. Despite its major importance, an understanding of embryo remodeling during the implantation stage has been lacking, due to the embryo’s inaccessibility within the mother. Motivated to understand this process, we have established a system permitting mouse and human embryogenesis beyond implantation in vitro. This allowed us to reveal steps of architectural remodelling and the importance of embryonic and extra-embryonic partnership in this process. Building upon this knowledge, we have attempted to mimic successive steps of this partnership with stem cells in vitro. To achieve this, we have combined mouse embryonic stem cells (ESCs) and extra-embryonic trophoblast stem cells (TSCs) in a 3D scaffold of extra-cellular matrix (ECM) that allowed us to generate structures whose morphogenesis is remarkably similar to natural embryos. By using genetically-modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos, ETS-embryos, depends on crosstalk involving Nodal signalling. When ETS-embryos develop further, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic – extra-embryonic border, in response to Wnt and BMP signalling. This study demonstrates that enabling crosstalk between embryonic and extra-embryonic stem cells in a 3D ECM scaffold is sufficient to trigger self-organization recapitulating spatio-temporal events and leading to faithful reconstruction of embryo architecture and patterning. This stem cell model of mammalian embryogenesis, in combination with genetic manipulations, might provide a powerful platform to dissect physical and molecular mechanisms that mediate natural embryogenesis.

PL10 Can enzymes be used to treat difficult cancers?

Thomas Leung The Hong Kong Polytechnic University Can enzymes act as specific and potent drugs? The answer is “Yes”. Enzymes can bind and act on their targets with great affinity and specificity. Many enzyme drugs have been successfully developed for a wide range of disorders. In 1982, insulin was marketed as the first recombinant protein drug. In 1987, recombinant human tissue plasminogen activator was approved by the Food and Drug Administration (FDA) as the first recombinant enzyme drug. In 1990, bovine adenosine deaminase (ADA) was successfully conjugated with polyethylene glycol (PEG) and approved to treat severe combined immunodeficiency disease (SCID). More importantly, PEGylated bacterial asparaginase is an enzyme already in use in the clinic. It is for the treatment of children with acute lymphoblastic leukemia (ALL). Normal cells are able to synthesize asparagine but cancer cells cannot do so and die in the presence of the asparagine-degrading enzyme. PEG-asparaginase has longer circulating half-life, lower immunogenicity and the overall cost of the treatment is very similar to the one with the native enzyme. Pharmaceutical companies can now produce safer and cheaper enzymes with enhanced potency and specificity. Furthermore, changes in orphan drug laws have facilitated efforts to develop enzyme drugs.

There are other examples of the use of enzyme therapeutics for treating cancer. PEGylated bacterial arginine deaminase (PEG-ADI) is an arginine-degrading enzyme.

It inhibits human melanoma and some liver cancers, which lack argininosuccinate synthetase (ASS) activity. Many cancer types are believed to be auxotrophic for arginine through the lack of expression of ASS. This has opened up new possibilities for effective cancer therapy. Nevertheless, many ASS-positive liver cancer cell lines are found to be resistant to ADI treatment. Our team is the first to discover an arginine-depleting enzyme for killing ASS-deficient as well as ASS-positive tumors. We have demonstrated that recombinant human arginase (rhArg) inhibits ASS-positive liver cancer cell lines, which were sensitive to rhArg but ADI had virtually no effect on these cells.

They all expressed ASS, but not ornithine transcarbamylase (OTC), the enzyme that converts ornithine, the product of degradation of arginine with rhArg, to citrulline, which is converted back to arginine via ASS. We further demonstrated that OTC expression alone is sufficient to induce rhArg resistance in ASS-positive cancer cells.

Therefore, pretreatment tumor gene expression profiling of ASS and OTC could aid in predicting tumor response to arginine depletion with arginine-depleting enzymes. We have also shown that the rhArg native enzyme and PEGrhArg gave similar anticancer efficacy in vitro. Furthermore, the growth of the OTC-deficient liver tumor cells (ASS-positive and ADI-resistant) in mice was inhibited by treatment with PEG-rhArg, which is active alone and is synergistic in combination with 5-fluorouracil. Thus, our data suggest that PEG-rhArg is a safe agent for effective cancer therapy.

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Abstracts 47

PL11 Why so many novel treatments work great in animals but not patients, and what you can do about it Jonathan Kimmelman Associate Professor, Biomedical Ethics Unit / Experimental Medicine, McGill University, Montreal, Quebec, Canada The vast majority of novel treatments introduced into randomized trials fail to demonstrate the promise suggested in animal experiments and early phase trials. In what follows, I suggest that some of the reasons that explain why so many drugs fail to progress into effective treatments get to the heart of what drug development is about. Other factors that explain why drugs fail, however, relate to inadequate design, reporting, and interpretation of animal and clinical studies. After describing some of the deficient practices I have uncovered in my own research, I explain what is morally at stake in improving the way we develop new drugs. I close by offering a series of recommendations at the individual and institutional level.

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Publication Workshop, Student session PW.1.

PW.2

How to present your work at a scientific meeting

How to increase the chance that your paper will be accepted

Jeff Cole

Agata Tyczewska

School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Vice-editor of quarterly BioTechnologia. Journal of Biotechnology, Computational Biology and Bionanotechnology.

Publications Workshop It was Nobel Prize winner Hans Krebs whose mantra was “Research that never gets published is worthless”. For young staff in many institutions there remains the underlying threat: Publish or perish. Despite these pressures and the training available to graduate students, journal editors never cease to be amazed by the stupidity shown by some authors who are keen to publish their results. The first aim of this Publications Workshop will be to provide examples of “How to ensure that your paper will be rejected”. The second aim immediately follows from the first: it will be to show “How to increase the chance that your paper will be accepted”. The third and final aim will focus on “How to interact with journal editors and reviewers”. This will remind you that your manuscript will be read by colleagues, volunteers who often will be working in their leisure time when they would prefer to be watching the soccer match on their television. If you make life difficult for them, they will be less motivated to help you! The workshop Convener will then suggest some “golden rules” that have helped many generations of university students write scientific results in a way that can be understood by people whose first language is not English.

The three short presentations will followed by ample time for questions from the audience. All three people presenting short talks are experienced editors or publishers who are well aware of the publishing process. All of us have experienced the disappointment of having excellent work rejected by a journal. Are you well prepared to cope with the consequences?

BioTechnologia is a scientific journal with a long history. It was intiated in 1988. For many years it was being published in Polish and served as a source of review papers on various biotechnological topics, covering research as well as industrial applications. It was always high-ranked and found very valuable by researchers, but also by students as it provided up-to-date information that scientific textbooks could not. In 2011 BioTechnologia went through significant changes to transform it into an international journal. The changes were not only focused on the layout but also the scope of interest shifted onto new disciplines developing at the boundary of biology, technology and computational sciences.

My adventure with BioTechnologia started in 2009, when I became a co-editor of a special issue on protein biosynthesis, which was followed by another special issue on small non-coding RNAs in 2010. Few years later I became a vice-editor of BioTechnologia. From this standpoint I wish to share my observations an reflections on how to prepare manuscripts so that they do not become reviewers’ and editors’ worst nightmares.

Short workshop on “How to present your work at a scientific meeting” In the next three days you will hear many excellent talks by eminent scientists. Sadly you will also hear talks by people who spoil their presentations by stupid mistakes, especially with their slides. This final session will alert you to the most common mistakes – and give you a framework within which to judge the quality of the talks during Eurobiotech 2017.

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Abstracts 49

Nucleic Acids Tools for Regulation of Gene Expression Keynote ecture

Lectures:

L1.1

L1.2

RNA modifications and NGS: It’s not a bug, it’s a feature

Pioneering ribozyme-based strategy for mitochondria transformation and gene regulation

Anna M. Kietrys, Eric T. Kool

Katarzyna Rolle1, Laure D. Sultan 2, Daria Mileshina3, Paweł Głodowicz 1, Oren Ostersetzer-Biran2, Jan Barciszewski 1, André Dietrich 3

Department of Chemistry, Stanford University, Stauffer I, Stanford, CA 94305 In last decade, the presence of modifications in RNAs has been revealed as a factor playing regulation role in a variety of biological processes: splicing, translation, cellular localization, RNA lifetime, and dynamic structure changes. Better understanding of modifications’ roles has become possible thanks to a combination of deep sequencing, and base-specific biochemical methods. Despite this progress, scientists need to face possible bias in sequencing data from the reverse transcription step. Reverse transcriptases may not read modified nucleotides as well as unmodified, and this may affect assignment of mutations as well as modified bases.

Our project targets this bias in RNA sequencing results, and makes use of it to help understand modification-related patterns in sequencing data. We present methods of interpreting the bias to identify modifications. We use deep sequencing patterns, including miscoding, indels, and truncations, as fingerprints to indicate the presence of specific modified bases.

We designed a set of 10 pairs of synthetic RNA sequences containing modified nucleotides: m6A, Φ, I, m1G, m6G, m1A, m66A, m3U, m5C, and m5U. Paired RNAs consist of an oligoribonucleotide containing consensus biological motif with one modified base, and a control unmodified oligonucleotide. We processed samples with adapters and primers from Illumina TruSeq® Small RNA kit, and the split-adapter library preparation method. We identified modifications’ profiles based on frequency of the truncation, insertion, deletion, and mutation frequencies. Using quantitative methods and principal component analysis, we discriminated unique fingerprints of several specific modifications. The results can be used in RNA-seq data interpretation to identify modified bases and percentage of their occurrence.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland; 2Department of Plant and Environmental Sciences, The Alexander Silberman Institute of Life Sciences The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel; 3 Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général 12 Zimmer, 67084 Strasbourg, France

1

The aim of the work was to develop a new efficient tool to down regulate mitochondrial genes with specific catalytic ribozymes.

We designed the transcriptional platform with trans-cleaving hammerhead ribozyme (HH) and the tRNA-like shuttle, expressed in plants and imported into the organelles with the first directed knockdown of a mitochondrial RNA in eukaryote.

With our aprroach we designed the constructs with HH ribozymes against matR – mitochondrion-encoded maturase mRNA and introduced them into A.thaliana. For the first time we generated the mitochondrial loss-of-function plant, followed by the functional characteristics of mitochondrial gene. These results showed the function of MatR, but most of all the strategy opens the way for RNA-directed manipulation of the mitochondrial genetic system for both fundamental investigations and biotechnology.

Acknowledgements This work was supported by the National Science Center grant UMO-2013/09/B/ NZ1/03359.

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L1.3

L1.4

Transcriptional silencing of the CD146 expression in breast cancer cells by promoter methylation

Landscape of RNA and protein processing of TMEMs in clear cell renal cell carcinoma

Paulina Dudzik, Kinga A. Kocemba-Pilarczyk, Barbara Ostrowska, Joanna Dulińska-Litewka, Piotr Laidler

Natalia Pstrąg1, Arkadiusz Kajdasz1, Hans Bluyssen2, Joanna Wesoły1

Chair of Medical Biochemistry, Jagiellonian University Medical College, Krakow, Poland Activation of epithelial to mesenchymal transition (EMT) in cancer cells leads to more aggressive fenotype, invasion and migration to the distant organs. Recently, the high expression of CD146 has been identified as a progression marker of several cancers. Despite the significant correlation between the overexpression of CD146 protein and a mesenchymal phenotype of cancer the molecular basis for the high level of this protein has not yet been elucidated. By modulating signalling pathways, often deregulated in tumour cells, we attempted to induce the expression of CD146 in epithelial type breast cancer cells (MCF7), that initially presented low expression of CD146 on mRNA and protein level. Of all methods applied only demethylation by 5-aza-deoxycytidine triggered the increase of CD146 expression. Next, by analyzing DNA sequence of CD146 gene, using MethPrimer program, we found CpG island composed of 594 bp overlapping an exon 1 and the region of −106 to +22 bp, described previously as minimal promoter region predominantly controlling the CD146 gene expression. Analysis of the CD146 promoter, by methylation-specific PCR (MSP), revealed CpG island methylation in MCF7 cancer cells. Moreover, treatment with 5-aza-deoxycitidine led to meaningful increase of CD146 expression on mRNA and protein level in three additionally analysed breast cancer cell lines. Thus, taken together, our data identified methylation as a cause of CD146 transcriptional silencing in breast cancer cells.

Laboratory of High Throughput technologies, Faculty of Biology, A. Mickiewicz University in Poznań; 2 Department of Human Molecular Genetics, Faculty of Biology, A. Mickiewicz University in Poznań 1

Keywords: TMEM, ccRCC Clear cell renal cell carcinoma (ccRCC) is the most common of renal cancers. Our meta-analysis of gene expression data1 showed a significant de-regulation of several genes in ccRCC tumors, namely TMEM 30B, 72, 116, 207 and 21, belonging to a group of transmembrane proteins (TMEMs).

TMEMs are a heterogeneous group of proteins that make up ~30% of the proteome, predicted to be located in cellular membranes and essential for the cell function. They are involved in many biological processes such as energy transduction and transport. Their role in healthy cells and the mechanisms of their de-regulation in ccRCC are unknown. We investigate those mechanisms in case of 5 TMEMs with highest down-regulation in ccRCC in combination with the role of the tissue specific splice variants of TMEMs. We found that TMEM116 possesses multiple isoforms, unequally expressed in healthy patients, tumor tissue and cell lines. Ultimately, we would like to investigate in depth the impact of TMEMs on cancerogenesis and their function in healthy cells.

References [1] Wrzesiński T et al (2015)BMC Cancer 5: 518

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Abstracts 51

L1.5

L1.6

Hydrolytic activity of the hNudt16 towards oligonucleotides bearing different native cap structures

Vitamin B12 as a carrier of peptide nucleic acid oligomers into bacterial cells

Renata Grzela , Karolina Nasilowska , Edward Darżynkiewicz1,2 1

2

1 Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland; 2Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland

Keywords: hNudt16, cap structure, mRNA The mature eukaryotic mRNA molecule contains a cap structure at its 5’ end. The cap structure plays an important role in many cellular processes, including protection of the mRNA molecule from 5’–3’ exonucleolytic degradation and consequently regulates its stability. One of the proteins responsible for hydrolysis of the cap at the 5’ end of the mRNA is the hNudt16 enzyme. hNudt16 protein catalyzes hydrolysis of both nuclear and cytoplasmic mRNA caps. The catalytic activity of hNudt16 depends on a conserved Nudix motif and is influenced by the presence of metal ions: Mg2+, Mn2+ and Co2. Here we will present hNudt16 enzyme substrate specificity towards oligonucleotides of different chain lengths (16 nt and 34 nt), terminated with the following cap structures: m7GpppG, m7GpppA, m7GpppGm, m7GpppAm m32,2,7GpppG, m32,2,7GpppA and GpppG and GpppA. Obtained data indicate that the type of the nucleobase in the first transcribed nucleoside and the presence of guanine methylation at N7 position have crucial effect on efficiency of the hNudt16 hydrolysis.

Moreover, it was observed that the length of the transcript has also some effect on the hNudt16 catalytic activity.

Acknowledgements This work was supported by National Science Centre grant no. UMO-2013/08/A/ NZ1/00866 (Poland) and The National Centre for Research and Development grant no. STRATEGMED1/235773/19/NCBR/2016

Marcin Równicki1,2*, Monika Wojciechowska2,, Aleksandra Wierzba3, Jakub Czarnecki4, Dariusz Bartosik4, Dorota Gryko3, Joanna Trylska2 College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Żwirki i Wigury 93, 02-089 Warsaw, Poland; 2 Centre of New Technologies, S. Banacha 2c, 02-097 Warsaw, Poland; 3 Institute of Organic Chemistry, Polish Academy of Sciences, M. Kasprzaka 44/52, 01-224 Warsaw, Poland; 4 Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096, Warsaw, Poland 1

Rapid growth of antimicrobial resistance and insufficient progress in the fight against pathogens has made the development of novel strategies to fight bacteria an urging matter. The use of short, modified oligonucleotides as inhibitors of bacterial gene expression seems a promising strategy. However, the main and yet unsolved problem precluding the use of such oligonucleotides is their efficient delivery to bacterial cells. We examined vitamin B12 as a natural carrier for peptide nucleic acid (PNA) oligomers into Escherichia coli and Salmonella Typhimurium cells. To provide a convenient system to monitor the effect of antisense PNA in E. coli and S. Typhimurium, we constructed a reporter vector expressing Red Fluorescent Protein (RFP). We designed and synthesized an antisense PNA sequence (anti-rfp PNA) targeted at the mRNA transcript encoding RFP. To evaluate the potential of anti-rfp PNA to inhibit the production of RFP, we grew bacteria in the presence of anti-rfp PNA covalently linked to five different vitamin B12 derivatives. We examined different types and lengths of the spacer between vitamin B12 and PNA. As a control we used PNA conjugated to the most widely used cell-penetrating peptide (KFF)3K. We found that vitamin B12 transports antisense PNA to the E. coli more efficiently than the (KFF)3K peptide. We also found that the structure of the linker impacts the antisense effect. Our study provides the foundation for further developing vitamin B12 as a carrier for PNA oligonucleotides to bacterial cells.

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52 Abstracts

Posters

P1.2

P1.1

Effect of His-tag sequence location in Decapping Scavenger enzymes on their hydrolytic activity towards dinucleotide cap analogs

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Trichophyton rubrum A. Ciesielska, P. Stączek

Aleksandra Ferenc-Mrozek1,2, Elżbieta Bojarska1, Maciej Łukaszewicz 2, Edward Darżynkiewicz1,2 Centre of New Technologies, University of Warsaw; Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw

1

University of Łódź, Faculty of Biology and Environmental Protection, Department of Microbial Genetics

2

The selection of reference genes used for data normalization to quantify gene expression by qRT-PCR is crucial step of this technique. Moreover, little information regarding such genes is available for gene expression analyses in dermatophytes. Thus, we investigated the suitability of sic candidate reference genes in isolate of Trichophyton rubrum subjected to different environmental stimuli typical for the stage of host infection such as different carbon sources, pH shifts, growth phase. The stability of these genes was determined by NormFinder, geNorm and BestKeeper software. Tthe results of these studies should permit further analysis of Trichophyton rubrum gene expression under several conditions.

Keywords: DcpS, cap analogues, mRNA degradation, enzyme kinetics Decapping scavenger enzyme (DcpS) participates in 3’–5’ mRNA degradation pathway, following deadenylation and exosome-mediated digestion. Human DcpS is a nucleocytoplasmic shuttling protein. Among lower eukaryotes the function of DcpS is limited to the cytoplasm. DcpS is a member of the HIT family of pyrophosphatases and uses a histidine triad to carry out catalysis, releasing m7GMP and diphosphate terminated short oligonucleotide or dinucleotide. DcpS enzymes are homodimers with two independent active sites and two structural domains in each subunit: highly conserved C-terminal domain containing HIT motif and more variable N-terminal domain. Most recombinant DcpS used in biochemical and biophysical studies were obtained as N-terminal His-tagged proteins.

In order to reveal how nature adjusted short mRNA homeostasis within lower and higher eukaryotes we used DcpS proteins from human and C. elegans. We examined hydrolytic activity of N-terminally and C-terminally Histagged DcpS enzymes towards dinucleotide cap analogs (m7GpppG, m32,2,7GpppG, GpppG). For comparison, we also studied native enzymes, without any tag. Our data demonstrate that the three proteins have the same specificity towards tested dinucleotides, hydrolyzing triphosphate bridge between β and γ phosphate. However, we observed significant differences in the reaction efficiency when different forms of DcpS were used in enzymatic assays.

Acknowledgements This work was supported by grant 2014/13/B/NZ7/02307 from National Science Centre, Poland. Research was partly conducted in the Laboratory of Microscopic Imaging and Specialized Biological Techniques, Faculty of Biology and Environmental Protection, University of Łódź, Poland.

Acknowledgements Work was supported by a project UMO-2013/08/A/NZ1/00866 (NCN, Poland)

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Abstracts 53

P1.3

P1.4

Section: Nucleic Acid Tools

Changes in transcriptome and epigenome in prostate malignant transformation model

Development of microsatellites additional panel for horse parentage testing based on Polish population Fornal Agnieszka, Radko Anna, Piestrzyńska-Kajtoch Agata National Research Institute of Animal Production, Department of Animal Genomics and Molecular Biology, Poland Key words: microsatellite markers, STR, parentage testing, horse The Microsatellite 15 TKY System is an additional panel used in horse parentage verification and it appear to be a very effective tool especially for resolving cases like for example single-locus excludes of the alleged parentage. In this study we have modified TKY primers (dye change) for fluorescent PCR assays. Next, primers with four different fluorescent dyes (FAM, VIC, NED, PET) were combined in one multiplex reaction. Products of multiplex PCR amplification were analysed on 3130xL and 3500xL Genetic Analyzers (Applied Biosystems). For STR profile determination GeneMapper Software v4.0 was used. The analyses were carried out on Polish horse individuals and reference samples with known TKY STR profiles.

TKY STR profiles were obtained for all studied samples regardless of various DNA quality and amount. Some nonspecific artefacts were found in DNA profiles, but they did not disturb microsatellites genotyping. In our preliminary study we obtained PCR products ranging from 90 bp to 255 bp. Our results suggested that TKY STR panel amplified in one multiplex PCR could be used as quick method for single-locus exclusion’s validation. In further research the TKY STR set would be tested on a larger population using five horse breeds in comparison to the reference samples.

Katarzyna Kamińska1,2, Janusz Kowalewski2, Sui Huang3, Marzena A. Lewandowska1,2 1 Department of Molecular Oncology and Genetics, Innovative Medical Forum, The F. Lukaszczyk Oncology Center, Bydgoszcz, Poland; 2Department of Thoracic Surgery And Tumors, The Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, Torun, Poland; 3 Department of Cell and Molecular Biology, Northwestern University, Feinberg School of Medicine, USA.

Introduction: Prostate cancer (PCa) cell lines derived from PC3: PC3M, PC3MLN4, PC3MPro4 represent model of malignant transformation and differ in perinucleolar compartment (PNC) prevalence. PNC is a subnuclear body associated with malignancy, forming mainly in cancer cells both in vitro and in vivo [1].

Aim: The aim of our research was to evaluate genes expression and epigenetic changes in PCa cell lines and WT skin fibroblasts.

Methods: We used qRT-PCR to evaluate 28 genes expression and 2 miRNAs. Methylation analysis was performed using methylation sensitive and dependent endonucleases followed by qPCR. Results and Conclusions: We identified genes downregulated (e.g. DKK3, MGMT) and upregulated (e.g. CDKN2A, CCNA1) in PCa cell lines. Majority of studied genes had silenced expression by hypermethylation of CpG islands in promoters regions. Summarizing we identified 11 genes regulated by hypermethylation: DKK3, MGMT, TNFRSF10D, GSTP1, PDLIM4, APC, AR, GPX3, PTGS2, RASSF1, TIMP2. First five showed correlation between gene expression, methylation and PNC prevalence. Futhermore, we identified that CCNA1 is regulated by miR30e-5p.

Acknowledgements Funding: gene expression and methylation: HOMING PLUS/2010-2/7; miRNA epression: ICGEB CRP/13/024 References [1] Wen Y et al (2013) Frontiers in Biology, 8(4)

6th Central European Congress of Life Sciences Eurobiotech 2017

54 Abstracts

P1.5

P1.6

RNA modifications and NGS: It’s not a bug, it’s a feature

Spatial chromosomal organization of ROD1 regulates gene expression in prostate cancer cell lines.

Anna M. Kietrys, Eric T. Kool

Marta Kubiak1, Sui Huang2, Marzena A. Lewandowska1,3

Department of Chemistry, Stanford University, Stauffer I, Stanford, CA 94305

1 Innovative Medical Forum, The F. Lukaszczyk Oncology Center, Department of Molecular Oncology and Genetics, Bydgoszcz; 2 Northwestern University Feinberg School of Medicine, Department of Cell and Molecular Biology, Chicago; 3The Ludwik Rydygier Collegium Medicum, Nicolaus Copernicus University, Department of Thoracic Surgery and Tumors, Bydgoszcz

In last decade, the presence of modifications in RNAs has been revealed as a factor playing regulation role in a variety of biological processes: splicing, translation, cellular localization, RNA lifetime, and dynamic structure changes. Better understanding of modifications’ roles has become possible thanks to a combination of deep sequencing, and base-specific biochemical methods. Despite this progress, scientists need to face possible bias in sequencing data from the reverse transcription step. Reverse transcriptases may not read modified nucleotides as well as unmodified, and this may affect assignment of mutations as well as modified bases.

Our project targets this bias in RNA sequencing results, and makes use of it to help understand modification-related patterns in sequencing data. We present methods of interpreting the bias to identify modifications. We use deep sequencing patterns, including miscoding, indels, and truncations, as fingerprints to indicate the presence of specific modified bases.

We designed a set of 10 pairs of synthetic RNA sequences containing modified nucleotides: m6A, Φ, I, m1G, m6G, m1A, m66A, m3U, m5C, and m5U. Paired RNAs consist of an oligoribonucleotide containing consensus biological motif with one modified base, and a control unmodified oligonucleotide. We processed samples with adapters and primers from Illumina TruSeq® Small RNA kit, and the split-adapter library preparation method. We identified modifications’ profiles based on frequency of the truncation, insertion, deletion, and mutation frequencies. Using quantitative methods and principal component analysis, we discriminated unique fingerprints of several specific modifications. The results can be used in RNA-seq data interpretation to identify modified bases and percentage of their occurrence.

Introduction: We are interested in ROD1 gene expression regulation – as different expression was observed in prostate cancer transformation model cells. We proposed that ROD1 expression is controlled by epigenetic mechanisms of three dimensional chromosome organization.

Aim: Our aim was to evaluated spatial organization and histone modifications in ROD1 locus and determined that chromatin architecture are affected in ROD1 gene expression regulation in prostate cancer cells.

Methods: We used Chromosome Confromation Capture (3C) to study chromatin architecture of ROD1 locus in vivo and Chromatin Immonoprecipitation (ChIP) to confirm histon acetylation in selected cell lines.

Results: We observed chromatin conformation changes in ROD1 gene. We detected chromatin looping between promotor and -63kbp and +48kbp loci of ROD1 in prostate cancer cell lines with overexpression of ROD1 but not in control fibroblasts with no significant expression of gene.

ChIP results confirm that in detected looping positions (-63kbp; + 48kbp) occurs acetylation of histone H3 (H3K27Ac), which is known as chromatin regulators of active enhancer.

Conclusions: Fort he first time we propose three-dimensional structure model for ROD1 gene activation in prostate cancer cell lines.

Acknowledgements Grant Fundacji na rzecz Nauki Polskiej HOM lNG PLUS/2010-2/7

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 55

P1.7

P1.8

Assessment expression of non-coding RNAs in patients with non-small cell lung cancer

Point mutations of APP gene (Amyloid beta precursor protein) as a target for SNPselective RNA degradation by ribonuclease H using modified antisense oligonucleotides

Konrad Kwaśniak1,2, Justyna Czarnik-Kwaśniak1, Katarzyna Pogoda1, Maria Pyszniak1,2, Paweł Rybojad3,Jacek Tabarkiewicz1 University of Rzeszow, Medical Faculty, Centre for Innovative Research in Medical and Natural Sciences, Department of Immunology, 1a Warzywna St., 35-310 Rzeszow, Poland; 2 Medical University of Warsaw, Postgraduate School of Molecular Medicine, 02‑091 Warsaw, Poland; 3Medical University of Lublin, Department of Thoracic Surgery, 20‑097 Lublin, Poland

1

Most of the human genome encodes RNAs which do not code proteins. Non-coding RNAs may affect gene expression in normal cells and influence disease progression. In this study, we focused on long non-coding RNAs which belong to a class of non-coding RNAs (ncRNAs) larger than 200 nucleotides and large intergenic non-coding RNAs (lincRNAs) which are transcribed from sites between coding protein sites. In this study, we evaluate the levels of eight selected ncRNAs (XLOC_005677 (linc-POU5F1_1), XLOC_002334 (linc-DARS), NONHSAT143046 (LNCCDH16-2_1), NEAT1_1, NEAT1_2, lncTCF7, DLX6-AS1, BANCR). Three of these ncRNAs, namely linc-POU5F1_1, linc-DARS and LNC-CDH16-2_1 are described for the first time. The rest of the mentioned ncRNAs are known and have influence on tumorigenesis and cell proliferation in non-small cell lung cancer. Expression of ncRNAs was evaluated in 18 patients with non-small cell lung cancer and were correlated to CXCR-4, EPCAM, TRIM 44, E-cadherin, CPEB-4 and OCT-4 genes. Moreover, we compared our results in expression level of genes and ncRNAs to results obtained in two cell lines A549 and H1975. Endogenous controls in this study were GAPDH and HPRT. Additionally, we evaluated our results within the framework of the TNM staging system.

The results show an influence of lncRNAs on tumorigenesis and can lead to development of new cancer biomarkers for diagnosis, prognosis and therapeutics.

Dorota Magner, Ryszard Kierzek Institute of Bioorganic Chemistry Polish Academy of Sciences, Department of Structural Chemistry and Biology of Nucleic Acids, Poznań, Poland Amyloid β precursor protein is an integral membrane protein and occurs especially abundant in the synapses of neurons. It undergoes regulated intramembrane proteolysis that generates beta amyloid polypeptide (Aβ) whose fibrillar form plays a major role in the pathogenesis of Alzheimer’s disease. Exons 16 and 17 of the APP gene encode the Aβ peptide. Point mutations of these exons affect metabolism and stability of the Aβ, being the cause of some percentage of familial dominant AD. In the presented study, three cases of familial, dominant AD-causing mutations: London (V717I), Flemish (A692G) and Arctic (E693G) were investigated as a targets for alleleselective RNA degradation by cellular ribonuclease H. Two different and new approaches using modified antisense oligonucleotides were applied. In total, about 90 antisense oligonucleotides, carrying different nucleotide and non-nucleotide modifications, were tested to activate RNase H to selectively cut the mutant RNA without affecting the wild type form. The highest selectivity of degradation was observed for Flemish RNA variants, that include C-to-G nucleotide substitution. Among 44 designed oligonucleotides to this particular case, 20 caused selective degradation of mutant RNA variant in in vitro assay, but only 8 differentiated the alleles ratio in HeLa cells.

References: Magner, D., Biala, E., Lisowiec-Wachnicka, J., Kierzek, E. & Kierzek, R. A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides. PLoS One 10, e0142139 (2015)

6th Central European Congress of Life Sciences Eurobiotech 2017

56 Abstracts

P1.9

P1.10

Bacterial growth inhibition by peptide nucleic acids complementary to ribosomal RNA.

Analysis of Nudt16 localization and colocalization using confocal microscopy

Agnieszka Markowska-Zagrajek1,2, Renata Grzela2, Joanna Trylska2

Beata Miedziak1, Renata Grzela1, Edward Darżynkiewicz1,2

Faculty of Biology, University of Warsaw, Poland; 2Centre of New Technologies, University of Warsaw, Poland

Centre of New Technologies, University of Warsaw; Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw

1

The ribosome is one of the bacterial targets for clinically used antibiotics. However, the ribosome can be also sterically blocked by complementary oligomers for example peptide nucleic acids (PNA). PNAs are synthetic analogues of DNA with a neutral peptide-mimicking backbone. Importantly, they are not digested by enzymes such nucleases and proteases. The main goal of this research was to design PNA sequences that would bind to bacterial rRNA and inhibit bacterial growth. We present the results for one PNA oligomer complementary to Helix 69 in 23S rRNA. Helix 69 is a part of the bridge joining the small and large ribosomal subunits, and also a secondary binding site for aminoglycoside antibiotics. We verified the sequence-specific hybridization of PNAs with isolated Helix 69 from Escherichia coli and lack of such hybridization with the human variant of Helix 69. We determined that PNA inhibits translation in a bacterial cell-freetranslation/ transcription system. Then, we showed that PNA (conjugated with a cell penetrating peptide for delivery) targeted at Helix 69 inhibits the growth of E. coli cultures. Furthermore, we proved by agarose gel electrophoresis that PNA binds to 23S rRNA in isolated total RNA, and also binds to 70S ribosomes.

The results of the study indicate that targeting rRNA with PNA could be a promising way to inhibit bacterial growth and to search for new target sites in bacterial RNA.

1 2

Keywords: Nudt16, cap structure, mRNA, P-bodies, EDC4 Degradation of mRNA plays important role in the regulation of gene expression. In eukaryotic cells, mRNAs have a 5’ monomethyl guanosine cap structure and a 3’ poly(A) tail which are significant for mRNA translation and stability. It is known that mRNA decapping and 5’ to 3’ degradation take place in discrete cytoplasmic foci, which are called P-bodies. Translational inhibitors blocking mRNA degradation induce a decrease in the number of P-bodies. Cycloheximide inhibits mRNA translation at the elongation step, what prevents mRNA degradation. Treatment with one of the translation inhibitors – cycloheximide – leads to the release of decapping factors from P-bodies. Nudt16 was initially identified in Xenopus as a U8 snoRNA binding protein, called X29, which has a decapping activity. This enzyme belongs to a large conserved family, Nudix hydrolases, which function as nucleotide diphosphatases acting upon various substrates.

In our research we checked the colocalization of new decapping enzyme Nudt16 with EDC4, marker of P-bodies.

Under treatment of cycloheximide we observed reduced quantity of both EDC4 and Nudt16 proteins accumulated in granules. Future studies are necessary to understand nature of this interaction.

Acknowledgementss This work was supported by National Science Centre grant no. UMO-2013/08/A/ NZ1/00866 (Poland) and The National Centre for Research and Development grant no. STRATEGMED1/235773/19/NCBR/2016

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 57

P1.11

P1.12

The cleavage of bacterial mRNA by a hammerhead ribozyme

Effect of GPAT2 gene polymorphism on meat texture parameters in pigs

Joanna Miszkiewicz1,2, Ksenia Maximova2, Maciej Łukaszewicz3, Edward Darżynkiewicz2,3, Joanna Trylska2

Ilona Mitka1, Katarzyna Ropka-Molik 2, Mirosław Tyra1

1 Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Poland; 2Centre of New Technologies, University of Warsaw, Poland; 3 Institute of Experimental Physics, Division of Biophysics, Faculty of Physics, University of Warsaw, Poland

Nowdays, many bacteria acquire resistance to existing antibiotics. Therefore, there is a need to find new ways to destroy these pathogens. One of yet unexplored methods to fight bacteria could be by usign RNA enzymes known as ribozymes. These enzymes first recognize and next cleave specific RNA sequence in trans for example during transesterification of RNA.1,2 The cleavage of pathogenic or oncogenic RNA hinders translation and in the end suppresses the progression of various diseases.3 For the first experiments we chose two model organismes. The hammerhead ribozyme which is the simplest natural ribozyme and a fragment of mRNA of Escherichia coli K-12 MG1655 strain, because this is the most intensively studied bacterial strain.4 The selected mRNA sequence encodes the acyl carrier protein (ACP). A cleavage of this mRNA by a ribozyme should preclude the translation of ACP and as a result inhibit bacterial growth.

The main goal of this study was to verify the cleavage of mRNA fragment by an artificial ribozyme. We designed the sequence of the hammerhead ribozyme to bind and hydrolize a selected E.coli mRNA fragment as a substrate.

We found that such synthetic RNA molecule cleaves RNA substrate so this approach appears workable. The results of this study would help design next ribozymes for many specific RNA sequences.

National Research Institute of Animal Production, 1 Department of Genetics and Animal Breeding; 2 Department of Animal Genomics and Molecular Biology, Poland GPAT2 gene encodes a mitochondrial NEM-sensitive glycerol-3-phosphate acyltransferase 2 enzyme, which catalyze the first step in de novo synthesis of triglycerides.

The aim of this study was to analyse the g.48497102G>A polymorphism of the NC_010445.3 sequence and to estimate its association with meat texture parameters in pigs. Analysis was performed on five pig breeds (Polish Landrace, Polish Large White, Puławska, Pietrain, Duroc) maintained in the Pig Performance Testing Stations (SKURTCh) in Poland, in total on 708 pigs. The analysis of NC_010445.3:g.48497102G>A genotypes was performed using PCR-RFLP method (with the use of Tsp45I endonuclease). The association between meat texture as well as meat quality parameters and GPAT2 genotypes was performed using GLM procedure (SASv. 8.02). The frequency of following genotypes AA, GA, GG was 18%, 36%, 46%, respectively. The Tsp45I g.48497102G>A affected few texture parameters in longissimus dorsi muscle and one in semimembranosus muscle. Meat from pigs with AA genotype was characterized by higher firmness and toughness parameter measured in raw loin compare to GG genotypes (p<0.05). eat of pigs with GA genotype was characterized by significantly higher value for resilience (p<0.01) compare to AA genotypes in cooked hamurrmore, regarding meat quality parameters, homozyots G n heterozygotes GA were characterized by the highest values of intramuscular fat content (p<0.05) while opposite homozygotes AA by the lowest.

References [1] Zhang B et al (1997) Nature 390: 96–100 [2] Przybilski R et al (2006) ChemBioChem 7: 1641–1644 [3] Mulhbacher J et al (2010) Curr Opin Pharmacol 10: 551–6 [4] Jackowski S et al (1987) J Bacteriol 169: 1469–1473

6th Central European Congress of Life Sciences Eurobiotech 2017

58 Abstracts

P1.13

P1.14

Effect of selected ACTN2 gene polymorphisms on meat quality in pigs

Searching for the right tool for heme oxygenase-1 inhibition – the comparison of small-molecule inhibitors, shRNA and CRISPR/Cas9 system

Mirosław Tyra1, Katarzyna Ropka-Molik 2, Katarzyna Piórkowska2, Ilona Mitka1 National Research Institute of Animal Production, 1 Department of Genetics and Animal Breeding; 2 Department of Animal Genomics and Molecular Biology, Poland ACTN2 gene encodes an α-actinin-2 protein which belongs to the family of spectrins. The protein affects stabilization and organization of actin filaments in the sarcomer bundles, in which it anchors the thin filaments of the Z-disc and plays a very important role in the process of muscle contraction. The aim of this research was to determine the effect of two selected polymorphisms within porcine ACTN2 locus on pork quality traits. A total of 777 pigs raised in Poland (represented by five breeds: Polish Landrace, Polish Large White, Puławska, Pietrain, Duroc) were used in this study. The frequency of selected SNPs localized in the 3’UTR region: ACTN2: c.*1657G>A and c.*1595A>G were estimated using PCR-RFLP method (with the use of FokI endonuclease). The association of meat quality traits and different SNPs was evaluated using GLM procedure (SASv. 8.02). The frequency of following genotypes AA, GA, GG was 62%, 24%, 14% for first, and 58%, 28%, 14% for second analyzed polymorphism, respectively. The present research showed the highly significant association of both analyzed polymorphisms and pH45 as well as IMF content. Pigs with GG genotype concerning analyzed polymorphisms were characterized by the highest value for pH45 and intramuscular fat content compare to these with AA as well as AG genotypes (p<0.01). Furthermore, AG pigs according to ACTN2: c.*1657GA polymorphism had the hihest value for meat colour (a*) (p<0.01).

Olga Mucha1, Paulina Podkalicka1, Maria Czarnek 2, Anna Biela1, Mateusz Mieczkowski1, Szymon Czauderna1, Neli Kachamakova-Trojanowska1, Jacek Stepniewski1, Alicja Jozkowicz1, Agnieszka Loboda1, Jozef Dulak1 1 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Krakow, Poland; 2Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biochemistry, Krakow, Poland

Keywords: HO-1, heme oxygenase-1, inhibitors, shRNA, CRISPR/Cas9 Genes knock-down (KD) through RNA interference as well as gene knock-out (KO) through CRISPR/Cas9 system serves as invaluable tool to study gene functions and provides a complementary view to traditional pharmacological approaches like small-molecule inhibitors. In our work we compared chemical inhibitors and genetic tools, including shRNA and CRISPR/Cas9 systems to knock-down/knockout heme oxygenase-1 (HO-1), the enzyme with cytoprotective, anti-apoptotic and anti-inflammatory properties.

In our hands, pharmacological inhibitors, tin and zinc protoporphyrins (SnPP and ZnPPIX, respectively) decreased HO activity but at the same time strongly induced HO-1 mRNA and protein level in 293T cells. On the other hand, basal and hemin-induced HO-1 inhibition in shRNA KD 293T cell lines (developed using different shRNA sequences against HO-1 delivered by lentiviral vectors) was confirmed on mRNA, protein and activity level. However, the effect was much stronger when CRISPR/Cas9 mediated knock-out was performed. Most of the clones did not express HO-1 as showed by western blot and failed to increase bilirubin concentration after hemin treatment.

Furthermore, HO-1 inhibition affected 293T cells proliferation, viability and clonogenic potential.

In conclusion, we have shown that all technologies can be used for inhibition of HO-1 activity in vitro, however, genetic tools (especially CRISPR/Cas9 system) allow us to affect not only HO activity but also mRNA/protein level.

Acknowledgements This work was supported by the grants from the National Science Centre (2014/14/M/NZ1/00010 and 2016/21/B/NZ1/00293) and National Centre for Research and Development (PBS2/B7/15/2014).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 59

P1.15

P1.16

Characteristic of miRNAome in three different tissues of pigs

qPCR validation of RNA-seq analysis of backfat transcriptome changes after different diets in pigs

Maria Oczkowicz1, Klaudia Pawlina-Tyszko1, Małgorzata Świątkiewicz2, Joanna Warzecha1.

Maria Oczkowicz1, Małgorzata Świątkiewicz2, Klaudia Pawlina-Tyszko1, Joanna Warzecha1.

National Research Institute of Animal Production, Departament of Genomics and Molecular Biology of Animals; 2National Research Institute of Animal Production,Departament of Animal Nutrition and Feed Science

National Research Institute of Animal Production, Departament of Genomics and Molecular Genetics of Animals, Departament of Animal Nutrition and Feed Science

1

The aim of our study was to characterize porcine miRNAome in three tissues (muscle, backfat, liver). In total, 74 samples (24 samples of muscle, 25 samples of liver and 25 samples of backfat) were collected from animals maintained for the nutritional experiment (grant no 2014/13/B/ NZ9/02134). Total RNA was isolated from the tissues using Direct-zol RNA kit (Zymo Research) according to the attached protocol. Next, miRNA libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1) (New England Biolabs). After clustering on cBot instrument (Illumina), libraries were sequenced on HiScan 2000 System (Illumina). In total, we obtained 264740935 raw reads (mean 3577580 per sample). The raw reads were filtered by several criteria (sequence length, low complexity, min/max abundance, invalid sequences, etc). Finally, 1041861 distinct reads were obtained. In all the analyzed tissues the most prevailing were 22 nucleotide sequences, however, predominance of 22 nt sequences was higher in muscle than in liver and backfat.

Most of the identified miRNAs have not been annotated: 511 versus 188 previously known. The 10 most abundant miRNAs were in the liver: ssc-miR-122, ssc-miR-148a-3p, ssc-miR-26a, ssc-miR-21, ssc-miR-126-3p, ssc-miR-1433p, ssc-let-7f, ssc-let-7g, ssc-miR-101, ssc-miR-99a, in the muscle: ssc-miR-133a-3p, ssc-miR-26a, ssc-miR-206, sscmiR-378, ssc-miR-10b, ssc-miR-27b-3p, ssc-miR-148a-3p, ssc-miR-21, ssc-let-7a, ssc-miR-381-3p, and in the backfat: ssc-miR-148a-3p, ssc-miR-143-3p, ssc-miR-26a, ssc-miR126-3p, ssc-miR-99a, ssc-miR-10b, ssc-let-7a, ssc-let-7g, ssc-miR-199b-3p, ssc-let-7i.

Acknowledgements This work was financed by National Science Center Poland, grant no 2014/13/B/ NZ9/02134.

cDDGS (corn Dried Distilled Grain with Soluble) is easily available and relatively cheap replacement for corn or soybean meal in pig feeding. However, it was shown that high amounts of cDDGS in the feedstuff worsen the quality of porcine backfat. We performed RNA-seq analysis of samples of backfat obtained from animals fed four different diets, which differed among each other in terms of the presence of 20% cDDGS (group I (–DDGS+rapeseed oil)) and source of fat: 3% rapeseed oil– (groups I and II (-DDGS+rapeseed oil, +DDGS+rapessed oil)), 3% beef tallow (group III (+DDGS+beef tallow)), 3% coconut oil – group IV (+DDGS+coconut oil)). The RNA-seq analysis revealed upregulation of 49 genes in the (–DDGS+rapeseed oil) group when compared to the (+DDGS+rapeseed oil) group. We selected six genes (ACACA, ACLY, FASN, ALAS1, HK1, FITM2) to validate RNA-seq results by qPCR. qPCR was performed in duplicate on a QuantStudio 7 –flex Instrument (Thermo Fisher Scientific, Waltham, MA) under the fast thermal profile, using GoTaq® qPCR Master Mix (Promega Corporation, Madison, WI) and TaqMan assays for OAZ1(endogenous control) and the target genes. Relative quantity (RQ) for each sample was calculated basing on ΔΔCt method, using QuantStudio Real-Time PCR Software. Pearson correlation between RNA-seq read counts of each sample and relative quantity (RQ) of qPCR was calculated using SAS software. The Pearson coefficiency (R2) was 0.3 p<0.0003 for all genes together. When each gene was analyzed separately the highest R2 was obtained for ACLY (R2=0.882, p<0.0001), ACACA (R2=0.66, p<0.0006) and FASN gene (R2=0.66, p<0.0005). For FITM2, ALAS1, HK1 Pearson coefficiency was relatively low (R2=0.277, p<0.2, R2=0.436, p<0.037, R2=0.378 p<0.0747, respectily).

Acknowledgements This work was supported by National Science Center, Poland, grant no 2014/13/B/ NZ9/02134.

6th Central European Congress of Life Sciences Eurobiotech 2017

60 Abstracts

P1.17

P1.18

Section: Nucleic Acid Tools

Gene expression changes in fast-growing chicken hypothalamus during ontogenesis

PRNP gene polymorphism in red deer and roe deer Agata Piestrzynska-Kajtoch, Anna Radko, Agnieszka Szumiec, Mariusz Kościelny National Research Institute of Animal Production, Department of Animal Genomics and Molecular Biology, Poland Keywords: CWD, red deer, roe deer, PRNP, polymorphism Chronic wasting disease (CWD) is one of the prion diseases and belongs to a group of transmissible spongiform encephalopathies (TSE). Its development is connected with pathogenic isoform (PrPCWD) of normal, cellular prion protein (PrPC), which is encoded in host by PRNP gene. PrPCWD accumulation in central nervous system cause progressive neurodegeneration and as a result animal death. PRNP gene polymorphism was associated with CWD occurrence and incubation period. The disease occur in free-living deer, elks and moose in North America. However, in 2016 first case of CWD was reported in free-ranging reindeer and European elk from Norway.

We amplified the fragment of PRNP gene coding region (596 bp; including codons 85 to 228) using DNA samples isolated from blood and other tissues of red deer and roe deer (Sothern Poland populations). Next, we sequenced (BigDye Terminator Cycle Sequencing Kit v3.1, Genetic Analyzer 3500xL; Applied Biosystems) and analysed (Variant Reporter Software 2; Applied Biosystems) PCR products. We compared the sequences to red deer and roe deer sequences deposited in GenBank (BLAST, ClustalW).

Several polymorphic sites were observed including nonsynonymous substitutions and silent mutations. Our results showed PRNP gene variants in Polish red deer and roe deer population. Further analysis would include the whole PRNP gene coding region.

Katarzyna Piórkowska1, Kacper Żukowski1, Katarzyna Połtowicz1, Joanna Nowak1, Katarzyna Ropka-Molik1, Dorota Wojtysiak3, Natalia Derebecka2, Joanna Wesoły2 National Research Institute of Animal Production, Animal Genomics and Molecular Biology, Cracow, Poland; 2National Research Institute of Animal Production, Animal Genetics and Breeding, Cracow, Poland; 3Agricultural University of Cracow, Institute of Veterinary Sciences, Department of Animal Anatomy, Cracow, Poland; 4Adam Mickiewicz University, Faculty of Biology, Laboratory of High Throughput Technologies Institute of Molecular Biology and Biotechnology, Poznań, Poland; 1

The goal of the present study was to indicate the differentially expressed genes of hypothalamus between two point time of ontogenesis in broiler chickens.

The hypothalamus samples were collected from twoweek-old and four-week-old fast-growing cockerels of Ross 308 line. They were isolated by using magnifier Benefit 130E Pro7 and minitweezer. The RNA-seq analysis was performed in 100 single-end cycles on HiScanSQ platform (Illumina) and differentially expressed genes (DEGs) were determined using Deseq2 software.

The preliminary study showed that in younger broilers the highest 22-fold change of expression was observed for LECT2 that stimulates the growth of chondrocytes and osteoblasts. The overexpression was detected also for few genes encode protein that regulate hormones such as CGA (11-fold change), GHRH (2.3 fold change) and POMC (3fold change). The metabolic pathways which were more active in younger broiler group: ECM-receptor interaction (COL1A2, ITGB3, COL4A6, COL6A3, COL9A1, ITGA8, THBS4, LAMB2, LAMB1, COL6A1, FN1), p53 signaling pathway (CDK1, RRM2, FAS, CCNB3, PERP2, GTSE1) and TGF-beta signalling (FST, BMP4, BMP5 and DCN).

In turn, in 4 weeks old broilers differentially expressed genes were implicated in neuroactive ligand-receptor interaction (FP, CYSLTR1, GRM2, NTSR1, GRIN2A, GABRR1, mGluR5a, VIPR1, BRS3, GPC5 and CHRNB3).

Acknowledgements The study was supported by statutory activity of National Research Institute of Animal Production (no. 01–013.1) National Multidisciplinary Laboratory of Functional Nanomaterials NanoFun nr POIG.02.02.00-00-025/09 (Innovative Economy Operational Programme, Priority Axis 2: R&D Infrastructure, Action 2.2: Support of Formation of Common Research Infrastructure of Scientific Units

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 61

P1.19

P1.20

Section: Nucleic Acid Tools

Enantiomeric catalytic nucleic acids in downregulation of gene expression

Use of multiplex microsatellite panel for assessment of polymorphism domestic cats and free-living cats

Belter A., Wyszko E., Rolle K., Barciszewski J.

A. Podbielska1, A. Radko1, W. Niżański2, J. Kochan3, A. Nowak3, M. Bugno-Poniewierska1

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61 704, Poznan, Poland

1 Department of Animal Genomics and Molecular Biology, National Research Institute of Animal Production, Balice, Cracow, Poland; 2Department of Reproduction and Clinic of Farm Animals, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland; 3Faculty of Animal Sciences, Institute of Veterinary Science, University of Agriculture in Cracow, Cracow, Poland

We aim to apply new catalytic nucleic acid-based technology for down-regulation of GBM-specific targets. To handle with previously reported limitations of ribozymes, such as their limited stability and specificity and problems with their delivery, we decided to deliver mirror image ribozymes (Spiegelzymes). They contain L-ribose instead of the naturally occurring D-ribose and thus exhibit extra ordinary stabilities, are non-toxic and non-immunogenic, which makes our investigation promising.

Based on our own experience in a field of glioblastoma (GBM), the multi-target approach seems to be the most beneficial in terms of potential therapy. We propose two different kinds of targets: tenascin C – an extracellular matrix glycoprotein and on the other hand small regulatory RNA, miR-21, both strongly up-regulated in GBM tissues. Since involved in cancer progression, they are believed be good therapeutic targets.

The aim of the study was a preliminary assessment of polymorphism domestic cats and free-living cats by using multiplex microsatellite panel recommended by ISAG for cat identification and parentage control. Forty-four individuals were tested in the family Felidae of species: different breeds a domestic cat (Felis catus), five tiger (Panthera tigri), two lynxes (Lynx lynx) and one of animal, which represent the species wildcat (Felis silvestris), manul (Otocolobus manul), clouded leopard (Neofelis nebulosa), and snow leopard (Panthera uncia). PCR products for all markers were obtained for a domestic cat. Studied wild cats, except for a wildcat and snow leopard were different than domestic cats. PCR products were not observed for FCA441 marker in clouded leopard and lynx. The same results obtained for FCA149 marker for manul, clouded leopard and tigers. Identified alleles of the lynx were out of range in FCA220 marker. The same situation was with manul alleles in FCA441 marker. Additionally, in manul we observed different variant in AMEL marker, which produced a 216-bp X allele, while others species had 214-bp X allele. We conclude, based on the presented studies that we should apply other known markers to the assessment of biodiversity free-living cats. Our preliminary studies allow stating that manul is the most different feline but we need further study on much larger population.

Acknowledgements This work was supported by the National Science Center grant UMO-2015/17/B/ ST5/01467

Acknowledgements Financed by: NCBiR PBS3/B8/16/2015.

6th Central European Congress of Life Sciences Eurobiotech 2017

62 Abstracts

P1.21

P1.22

Exercise-related modification of the PLIN2 and FASN genes expression in Arabian horses skeletal muscle

Whole transcriptome analysis of porcine muscle tissue in the terms of muscle growth and development

Katarzyna Ropka-Molik1, Monika Stefaniuk-Szmukier3, Kacper Żukowski2, Katarzyna Piórkowska1, Monika Bugno-Poniewierska1 National Research Institute of Animal Production, Department of Animal Genomics and Molecular Biology, Sarego 2, 31-047 Cracow; Poland; 2National Research Institute of Animal Production, Department of Animal Genetics and Breeding; Sarego 2, 31-047 Cracow; Poland; 3 Department of Horse Breeding, Institute of Animal Science, University of Agriculture in Cracow; Poland 1

The intensive training is recognized by organism as stress factor. Regulation of metabolic processes is critical during adaptation to training and recovery of body homeostasis. The aim of present study was to evaluate the changes of transcript level of two genes -PLIN2 and FASN – strongly related with lipid metabolism in Arabian horses muscle tissue.

The analysis was performed on gluteus medius muscle collected from Arabian horses at three time points of oneyear training schedule: untrained horses (n=5), horses after intense gallop phase (n=10) and at the end of the racing season (n=8). Muscles were collected using a biopsy method. The expression profile of analyzed genes were estimated using real-time PCR method. qPCR analysis was performed using EvaGreen® qPCR Mix Plus (ROX) and in three technical replicates for each sample and for two analyzed genes and endogenous control gene (GAPDH).

Both PLIN2 and FASN genes are involved in triacylglycerol synthesis and secretion. Our analysis showed the significant increase expression level of PLIN2 gene (fold change 3.39; p≤0.01) and decrease of FASN gene (fold change 1.44; p≤0.05) in horses after intense gallop phase compared to untrained horses. These results can indicated on activation of lipid metabolism processes during exercise. The up-regulation of PLIN2 gene can suggest that during the most intense training period occurs secretion of triglycerides resulting from intensified lipolysis processes. In turn, the down-regulation of FASN gene in intense training period can pinpointed on the inhibition of fatty acid synthesis due to the increased use of fatty acids on energy processes.

Acknowledgements The study was supported by the Polish Ministry of Science and Higher Education (project no. 2014/15/D/NZ9/05256).

Katarzyna Ropka-Molik1, Kacper Żukowski2, Katarzyna Piórkowska1, Grzegorz Żak 2, Justyna Dulska2, Natalia Derebecka 3, Joanna Wesoły 3, National Research Institute of Animal Production, Department of Animal Genomics and Molecular Biology, Sarego 2, 31-047 Cracow; Poland; 2National Research Institute of Animal Production, Department of Animal Genetics and Breeding; Sarego 2, 31-047 Cracow; Poland; 3Adam Mickiewicz University, Faculty of Biology, Laboratory of High Throughput Technologies Institute of Molecular Biology and Biotechnology, Wieniawskiego 1, 61-712 Poznań; Poland 1

The aim of present research was to identify genes potentially related with muscle growth in pig. RNA-seq method was applied to compare whole gene expression prolife of muscle tissue collected from two sire-line pig breeds, which are characterized by high muscularity.

RNA-seq analysis was performed on longissimus lumborum samples collected from 13 pigs representing two breeds – Pietrain and Hampshire. Pigs were selected from a larger population based on weight of loin muscle and meat content in carcasses in order to obtain the most extreme groups in each breed. The RNA-seq was performed in 101 single-end cycles on HiScanSQ platform (Illumina) and differentially expressed genes (DEGs) were determined using Deseq2 software.

RNA-seq approach allowed to identify 627 and 416 DEGs between pig groups with different loin weight (pvalue 0.05; fold change 1.5) for Pietrain and Hampshire; respectively. The comparison of obtained gene sets shoed 43 genes common for both breeds, which expression was associated with muscle weight. The Gene Ontology analysis (Panther software) confirmed that detected genes were involved in regulation of metabolic process (18 genes), developmental growth or skeletal system development (PCOLCE; BBS2; TBX2; RBBP6; ATAF5). According to David software these genes belong to PI3K-Akt signaling, Focal adhesion and ECM-receptor interaction pathways.

Obtained results allow to propose the panel of genes, which can be related with porcine muscle growth and development. Such analysis can be the basis of future research in terms of identification of candidate genes related with important production traits in pigs.

Acknowledgements The study was supported by statutory activity of National Research Institute of Animal Production (no. 04–014.1); National Multidisciplinary Laboratory of Functional Nanomaterials NanoFun nr POIG.02.02.00-00-025/09 (Innovative Economy Operational Programme, Priority Axis 2: R&D Infrastructure, Action 2.2: Support of Formation of Common Research Infrastructure of Scientific Units

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 63

P1.23

P1.24

Genome editing in tumor and iPS cells for the study of SNAI1 role in human diseases

Section: Nucleic Acid Tools

Klaudia Skrzypek, Aleksandra Ulman, Bogna Badyra, Maciej Sulkowski, Marcin Majka Department of Transplantation, Institute of Pediatrics, Jagiellonian University Medical College, Krakow, Poland Rhabdomyosarcoma (RMS) is a common tumor in children that originates from an impaired myogenic differentiation. Genome editing with transcription activator-like effector-based nucleases (TALENs) or clustered regularly interspaced short palindromic repeat / Cas9 RNA-guided nucleases: CRISPR/Cas9 allows to study genes role in human diseases.

The aim of our studies was to inactivate SNAI1 gene in both RMS and induced pluripotent stem (iPS) cells using designer nucleases for the study of SNAI1 role in pathological and normal myogenic differentiation.

We designed TALEN pairs and CRISPR/Cas9 nucleases targeting exon 1 or 2 of human SNAI1. Transfection with both CRISPR/Cas9 and TALENs nucleases led to SNAI1 allelic gene disruption in different cells, but CRISPR/Cas9 nucleases displayed higher cleavage activity. Consequently, we transfected both iPS and RMS cells with the nucleases targeting SNAI1 gene both in exon 1 and exon 2 to isolate and screen single cell clones. iPS cells with deletion of SNAI1 gene on both alleles were obtained as a model to study SNAI1 role in normal myogenesis. On the other hand, RMS cells with SNAI1 gene deletion were characterized in comparison to RMS cells transduced with lentiviral vectors encoding shRNA against SNAI1. SNAI1-deficient RMS cells displayed an enhanced expression of myogenic factors and microRNAs. Acknowledgements Financial support from from UJCM: K/DSC/003116 and from the National Science Centre in Poland: 2013/09/B/NZ5/00769 and 2015/17/D/NZ5/02202.

Identification of mutations in BMPR1B, BMP-15 and GDF-9 genes in Cakiel podhalański and Wrzosówka sheep breeds Grzegorz Smołucha, Agata Piestrzyńska-Kajtoch National Research Institute of Animal Production, Krakowska 1, 32-020 Balice n. Cracow; Department of Animal Genomics and Molecular Biology.

The aim of the study was BMP-15, GDF-9 and BMPR1B polymorphisms identification to determine the relationship of polymorphisms with high or low fertility in sheep. Analysis was performed using the PCR reaction, Sanger dideoxy sequencing and capillary electrophoresis.

Results: Analysis of the BMP-15 gene showed presence of SNP c.755T>C (g.6063) in Wrzosówka sheep breed. The identified mutation causes replacement leucine (L) to proline (P) p.L252P in the BMP-15 protein. Analysis of GDF-9 gene fragment showed two previous found by Hanrahan transition c.978A>G and c.994G>A in both breeds. Another mutation g.1203G>A was identified in the coding region of the mature GDF-9 protein in Wrzosówka and Cakiel podhalański sheep breeds. This mutation does not alter the encoded amino acid. Further analysis of GDF-9 gene sequences from Cakiel Podhalański sheep breed allowed to identification two mutation: g.1078T>C and g.1081T> C. G.1081T> C mutation, does not cause a change in the sequence of polypeptide chain while the g.1078T>C mutation change the coding amino acid from leucine to proline.

In order to exclude a known mutation in BMPR1B gene (c.746 A>G) affecting high fertility and fecundity in sheep, the polymorphism analysis was performed using the restriction enzyme AvaII. No mutations (c.746 A>G) in the BMPR-1B gene were observed in the examined animals.

Acknowledgements The study, financed by BIOSTRATEG2/297267/14/NCBR/2016, has been continued.

6th Central European Congress of Life Sciences Eurobiotech 2017

64 Abstracts

P1.25

P1.26

Section: Nucleic Acid Tools

Peripheral blood microRNA as potential biomarker of significant carotid vessels stenosis

Polymorphism of the Growth Hormone (GH) and Myostatin (MSTN) genes in Kielecka and Landes geese Grzegorz Smołucha1, Anna Kozubska-Sobocińska1, Anna Koseniuk1, Mirosław Lisowski2, Bartosz Grajewski3 National Research Institute of Animal Production, Krakowska 1, 32-020 Balice n. Cracow; Department of Animal Genomics and Molecular Biology; 2National Research Institute of Animal Production, Krakowska 1, 32-020 Balice n. Cracow; Department of Animal Reproduction Biotechnology, Poland; 3National Research Institute of Animal Production, Experimental Station in Kołuda Wielka, Waterfowl Genetic Resources Station in Dworzyska, Poland 1

Growth hormone is a single polypeptide secreted by pituitary. It has many physiological functions in animals, such as promoting muscle growth, bone formation and regulating fat content, all of which are related to and affect the growth and development of animals. Myostatin is a major regulator of myogenesis and it is expressed predominantly in skeletal muscle and functions specifically as a negative regulator of skeletal muscle growth and development.The aim of the study was to identify the SNP polymorphisms in exon 2 of the growth hormone gene and myostatin gene to determine the relationship of polymorphism with production performance. Two geese breeds Kielecka and Landes were selected for the study. A total 88 samples were used: 44 from Kielecka and 44 from Landes geese. Analysis was performed using the PCR reaction, Sanger dideoxy sequencing and capillary electrophoresis.

Analysis of the GH gene revealed the presence of single nucleotide polymorphism (SNP) c.128C>T in both geese breeds. C>T transition resulted in an amino acid change from valine to alanine. All animals have TT genotype.

The identified SNP was previously described by DUAN (2010), and was associated with the production features of geese in China. One SNP was found in exon 3 MSTN gene c.1124C>A resulted in amino acid change to stop kodon.

Association studies between genotypes and production traits in those breeds has been continued.

Miłosz P. Kawa1, Anna Sobuś1, Arkadiusz Kazimierczak 2, Paweł Rynio2, Anita Rybicka2, Dorota Rogińska1, Katarzyna Babiak1, Zofia Litwińska1, Karolina Łuczkowska1, Anna Machalińska3, Piotr Gutowski2, Bogusław Machaliński1 1 Pomeranian Medical University, Department of General Pathology, Poland; 2Pomeranian Medical University, Department of Vascular Surgery and Angiology, Poland; 3Pomeranian Medical University, Department of Histology and Embryology, Poland

Keywords: MicroRNA, carotid stenosis, inflammation, oxidative stress, atherosclerosis; Recent studies suggest regulatory function for several microRNAs (miRNAs) in biology of carotid plaque, but their specific role is mostly unknown. The aim of our study was to investigate selected miRNA expression in CD34+ cells circulating in peripheral blood (PB) from patients with significant internal carotid artery stenosis (ICAS) collected before and after carotid endarterectomy (CEA) to associate miRNA expression with presence of ICAS. Methods: PB was collected from 24 ICAS patients before CEA and in 3rd month post-CEA to harvest CD34+ cells, which were source of miRNAs. Self-validated individual quantitative RT-PCR study was then performed to evaluate the expression levels of selected miRNAs. Results: miRNA expression analysis revealed decreased expression of selected inflammation and oxidative stress-related miRNAs, including miR-21-3p, miR23a-3p, miR-146a and miR155-5p, in ICAS patients after CEA. Conclusion: Differences in miRNA profile exist between ICAS patients before and after CEA. This study augments our knowledge about biological role of miRNAs in atherogenesis and vessel stenosis. This work was supported by grant No UMO-2012/05/B/NZ7/02299.

Acknowledgements The study, financed by research number 07-4.15.7.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 65

P1.27

P1.28

Preliminary study of training induced changes in transposable elements (TEs) expression in blood of Arabian horses

ACTN2 polymorphisms in pigs and its effect on meat texture parameters

Stefaniuk-Szmukier Monika , Ropka-Molik Katarzyna , Żukowski Kacper1, Piórkowska Katarzyna1, Monika Bugno-Poniewierska1 2

1

National Research Institute of Animal Production, Cracow, Poland; 2University of Agriculture in Cracow, Poland

1

The aim of the analysis was to study RNA-seq profiling to identified potential transposable elements (TEs) in blood of Arabian horses under racing training regimens.

From total 10 horses, 6 horses (GI) were sampling in September when they were 2.5-year-old before introduction to racetrack thereafter treated as controls. 4 horses (GII) were sampled after 24 weeks’ period which included conditioning exercise prior to the start of race training (12 weeks) and the further workload comprises heavy canters required to fitness sufficient to compete in races (12 weeks).

RNA-seq analysis was performed according to producer protocol. Statistical analysis was based on 1) trimming of raw reads (by using Flexbar software), 2) alignment to reference horse genome (by using STAR aligner) and 3) identification of differentially expressed genes (DEGs) between GI and GII samples (by using TEtranscripts software). The DEGs gene list was performed against transposable elements (TE).

The preliminary study showed that occurrence of training induced changes in TE expression. The most overrepresented TEs where those localised in: ENSECAG00000000971; ENSECAG00000008639; ENSECAG00000019629; ENSECAG00000023217 genes. The functional annotation of DEGsz was classified as positive regulation of collagen biosynthetic process, osteoblast differentiation, regulation of bone remodelling, muscle contraction, monocarboxylate transport and actin cytoskeleton organization.

Mirosław Tyra1, Katarzyna Ropka-Molik 2, Katarzyna Piórkowska2, Ilona Mitka1, Anna Bereta1, Magdalena Szyndler-Nędza1 National Research Institute of Animal Production, 1 Department of Genetics and Animal Breeding, 2 Department of Animal Genomics and Molecular Biology, Poland α-actinin-2, coded by ACTN2 gene, belongs to the spectrin family of cytoskeletal proteins. ACTN2 is a protein with multiple functions in different cells, including binding actin to the membrane of nonmuscle cells and anchoring the myofibrillar actin filaments in Z-disc as well as in analogous dense bodies. The aim of this work was an analysis of NC_010456.4: g.58645388C>T and g.58645450T>C within porcine ACNT2 locus and estimation of its effect on meat texture parameters is pigs. Analysis was performed on five breeds (Polish Landrace, Polish Large White, Puławska, Pietrain, Duroc) in total on 777 pigs, for which frequency of both genotypes was identified by PCR-RFLP method (with the use of FokI endonuclease).

The association between meat texture parameters and FokI: g.58645388C>T as well as FokI g.58645450T>C genotypes was performed using GLM procedure (SASv.8.02).

The genotype frequencies of following genotypes AA, AG, GG were 62%, 24%, 14% for FokI: g.58645388C>T, and 58%, 28%, 14% for FokI g.58645450T>C, respectively.

Meat of pigs with GG genotype concerning both analyzed polymorphisms was characterized by the highest firmness parameter (p<0.01) measured in cooked longissimus dorsi muscle compare to heterozygotes GA. The same trend was observed in cooked semimembranosus muscle. Furthermore concerning the second polymorphism meat of pigs with GG genotype also showed higher value for firmness parameter compared to AA genotype (p<0.01).

Acknowledgements The study was supported by the Polish Ministry of Science and Higher Education (project no. 2014/15/D/NZ9/05256).

6th Central European Congress of Life Sciences Eurobiotech 2017

66 Abstracts

P1.29

P1.30

Association of new PPARGC1 gene polymorphisms with meat texture parameters in pigs

Section: Nucleic Acid Tools

Mirosław Tyra1, Katarzyna Ropka-Molik 2, Katarzyna Piórkowska2, Ilona Mitka1, Magdalena Szyndler-Nędza1, Anna Bereta1 National Research Institute of Animal Production, 1 Department of Genetics and Animal Breeding; 2 Department of Animal Genomics and Molecular Biology, Poland Peroxisome proliferator-activated receptor gamma coactivator 1, encoded by PPARGC1 gene is responsible for mitochondrial biogenesis, glucose utilization, thermogenesis, angiogenesis and is a major factor that regulates muscle fiber type determination. The aim of this study was to determine the association of PPARGC1: g.del 2904-2956 (FR693835.1) and g.6293G>A (AH013726.2) polymorphisms with meat texture parameters in pigs. Research was conducted on five pig breeds Polish Landrace, Polish Large White, Puławska, Pietrain, Duroc) maintained in the Pig Performance Testing Stations in Poland, in total on 776 pigs. The analysis of PPARGC1: g.del 2904-2956 (FR693835.1) and g.6293G>A (AH013726.2) genotypes was performed using agarose gel electrophoresis of PCR product and PCR-RFLP method (with the use of FokI endonuclease), respectively. The association between meat texture parameters and different PPARGC1 genotypes was performed using GLM procedure (SASv.8.02). Results showed that meat of pigs with BB genotype concerning the first analyzed polymorphism was characterized by the highest chewiness parameter (p<0.01) measured in cooked semimembranosus muscle, compare to heterozygotes AB. In relation to the second analyzed polymorphism, heterozygotes AG were characterized by the highest value of firmness and toughness (p<0.01) in cooked ham and toughness in loin (p0.01).

Genetic diversity among different geese breeds based on molecular markers Joanna Warzecha, Agnieszka Fornal, Dominika Rubiś, Maria Oczkowicz, Monika Bugno-Poniewierska National Research Institute of Animal Production, Department of Animal Genomics and Molecular Biology, Poland Analysis of microsatellite loci and hypervariable region of a mitochondrial genome are the applicable tools used in evaluation of genetic diversity of the farm animals.

The aim of the present study was to preliminary characterise the genetic diversity based on microsatellite loci polymorphism and a fragment of mtDNA D-loop region.

The experimental material included 48 plumage samples of flocks of geese maintained in Poland: White Koluda®, Landes, Slowacka, Romanska, Kubanska and Garbonosa. The whole DNA was isolated with DNA Isolation Kit Sherlock AX (A&A Biotechnology). Based on D-loop sequence of Anser anser (GeneBank accession number NC_011196.1) primers were designed. MtDNA sequencing products were separated using the 3500xL Genetic Analyzer (Applied Biosystems). The 15 microsatellite primers were tested on the same group of 48 individuals (Bca µ1, TTUCG5, CKW21, Bca µ9, Bca µ8, Ans02, Ans18, Ans25, CAUD-G013, CAUD-G007, CKW47, Aal µ1, Afa35, CAUD-G012, Ans07) and were separated using the 3130xL Genetic Analyzer and genotyped. We observed the lack of genetic variability of fragment of mtDNA D-loop region in the tested group of individuals of goose. However, three microsatellite loci, TTUCG5, CKW21 and Aal µ1 were highly polymorphic. Our results suggest that microsatellite sequences are more useful for diversity studies in goose than mtDNA sequences.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 67

P1.31

P1.32

Section: Nucleic Acid Tools

The differences in CDC42BPA and TIMP3 gene expression in equine sarcoid and fibroblast in vitro cell cultures

Premature centromere division (PCD) as a possible cause of abnormal X chromosome morphology in a hucul mare Wojciech Witarski, Monika Bugno-Poniewierska, Katarzyna Kowalska National Research Institute of Animal Production, Department of Genomics and Animal Molecular Biology, Balice-Kraków, Poland Premature centromere division is a phenomenon of undefined molecular origin, linked directly to either general chromosome instability or abnormalities with mitotic spindle machinery. Distinctive features describing PCD include: a) X chromosome involvement, b) separated and rod-shaped chromatids, c) non-discernible centromeres d) age dependency of the PCD, e) unclear inheritance pattern f) presence of aneuploidy.

We identify PCD as a possible cause of abnormal X chromosome morphology in examined case. For the evaluation of the phenomena, the FISH technique was applied, using an equine X whole chromosome painting probe. The study revealed the presence of the group of white blood cells with prematurely separated X chromosome with a proper karyotype preserved. This particular pattern was observed in 82 cases, which comprised 9,53% of specimen’s examined population. Rest of the cell population as well as examined healthy controls showed normal metaphase chromosome spreads.

The mare was analysed cytogenetically due to reduced fertility. The direct link between X chromosome aneuploidy, as a form of leukocyte mosaicism, and aneuploidy within germinal lines has been well established. Different morphological, and probably functional, the status of the single X chromosome in the abnormal lymphocyte cells of the mare can be correlated with its abnormal phenotypic trait, however direct mechanism requires further investigation.

Wojciech Witarski, Przemysław Podstawski, Katarzyna Ropka-Molik, Monika Bugno-Poniewierska National Research Institute of Animal Production, DEPARTMENT OF GENOMICS AND ANIMAL MOLECULAR BIOLOGY, Kraków-Balice, Poland Sarcoid is a locally invasive equine skin benign neoplasm. In this study, two genes connected with cell motility and invasiveness were studied: CDC42 BPA and TIMP3.

CDC42BP alfa is a Ser/Thr kinase that bond to CDC42 and act as a downstream effector of actin remodelling and filopodia and invadopodia formation. TIMP3 act as an inhibitor of MMP3 activity. The balance between matrix metalloproteinases and their inhibitors define a structure of extracellular matrix and in its pathological state could support tumour invasiveness.RNA was isolated with PureLink RNA mini kit (Thermo Fisher) and underwent reverse transcription with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Gene expression levels were measured by an RT-PCR method using QuantStudio 7flex system.During the investigation of expression changes for CDC42BPA and TIMP3 genes, similar trends occurred. Expressions patterns for both genes were significantly (p≤0.01) higher in sarcoid cells. Despite significance was not present between sarcoid cell culture growing in 10% FBS compared to fibroblast 0.5% FBS groups, for both cell types higher expression level was observed for cells treated with medium containing 0.5% FBS content. A significant difference was observed only between TIMP3 gene expression in fibroblast cells. Given results suggested that cell type and its treatment have an impact on gene expression profile and may result in differences of functional behaviour.Funded: IZ-PIB 07-4.13.7

Acknowledgements NCBiR:BIOSTRATEG2/297267/14/NCBR/2016

6th Central European Congress of Life Sciences Eurobiotech 2017

68 Abstracts

Stem cells Keynote lecture L2&5.1 Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency Zipora Yablonka-Reuveni, Andrew Shearer, and Pascal Stuelsatz

EOM sparing. Collectively, new insights into unique features of EOMs and their myogenic progenitors can reveal intrinsic properties that contribute to selective protection of this muscle group in dystrophin deficiency and advance our knowledge of both pathogenesis and potential treatment of muscular dystrophy.

Lectures

University of Washington, Seattle, Washington, USA The human body contains over 600 individual skeletal muscles whose molecular and anatomical features can differ widely in accordance with their specific function. Extraocular muscles (EOMs) comprise a group of highly specialized skeletal muscles that control eye movements. The EOMs represent a unique skeletal muscle phenotype based on a range of properties, including their embryonic origin (i.e., head mesoderm) and their altered susceptibility to diseases (i.e., being preferentially involved or spared in a variety of metabolic and neuromuscular disorders). Especially intriguing for muscular dystrophy research, EOMs are uniquely spared in Duchenne muscular dystrophy and other dystrophies associated with impairments in the dystrophin complex. The mechanism behind EOM sparing remains unresolved. Specific traits of myogenic progenitors may be involved in EOM sparing, but very little is known about the myogenic cells in this muscle group. To this end, we have investigated lineage origin, antigen signature and function of satellite cells (the adult myogenic progenitors) from mouse EOM vs. the prototypic somite-derived limb and diaphragm muscles. Previously, we showed that EOMs harbor satellite cells that share a common Pax7+ signature with their limb and diaphragm counterparts, but are remarkably endowed with a high proliferative and renewal potential. Specifically, we demonstrated that in adult as well as in aging mice, EOM satellite cells possess a superior expansion capacity, contributing significantly more proliferating, differentiating and renewal progeny than their limb and diaphragm counterparts as revealed in cell culture assays. These robust growth and renewal properties were maintained by EOM satellite cells isolated from dystrophin-null (mdx) mice, while satellite cells from muscles affected by dystrophin deficiency (i.e., limb and diaphragm) expanded poorly in vitro. EOM satellite cells also retained higher performance in cell transplantation assays in which donor cells were engrafted into host mdx limb muscle. More recently, using a Cre/loxP reporter approach, we have discovered a novel myogenic lineage that specifies the EOM orbital layer following birth, establishing myofibers and satellite cells that persist throughout adult life. This discovery introduces a new concept regarding the EOM organization into orbital and global layers, an anatomical feature critical for fine-tuning of eye movement and binocular alignment. Furthermore, while in wildtype EOMs this secondary myogenic lineage primarily occupies the orbital layer, our pilot study suggests that in dystrophin-null mice this lineage occupies both the orbital and global layers, and thus, could potentially be involved in

L2&5.2 New modifiers of Duchenne muscular dystrophy Magdalena Kozakowska, Katarzyna PietraszekGremplewicz, Iwona Bronisz, Olga Mucha, Paulina Podkalicka, Krzysztof Szade, Alicja Józkowicz, Agnieszka Łoboda, Józef Dulak Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland Incurable DMD is caused by mutation in dystrophin gene. However, disease severity can be modified by influence on muscle damage and inflammatory reaction. In this regard, we have tested the effect of heme oxygenase-1 (HO-1), an important anti-inflammatory enzyme, known also to influence microRNA-mediated satellite cells (SCs) differentiation. In dystrophin-deficient mdx miceHO-1 is strongly induced in the muscle fibers and inflammatory cells. Systemic inhibition of HO-1 either by pharmacological compounds or by genetic knockout of HMOX1 gene aggravates the disease, impairing exercise capacity, augmenting muscle damage and inflammatory reaction. Interestingly, SCs of mdx mice demonstrate lower HO-1 expression than SCs from wild-type mice and revealed enhanced and disturbed differentiation which can be normalized by CO, one of the product of HO-1 activity. Thus, increasing the expression of HO-1 can potentially ameliorate muscle damage and disturbed differentiation of SCs. Interestingly, the knockout of muscle-abundant miR378 improved significantly the exercise capacity of the mdx mice, decreased inflammation, muscle fibrosis and also attenuated the disturbed differentiation of mdx satellite cells. The data indicate that lack of miR-378 changes the metabolism of the muscles.

In sum, the inflammation and muscle damage in DMD can be modified by increasing HO-1 or decreasing the expression of miR-378a.

Acknowledgements Supported by NCN Maestro grant (2012/06/A/NZ1/00004).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 69

L2&5.3

L2&5.4

TGF-1 pathways and MMPs activity in myoblasts differentiation

Role of Nrf2 in muscle satellite cells differentiation and progression of Duchenne muscular dystrophy

Paulina Kasprzycka, Paulina Kołodziej, Igor Stepaniec, Władysława Stremińska, Katarzyna Ilach, Maria A. Ciemerych, Małgorzata Zimowska

Iwona Bronisz, Magdalena Kozakowska, Katarzyna Pietraszek-Gremplewicz, Magdalena Madej, Alicja Jozkowicz, Jozef Dulak

University of Warsaw, Faculty of Biology, Poland

Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Krakow, Poland

Keywords: MMP-9, MMP-2, TGFβ1 pathways, skeletal muscle regeneration Skeletal muscles regenerate after injury. The significant role during repair is played by muscle-specific stem cells, i.e. satellite cells. Satellite cells activation and myoblasts differentiation is controlled by transcription factors – Myf5, MyoD, and myogenin, and also by extracellular matrix (ECM) components that can either trigger or foil muscle regeneration. Among them are cytokines, such as TGFβ1, and enzymes, such as matrix metalloproteases (MMP). MMP-2 and MMP-9, were shown to be the key enzymes involved in the repair of skeletal muscles. TGFβ family members control expression of ECM degrading enzymes or proteinase inhibitors. However, the relationship between TGFβ1 and MMP-2, MMP-9 in myoblasts has not been established, yet. In our previous study, we showed that the inhibition of TβR1 reduced MMP-2 and MMP-9 activity in differentiating myoblasts isolated from slow twitch muscle (Soleus). Nevertheless, as TGFβ1 uses different signaling pathways i.e. Smad dependent or non-Smad pathways, the signaling pathways involved in regulating MMPs activity are not characterized. In our current study we tested whether the Smad dependent pathway or the MAPKpathways control MMPs in context of myoblasts differentiation. To achieve this, we inhibited Smad3 phosphorylation using SIS3 or halofuginon. We also inhibited MKK using U0126 or PD98059. U0126 inhibits MKK1, MKK2, MKK5. PD98059 inhibits MKK1, MKK2. We inhibited p38 MAPK using SB202190 which inhibits the p38α and β isoforms.

We have previously shown that overexpression of heme oxygenase-1 (HO-1) inhibits myoblasts differentiation (Kozakowska et al, 2012). Here we demonstrate the role of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) – transcription factor known as a master regulator of genes encoding oxidative stress-response and phase II detoxifying enzymes such as NQO1(NAD(P)H dehydrogenase (quinone) and heme oxygenase-1 (HO-1).

In order to evaluate the role of Nrf2 in muscle satellite cells (MuSCs) differentiation, MuSCs (CD31-CD45-Sca1-α7integrin+) were isolated from Nrf2+/+ mice and in vitro differentiation was performed with the addition of Nrf2 activator – sulforaphane, what resulted in impaired differentiation.

To investigate the role of Nrf2 in the progression of Duchenne muscular dystrophy (DMD) double knockouts (mice lacking both dystrophin and Nrf2) were generated.

Firstly, mice were subjected to a treadmill test to assess muscle functionality and performance. Nrf2-/-mdx mice showed partially impaired exercises capacity in comparison to mdx (dystrophin-deficient) mice. Surprisingly, level of inflammation and amount of regenerating fibers were reduced in double knockouts in comparison to mdx mice, what may indicate the presence of some compensatory mechanism.

In conclusion, our data indicate that Nrf2 affects muscle satellite cells differentiation. Moreover, Nrf2 may have an impact on the regulation of inflammation in dystrophic muscles.

Acknowledgements Supported by National Science Center (NCN) grant MAESTRO (2012/06/A/ NZ1/00004)

6th Central European Congress of Life Sciences Eurobiotech 2017

70 Abstracts

L2&5.5

L2&5.6

ADAMTS5 knockdown impairs murine adipogenesis

From pluripotency to myogenesis

Roger H. Lijnen, Dries Bauters

Maria Anna Ciemerych-Litwinienko

Center for Molecular and Vascular Biology, KULeuven, Belgium

Department of Cytology, Institute of Zoology, Faculty of Biology, University of Warsaw, Poland

Adipose tissue (AT) expansion involves adipocyte hypertrophy, hyperplasia (via adipogenesis), angiogenesis and extracellular matrix remodeling. Enhanced expression of ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs; member 5) has been observed in AT of obese rodents. To elucidate the role of ADAMTS5 in adipogenesis, in vitro differentiation of precursor cells into mature adipocytes was studied using embryonic fibroblasts (MEF) derived from wild-type (Adamts5+/+) and ADAMTS5 deficient (Adamts5-/-) mice, or 3T3-F442A preadipocytes with stable shRNA-mediated Adamts5 gene silencing. Adamts5-/- MEF, as well as 3T3-F442A preadipocytes with Adamts5 knockdown, showed significantly reduced differentiation as compared to control cells. This was shown by reduced Oil Red O staining (intracellular lipids), lower expression of the adipogenic markers aP2 and PPARγ, and higher expression of the preadipocyte marker Pref-1. In Nude mice, de novo fat pads formed 4 weeks after injection of 3T3-F442A cells with Adamts5 knockdown were significantly smaller as compared to controls, associated with higher expression of Pref-1. Histological analysis did not reveal marked differences in size or density of adipocytes or blood vessels. Our data thus support the concept that ADAMTS5 promotes adipogenesis in vitro and in vivo, and suggest that its knockdown may present a strategy to impair AT expansion.

Keywords: pluripotent stem cells, differentiation, myogenesis Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, are an important tool in the studies focusing at the differentiation of various cell types, including skeletal myoblasts. They are also considered as a source of the cells that due to their pluripotent character and availability could be turned into any required tissue and then used in future regenerative medicine. For this reason, the methods of the derivation of some of cell types from pluripotent cells are still being perfected. Importantly, pluripotent stem cell lines and currently available protocols of their myogenic differentiation, allow to carefully analyze the function of various factors regulating myogenesis, including microRNAs.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 71

Keynote lecture

L2&5.8

L2&5.7

The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin

iPS cell-derived myogenic progenitors for the treatment of muscular dystrophies: how far are we?

Rita C. Perlingeiro, Ph.D. Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis-MN, USA Pluripotent stem cell-derived myogenic progenitors represent an attractive therapeutic approach to promote muscle regeneration in muscular dystrophy patients. A critical aspect before considering application of this technology to patients is the identification of strategies for the purification of myogenic progenitors from unwanted cells using clinically-compatible methods. We have documented the robust regenerative potential of human pluripotent stem cell-derived PAX7-induced myogenic progenitors, which engraft extensively, promote improved muscle contractility, and seed the stem cell compartment of transplanted dystrophic muscles. We have recently profiled PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment and identified CD54, α9β1 and SDC2 as novel candidate surface markers for the purification of human pluripotent stem cell-derived myogenic progenitors. These markers allow for the isolation of myogenic progenitors using both fluorescent- and magnetic-based sorting technologies, and importantly purified CD54+α9β1+SDC2+ cells contribute to long-term muscle regeneration in vivo. This is a critical step to enable translation of pluripotent stem cell-based therapies for muscle disorders. During this talk I will be discussing these findings, in addition to our new developments on induced pluripotent stem (iPS) cells, their potential for gene correction, and therapeutic application.

Jarosław Lewandowski1, Natalia Rozwadowska1, Tomasz Kolanowski1, Agnieszka Malcher1, Agnieszka Zimna1, Anna Rugowska1, Wojciech Łabędź2, Łukasz Kubaszewski2, Jacek Karczmarczyk 2, Katarzyna Chojnacka3, Katarzyna Bednarek-Rajewska3, Przemysław Majewski3, Maciej Kurpisz1 Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60-479, Poznan, Poland; 2 Department of Orthopedics and Traumatology, W. Dega University Hospital, University of Medical Science Poznan, 28 Czerwca 1956r, 61-545 Poznań; 3 Department of Clinical Pathomorphology, Karol Marcinkowski University of Medical Sciences, Przybyszewskiego 49, 60‑355 Poznań

1

Ischemic heart disease, also known as the coronary artery disease (CAD), poses a challenge to regenerative medicine. The iPSC technology might become a medical breakthrough due to possible differentiation of autologous descendent cells. Nevertheless, obtained in this way iPSC-CM like cells still demonstrate a fetal phenotype.

We attempted to acquire the adult human cardiomyocyte phenotype when studying 20th and 40th day of in vitro differentiation culture. Ontogenically close embryogenetic paths of skeletal myoblasts to cardiac myocytes and some evidences of ‘epigenetic memory’ in iPSC lines originated from myoblastoid cells could potentially better mimic myogenic cardiac cell properties than the other iPSCs. The genetic reprogramming of human skeletal myoblasts generated pluripotential stem cell line (SM-iPSC) of reproducible quality. Next, SM-iPSC line underwent successful pro-cardiac differentiation using BMP4 protein and chemical modulators of WNT pathway as well as Cardiomyocyte Differentiation Kit. Better developed myocardial stage (late stage of in vitro culture) was demonstrated by the presence of cardiac protein markers, sarcomere structure, progressive cellular hypertrophy, efficient intracellular calcium turnover reflected by better contractile performance, statistically significant elevation of CX43 gene expression similar to human myocardial ventricular cells, upregulated β-MHC expression, presence of MHC isoform switch and troponin I isoform transition.

6th Central European Congress of Life Sciences Eurobiotech 2017

72 Abstracts

L2&5.9

Posters

Role of heme oxygenase-1 in differentiation of human induced pluripotent stem cells to cardiomyocytes

P2&5.1

Mateusz Jeż1, Jacek Stępniewski1, Yuliya Chykunova1, Alan Kania2, Łukasz Chrobok 2, Marian H. Lewandowski2, Alicja Józkowicz1 and Józef Dulak1 1 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2 Department of Neurophysiology and Chronobiology, Institute of Zoology and Biomedical Research, Jagiellonian University, Kraków, Poland

Keywords: hIPSCs, cardiomyocytes , HO-1 Induced pluripotent stem cells (hiPSCs) are invaluable tools for investigating molecular mechanisms of differentiation.HO-1 is a cytoprotective enzyme which also affects stem cells differentiation. Recently we showed the significance of HO-1 in reprogramming of murine iPSC and their differentiation to cardiomyocytes (CM) (Stepniewski et al, in revision). Here we tested the role of HO-1 in human CMs differentiation.

We have generated hiPSCs from fibroblasts of healthy donor by transduction of Yamanaka’s factors with Sendai vectors. Obtained hiPSCs expressed markers of pluripotency (Oct4, Nanog, SSEA4, TRA-1-60 and TRA-1-81), differentiated spontaneously to all 3 germ layers in embryoid bodies and differentiated to CM with high efficiency (up to 85%), as evidenced by cardiac troponin T expression.

Obtained CM exhibited cardiac features i.e. expression of cardiac markers, synchronic contraction and electrophysiological potential.

To understand the role of HO-1 in cardiomyogenesis we have constructed plasmids with HO-1 with different cellular localization signals (nuclear, cytoplasmic and mitochondrial) and established HO-1 knockout hiPSCs employing CRISPR/CAS9 gene editing. Silencing of HO-1 gene was confirmed by Surveyor nuclease assay and by Western Blot. Effect of knockout out of HO-1 and knock in of HO-1 with different cellular localization will be checked in these cells.

Acknowledgements Supported by NCN grant Harmonia 2014/14/M/NZ1/00010.

Lack of heme oxygenase-1 induces inflammatory reaction and proliferation of muscle satellite cells after cardiotoxin-induced skeletal muscle injury Magdalena Kozakowska, Katarzyna Pietraszek-Gremplewicz, Maciej Ciesla, Marta Seczynska, Iwona Bronisz, Karolina Bukowska-Strakova, Alicja Jozkowicz, Jozef Dulak Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.

Keywords: heme oxygenase-1, muscle satellite cells, skeletal muscle regeneration Heme oxygenase-1 (HO-1, encoded by Hmox1 gene) is a heme-degrading enzyme regulating viability, proliferation and differentiation of many cell types. Among them it also strongly inhibits murine skeletal myoblast differentiation. Here we injected cardiotoxin into gastrocnemius muscle of Hmox1+/+ and Hmox1-/- animals and analysed muscle satellite cells (mSC) responsible for maturation of new myofibres, as well as inflammatory reaction, fibrosis and formation of new blood vessels.

Lack of HO-1 augments expression of MCP-1, IL-6, IL-1β and skeletal muscle injury. Disturbed proportion of M1/M2 macrophages, accompanied by enhanced formation of arterioles and expression of IGF-1 may be responsible for accelerated rate of regeneration observed in injured Hmox1-/- skeletal muscle. Importantly, HO-1-deficient mSC are prone to activation and proliferate more intensively upon injury. This effect can be partially mimicked by stimulation of Hmox1+/+ mSC with MCP-1, IL-6, IL-1β and is associated with increased MyoD expression, suggesting that Hmox1-/- mSC are shifted toward more differentiated myogenic population. Thus, multiple rounds of degeneration/regeneration in HO-1 deficient animals may lead to exhaustion of mSC pool. Accordingly, the percentage of mSCs is decreased also in old Hmox1-/- mice.

In summary, HO-1 modulates muscle repair mechanisms preventing its uncontrolled acceleration and attenuates age-dependent loss of mSCs. Acknowledgements Supported by the MAESTRO grant No 2012/06/A/NZ1/0004 from the National Science Center.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 73

P2&5.2

P2&5.3

The use of canine mesenchymal stem cells for the autologous treatment of osteoarthritis and for enhancing their therapeutic capacity

The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin

Gordana Nikcevic1, Sanja Srzentic Drazilov1, Vesna Spasovski1, Janko Mrkovacki2, Amira Fazlagic2 and Sonja Pavlovic1

Jarosław Lewandowski1, Natalia Rozwadowska1, Tomasz Kolanowski1, Agnieszka Malcher1, Agnieszka Zimna1, Anna Rugowska1, Wojciech Łabędź2, Łukasz Kubaszewski2, Jacek Karczmarczyk 2, Katarzyna Chojnacka3, Katarzyna Bednarek-Rajewska3, Przemysław Majewski3, Maciej Kurpisz1

1 Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Laboratory for Molecular Biomedicine, Serbia; 2Stem Art Ltd., Serbia

Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60-479, Poznan, Poland; 2 Department of Orthopedics and Traumatology, W. Dega University Hospital, University of Medical Science Poznan, 28 Czerwca 1956r, 61-545 Poznań; 3 Department of Clinical Pathomorphology, Karol Marcinkowski University of Medical Sciences, Przybyszewskiego 49, 60‑355 Poznań

1

Keywords: mesenchymal stem cells, canine, osteoarthritis, transgene, human IL-10 Mesenchymal stem cells (MSCs) hold enormous potential for cell-based therapy in the treatment of various diseases, particularly those which currently can not be cured and result in poor outcomes or invasive surgery. Here we present results of the application of autologous, culture-expanded, adipose tissue (AT)-derived MSCs for the osteoarthritis treatment of 10 canine patients. The stemness of isolated cells has been confirmed by their ability to differentiate into osteo- and chondro-cytic lineages. The clinical effect of a single injection of AT-MSCs into the symptomatic joints was evaluated by veterinarians for 5 disabilities characteristic of OA at 30, 60 and 90 days post treatment.

Functional outcomes improved markedly at the end of the evaluation compared with the baseline in the majority of dogs, with the highest scores detected for lameness at trot and pain on manipulation of the joints (improvement in 100% of dogs). In addition, for the experimental use, we genetically modified isolated canine AT-MSCs with the aim of enhancing their therapeutic potential. Cells were transfected with the plasmid carrying the human IL-10 cDNA, the potent immunosuppressive cytokine. The successful expression of transgene has been detected. Conclusions: The obtained results demonstrate the use of autologous AT-MSCs as a successful canine OA therapy approach. Also, a working platform for further studies related to the reinforcement of MSCs-mediated therapeutic impact has been established.

Ischemic heart disease, also known as the coronary artery disease (CAD), poses a challenge to regenerative medicine. The iPSC technology might become a medical breakthrough due to possible differentiation of autologous descendent cells. Nevertheless, obtained in this way iPSC-CM like cells still demonstrate a fetal phenotype.

We attempted to acquire the adult human cardiomyocyte phenotype when studying 20th and 40th day of in vitro differentiation culture. Ontogenically close embryogenetic paths of skeletal myoblasts to cardiac myocytes and some evidences of ‘epigenetic memory’ in iPSC lines originated from myoblastoid cells could potentially better mimic myogenic cardiac cell properties than the other iPSCs. The genetic reprogramming of human skeletal myoblasts generated pluripotential stem cell line (SM-iPSC) of reproducible quality. Next, SM-iPSC line underwent successful pro-cardiac differentiation using BMP4 protein and chemical modulators of WNT pathway as well as Cardiomyocyte Differentiation Kit. Better developed myocardial stage (late stage of in vitro culture) was demonstrated by the presence of cardiac protein markers, sarcomere structure, progressive cellular hypertrophy, efficient intracellular calcium turnover reflected by better contractile performance, statistically significant elevation of CX43 gene expression similar to human myocardial ventricular cells, upregulated β-MHC expression, presence of MHC isoform switch and troponin I isoform transition.

6th Central European Congress of Life Sciences Eurobiotech 2017

74 Abstracts

P2&5.4

P2&5.5

Biological activity of hematopoietic stem/ progenitor cells collected from patients with abnormal secretion of glucocorticoids

Adult stem cells from the porcine ovaries as a source of potential neural cells – in vitro studies

Miłosz P. Kawa , Anna Sobuś , Karolina Łuczkowska1, Lilianna Osowicz-Korolonek 2, Aneta Cymbaluk-Płoska3, Ewa Pius-Sadowska1, Dorota Rogińska1, Zofia Litwińska1, Edyta Paczkowska1, Elżbieta Dąbkowska1, Anhelli Syrenicz2, Bogusław Machaliński1 1

1

1 Pomeranian Medical University, Department of General Pathology, Poland; 2Pomeranian Medical University, Department of Endocrinology, Metabolic Diseases and Internal Diseases, Poland; 3Pomeranian Medical University, Department of Gynecological Surgery and Gynecological Oncology, Poland

Keywords: glucocorticoids, stem cells, cell cycle, apoptosis, proliferation We investigated effects of non-physiological high concentration of glucocorticoids (GCs) on hematopoiesis in adults with Cushing’s syndrome (CS).

Material: Peripheral blood (PB) was collected from 35 controls and 21 CS patients to isolate CD34+-enriched hematopoietic stem/progenitor cells (HSPCs). HSPCs were used to analyze GCs receptor (GR) expression, clonogenic growth, cell cycle and apoptotic activity.

Results: We found that HSPCs circulating in PB express active form of GR. Non-physiological concentration of GCs augmented the number of GR+CD34+ cells. High GCs levels resulted in decrease of clonogenic growth of hematopoietic lineages. These data were corroborated by expression analysis of mRNA for cyclin E and PCNA. Finally, GCs induced apoptosis in HSPCs as detected by increased expression of mRNA for BAX gene compared to controls.

Conclusion: This study augments our knowledge about biological effects of GCs on hematopoiesis in patients with CS. Acknowledgements This work was supported by grant No UMO-2011/01/B/NZ5/04224 and IP2011022771.

Kamil Wartalski1*, Gabriela Gorczyca1, Jerzy Wiater2 and Małgorzata Duda1 1 Department of Endocrinology, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków; 2 Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków; *kamil.wartalski@doctoral.uj.edu.pl

Stem cells from adult body – ASCs, probably are involved for tissue regeneration in mature organisms also nervous tissue.

The research material consisted of immature ovaries approximately 4–5 months old pigs collected from a local abattoir. Ovarian cortex was sliced and digested enzymatically. To isolate ovarian ASCs, the antibodies against CD34 conjugated to magnetic beads were used (EasySep CD34 Positive Selection Kit, StemCell Technologies). Cells were cultured for 48 h in DMEM/F12 with non serum supplement B-27 (ThermoFisher Scientific) and stem cells factor SCF to maintain cells in undifferentiated state. Then for the next 7 days, ASCs were differentiated using both forskolin (plant-derived substances, increasing the amount of cAMP) – 10 μM and fibroblast growth factor – bFGF – 10 ng/mL. After this time total protein were extracted from some of the cultured cells, the remaining ones were fixed for immunofluorescence studies.

In the part of cells stimulated for differentiation, cytoplasmic nestin and nuclear NeuN (neural markers) immunolocalization has been demonstrated. Moreover, both nestin and NeuN has been noticed also by Western blot analysis.

Nestin is an intermediate filaments protein and serves as key marker of neuronal cells. NeuN is a neuron-specific nuclear protein. Based on the results, we suggest that the ASCs differentiate into putative neural cells in the presence of forskolin and bFGF. Acknowledgements Supported by National Science Centre Poland: DEC-2016/21/N/NZ9/01205

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 75

P2&5.6 Does nuclear heme oxygenase-1 interact with G-quadruplexes and influence their stability?

Monika Zukowska, Alicja Czmoczek, Anna Konturek, Maciej Cieśla, Witold Nowak, Krzysztof Szade, Jozef Dulak, Alicja Jozkowicz Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Heme oxygenase-1 (HO-1) is a cytoprotective enzyme which catalyzes degradation of prooxidative heme to CO, iron ions and biliverdin. HO-1 enzymatic activity is connected with its well-described cytosolic form, however HO-1 can be also localized in the nucleus what is especially pronounced in hematopoietic stem cells. Our findings suggest that nuclear HO-1, apart from interacting with other proteins, can directly influence DNA G-quadruplexes (G4) and modulate their stability.

In our studies, we focused on visualizing protein-DNA interactions in hematopoietic stem and progenitor cells between HO-1 and G4 by applying proximity ligation assay (PLA). We show, that HO-1 and G4 are in close proximity (below 40 nm) what we additionally verified by colocalization stainings. Moreover, the substrate for HO-1 – heme is also a stabilizer of G4 in buffers and our FACS analysis reveal that cells lacking HO-1 possess more of these structures. This may indicate that enzymatic activity of HO-1 could possibly destabilize G-quadruplexes by degrading heme. We verify G4 stabilization with heme by stimulating cells with hemin and comparing it with known G4 stabilizer – pyridostatin. Additionally, we checked the influence of HO-1 on abundance of G4 structures in both RNA and DNA by treating cells with DNase and RNase.

Here, we demonstrate that HO-1 can directly interact with DNA. To best of our knowledge, for the first time we apply PLA staining to visualize protein-DNA interaction.

Our research lays groundwork for future studies on HO-1 influence on DNA structures and genome stability.

6th Central European Congress of Life Sciences Eurobiotech 2017

76 Abstracts

Plant biotechnology Keynote lecture

Lectures

L3.1

L3.2

Expanding biotechnology approaches to control insect pests

Reduction of Lignin Content in Rice Straw for Biofuel Production

Stanislaw Flasinski, Ph.D.

Sathish Kumar Ponniah1, Zhenhua Shang1, M. Aydın Akbudak 2,3, Vibha Srivastava3, Muthusamy Manoharan1

Monsanto, St. Louis, MO, USA Keywords: Insect control, RNAi, Insecticidal proteins, sustainable agriculture Insect pests caused significant economic losses in the United States and worldwide. Biotech crops are delivering substantial agronomic, environmental and economic benefits to farmers while making agriculture more sustainable. When expressed in plants Bacillus thuringiensis (Bt) proteins provide superior insect control solutions that are more, pest-selective and safer than conventional chemical insecticides. In order to maintain these sustainable insect control strategies integrated pest management system were developed that involve refuge strategy, pyramiding insect control toxin and discovery of new modes of action (MOA). New MOAs are used to supplement or replaced current biotech traits as resistance to older control measures emerges. RNAi technology is being utilized in a next generation product for Western corn rootworm control to be commercialized in 2018. Toxin engineering and retargeting with phage assisted continuous evolution (PACE) proved to be valuable technology to increase the potency of native proteins or to overcome field evolved resistance to Bt toxin. Modern sequencing technologies of microbes delivered new proteins with good activity on different insects. These new proteins have sufficient sequence and structural diversity to provide new MOAs for the control of selected insects. Many of the new proteins are structurally unrelated to Cry proteins in commercial products and showed good efficacy in diet bioassay on both fully susceptible insects as well as on a population that shows tolerance. Transgenic plants expressing these new proteins have demonstrated superior protection in greenhouse tests. These proteins provide new MOAs critical to the effective control of insect pests that have developed tolerance to the current traits.

Department of Agriculture, University of Arkansas, USA; 2Department of Agricultural Biotechnology, Akdeniz University, Turkey; 3Department of Crop, Soil & Environmental Sciences, University of Arkansas, USA 1

Rice straw is one of the largest biomass in the world that can potentially be exploited for bio-fuel. Nevertheless, the association of lignin with cellulose and hemicellulose has hindered the efficient utilization of rice straw for cellulosic bio-fuel. The objective of this study was, therefore, to down-regulate genes involved in lignin biosynthesis pathway, such as hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), cinnamoyl CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) through terminator-less construct to reduce lignin in transgenic rice straw for its use in cellulosic bio-fuel. Using terminator-less constructs, total lignin content was significantly reduced in seven lines (HCT-4, HCT-7, CAD-1, CAD-7, CCR-3, CCR-7, and CCR-12) from 4.6% to 10.8%.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 77

L3.3

L3.4

Improved production of dibenzocyclooctadiene lignans in elicited microshoot cultures of Schisandra chinensis

A dye degradation study: Green synthesis of Iron Nanoparticles

Agnieszka Szopa , Adam Kokotkiewicz , Agata Król2, Maria Luczkiewicz2, Halina Ekiert1 1

2

Chair and Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, ul. Medyczna 9, 30-688 Kraków, Poland; 2 Chair and Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Gdansk, al. gen. J. Hallera 107, 80-416 Gdańsk, Poland

1

Dibenzocyclooctadiene lignans (schisandra lignans), are a specific group of Schisandra chinensis (Turcz.) Baill. secondary metabolites with the large scope of pharmacological properties [1]. In our previous work the microshoot cultures of S. chinensis were demonstrated to produce substantial amounts of lignans [2]. The aim of the presented work was to improve the accumulation of these compounds in agitated cultures using elicitors.

The experimental agitated microshoot cultures of S. chinensis were maintained on MS medium supplemented with 3 mg/l BA and 1.0 mg/l NAA. The experiments included the testing of different concentrations of abiotic elicitor: CdCl2 (1, 10, 100 and 1000 µM), and biotic elicitors: chitosan (25, 50, 100 and 200 mg/l) and yeast extract (YeE) (0.1, 1.0, 3.0 and 5.0 g/l). The elicitors were added on the 1st, 10thor 20th day of the growth cycle. After 30 days, the microshoots were harvested and evaluated for growth parameters and for lignan content by LC-DAD method [2].

Performed research showed considerable enhanced of lignans production in microshoots of all of the applied elicitation schemes. The highest productivity, was confirmed for elicitation with YeE applied at 1.0 or 3.0 g/l on the 20th day; ca. 2.3 mg of lignans per flask (50 ml of medium).

The investigated cultures can be proposed as a plausible source of pharmacologically active lignans.

Demet Erdönmez1, Nihal Kenar1, Ömür Acet2, Mehmet Odabaşı2 Aksaray University, Faculty of Science and Letters, Department of Biology, Turkey; 2Aksaray University, Faculty of Science and Letters Department of Chemistry, Turkey

1

Keywords: Crataegus szovitsii Pojark., Dye decolorization, Iron nanoparticle, Reactive Blue 4 Dyes used in textile industry are produced 1 million tons more than a year. They give rise to ecological and environmental problems for aquatic ecosystems. Removal of textile wastewater within colorants is a high cost and effortful process. These removal processes through chemical methods cause accumulation of some chemicals in the medium. Therefore, eco-friendly approaches for removal of dyes have been in demand, recently. In this study, we produced iron nanoparticles via Crataegus szovitsii Pojark. which is rich in terms of polyphenolic compounds.

The characterization of these nanoparticles was carried out by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). It was determined that the nanoparticles complexes removed the color of Reactive Blue 4 approximately (monitored at 598 nm spectrophotometrically) at the rate of 95% and quickly (40–45 mins.). The usage of iron nanoparticles produced by plant-borne instead of physical and chemical methods for removal wastewaters will be a non-hazardous process for the environment and an alternative way economically.

References [1] Szopa A et al. Phytochem Rev, 2017,16,195-218. [2] Szopa A et al. Appl Microbiol Biotechnol 2016,100,3965–3977.

6th Central European Congress of Life Sciences Eurobiotech 2017

78 Abstracts

L3.5

L3.6

First report on the occurrence of Pectobacterium carotovorum subsp. brasiliense in Polish waterways

Useful catalogue of Candidate Gene Alleles established in different Guineensis, Oleifera and Hybrid Populations

Weronika Babinska1, Agata Motyka2, Sabina Zołedowska3, Wojciech Sledz4, Ewa Lojkowska5 Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk1, Laboratory of Plant Protection and Biotechnology2, Poland3 Pectynolytic bacteria cause blackleg and soft rot on various plants including vegetables. Their economic impact on potato production was estimated to reach 250 million euro. Among soft-rot Pectobacteriaceae (SRP) recently Pectobacterium carotovorum subsp. brasiliense(Pcbr) drew the scientific interest. Hot temperature origin and higher virulence in comparison to other soft rot pathogens, appearance of Pcbr under temperate climate gave rise to numerous concerns. In a view of global warming, field irrigation is becoming a necessity. The aim of project was to monitor the presence of SRP in Polish waterways, identify and characterise the isolates. During 6 months, water were collected at every 5 m depth from 10 lakes in Northern Poland by a scuba-diver. 100 µl of collected suspension was plated on Crystal Violet Pectate (CVP) medium and incubated at 28°C. Pectinolytic bacteria strains were identified to species level by multiplex PCR (Potrykus et al., 2014) or species-specific PCR. Characterisation was based on phenotypic features such as pectinase, cellulose, protease activities, motility, siderophore and their virulence on potato was tested. Phylogenetic analysis based on the comparison of the sequences of housekeeping genes: dnaX and recA of isolated and reference strains (Sławiak et al., 2009; Parkinson et al., 2009). In this study Pcbr was solely isolated from the lake coastal region, however.

We demonstrated for the first time the presence of Pcbr in Polish waterways.

Maider Astorkia1 , Baitha Santika2, Mónica Hernández1, Ana Herrán1, Leire Barandalla1, Fahmi Wendra S.2, Nathalie Quezada4, Pratiwi Erika2, Francisco Orellana3, Olga Leon3, Felix Vera3, Shone Morales4, Kevin Ponce4, Zulhermana S.2, Dwi Asmono2, and Enrique Ritter1 1 Department of Biotechnology, NEIKER-Tecnalia – Basque Institute of Agricultural Research and Development, P.O. Box 46, E01080 Vitoria, Spain, Email: mastorkia@neiker.eus; 2 Department of Research & Development, PT Sampoerna Agro Tbk., Jl. Basuki Rahmat No. 788 Palembang 30127, Indonesia; 3Energy & Palma SA, Av. Atahualpa E3-49 y Juan Gonzales Ed. Fundacion Perez Pallarez Officina 4A Quito, Ecuador; 4La Fabril SA, km 5.5 via Manta – Montecristi, Avenida 113, Manta, Ecuador

Oil palm is one of the most promising oil crops in the world due to its productivity (Elaeis guineensis Jacq.) and quality (Elaeis oleiferaKunth Cortes). A hybrid population derived from Guineensis and Oleifera population was reported to combine better palm oil quality with good productivity. Supporting the classical breeding, a molecular approach through marker assisted selection (MAS) can accelerate the genetic improvement for selecting desired palm genotypes to be crossed.

Partial CG amplicons of potential CG are studied. On one hand, the allelic variation of these CG amplicons (referred to “alleles”) in three different Guineensis, Oleifera, and hybrid populations is analyzed. On the other hand, the effects of particular CG alleles, SNPs and specific allele combinations on trait expression are analyzed by applying different association mapping techniques. In order to compare the effects in different populations, it is crucial to determine for each analyzed CG, the correspondence of the detected alleles and SNPs in each population.

A useful scheme for associating the detected alleles in the different populations including the identification of specific Oleifera alleles as a set of reference sequences for each CG have been developed. A catalogue of reference sequences for a growing number of CG is produced, following the needs of breeders and seed producers. These Elaeis reference sequences can be conveniently exploited for comparative effect analysis derived from association study, model building and for setting up efficient marker assisted selection systems to determine the most appropriate crosses.

The presented concept can be transferred in analogues way easily to any other crop species and its relatives.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 79

Posters

P3.2

P3.1

The use of Callitriche to phytoremediate chromium in bottom sediments of natural watercourses

Effect of Gene Order in DNA Constructs on Gene Expression upon Integration into Plant Genome M. Aydın Akbudak1, Vibha Srivastava2 1 Dept. of Agricultural Biotechnology, Akdeniz University, Turkey; 2Dept. of Crop, Soil & Environmental Sciences, University of Arkansas, USA.

No specific information is available on the effect of the order in which genes should be assembled in the construct to support optimum expression of each gene upon integration in the genome. While many factors including genomic position and the integration structure could affect gene expression, the investigators judiciously design DNA constructs to avoid glitches. However, the gene order in a multigene assembly remains an open question. This study addressed the effect of gene order in the DNA construct on gene expression upon site-specific integration in the rice genome using a simple design of two genes placed in two possible orders with respect to the genomic context. No significant effect of the gene order on gene expression was observed in the transformed rice lines containing precise site-specific integrations.

Joanna Augustynowicz1, Agnieszka Baran2, Ewa Sitek1, Beata Ostachowicz3, Tomasz Bryniarski1, Małgorzata Urbańska-Stopa1 University of Agriculture in Kraków, Institute of Plant Biology and Biotechnology, al. 29 Listopada 54, 31-425 Kraków, Poland; 2 University of Agriculture in Kraków, Faculty of Agriculture and Economics, Department of Agricultural and Environmental Chemistry, al. Mickiewicza 21, 31-120 Kraków, Poland; 3AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Department of Medical Physics and Biophysics, al. Mickiewicza 30, 30-059 Kraków, Poland 1

The aim of the work was to find out the suitability of Callitriche cophocarpa to remove Cr from polluted bottom-sediments under environmental conditions. This aquatic plant (macrophyte) was selected since it exhibits hyperaccumulation of Cr(III)/Cr(VI) in solutions on laboratory-scale studies. In his work we introduced Callitriche seedlings into the sediments of a watercourse extremely (up to 4 000 ppm) polluted by a tannery (Nowy Targ, Southern Poland). The plants grew vigorously under this unfavorable conditions, acclimatizing with no physiological disorders. Bioconcentration factors (BCFs), however, had low values. We found that chromium was associated with fractions in bottom sediments, in the following order [%]: oxidizable III (68.0) > residual IV (28.6) > reducible II (1.7) > exchangeable I (1.7). According to the Risk Assessment Code it was found low risk of Cr release from these sediments. Moreover, the measurements of redox potential (< ­-20 mV) and pH of water (alkaline) confirmed the lack of Cr mobility and biovalability under these conditions. Though, the change of the redox potential during i.e. sediment deposition on the land, may lead to changes in chemical forms of bound metals in solid phase of the sediments. This process is important when polluted sediments are subjected to oxidation, because it causes a release of toxic metals to the water. Our conclusion is that this species being outstanding Cr accumulator in waters can be useful in the reclamation of sediments under controlled, oxidising conditions.

6th Central European Congress of Life Sciences Eurobiotech 2017

80 Abstracts

P3.3

P3.4

First report on the occurrence of Pectobacterium carotovorum subsp. brasiliense in Polish waterways

Development of carotenoid gene knockouts in a model callus system using CRISPR/Cas9 vectors

Weronika Babinska1, Agata Motyka2, Sabina Zołedowska3, Wojciech Sledz4, Ewa Lojkowska5

Tomasz Oleszkiewicz1, Magdalena Klimek-Chodacka1, Levi G. Lowder2, Yiping Qi2,3, Rafal Baranski1

Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk1, Laboratory of Plant Protection and Biotechnology2, Poland3

1

Pectynolytic bacteria cause blackleg and soft rot on various plants including vegetables. Their economic impact on potato production was estimated to reach 250 million euro. Among soft-rot Pectobacteriaceae (SRP) recently Pectobacterium carotovorum subsp. brasiliense(Pcbr) drew the scientific interest. Hot temperature origin and higher virulence in comparison to other soft rot pathogens, appearance of Pcbr under temperate climate gave rise to numerous concerns. In a view of global warming, field irrigation is becoming a necessity. The aim of project was to monitor the presence of SRP in Polish waterways, identify and characterise the isolates. During 6 months, water were collected at every 5 m depth from 10 lakes in Northern Poland by a scuba-diver. 100 µl of collected suspension was plated on Crystal Violet Pectate (CVP) medium and incubated at 28°C. Pectinolytic bacteria strains were identified to species level by multiplex PCR (Potrykus et al., 2014) or species-specific PCR. Characterisation was based on phenotypic features such as pectinase, cellulose, protease activities, motility, siderophore and their virulence on potato was tested. Phylogenetic analysis based on the comparison of the sequences of housekeeping genes: dnaX and recA of isolated and reference strains (Sławiak et al., 2009; Parkinson et al., 2009). In this study Pcbr was solely isolated from the lake coastal region, however.

We demonstrated for the first time the presence of Pcbr in Polish waterways.

Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, Krakow, Poland; 2 Department of Biology, East Carolina University, NC, USA; 3 Department of Plant Science and Landscape Architecture, University of Maryland, MD, USA

In the carrot genome several single genes (CHXE, LCYE) of carotenoid pathway exist while other genes occur in two or more (PSY, ZDS, LCYB) paralogs. The significance of paralogs and a critical role of single genes requires verification to understand the complexity of carotenogenesis in carrot root. We developed a unique system with callus tissue that accumulate about ten fold more carotenoids than commonly induced carrot callus and at a similar level to carrot orange roots. These two callus types, orange and pale yellow, were genetically modified by inserting CRISPR/Cas9 constructs by A. tumefaciens-mediated transformation. The T-DNA contained Cas9 gene followed by short guide RNA sequences homologous to key carotenoid genes. Among developed transgenic calli we identified events of various color suggesting that some carotenoid genes were successfully targeted by Cas9::gRNA complexes and mutations were generated at that sites.

Sequencing of carotenoid genes in these calli and also in calli having no symptoms of color change confirmed that they were edited. Point and indel mutations shifting open reading frames thus amino acid composition were identified. These calli will be used to reveal the role of knockout genes and serve as a starting material for embryogenesis and mutant plant production.

Acknowledgements The financial support of the National Science Centre, Poland is acknowledged (UMO-2013/09/B/NZ9/02379).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 81

P3.5

P3.6

Antifungal activity of some steroidal glycoalkaloids extracted from species of the genus Solanum on Venturia inaequalisextension

Effect of thidiazuron on shoot induction of fibre hemp (Cannabis sativa L.)

Stelica Cristea , Mali S. Manole , Cristinel Zala , ȘtefanaJurcoane, 1 2, Silvana M. Dănăilă-Guidea1,2, Matei Florentina1,2, Brandușa Dumitriu3, Laura Olariu3 1

1

1

University of Agronomical Sciences and Veterinary Medicine, 59 Marasti Blvd., District 1, 011464, Bucharest, Romania; 2 Microbial Biotechnology Centre-BIOTEHGEN, 59 Marasti Blvd., District 1, 011464, Bucharest, Romania; 3 S.C. Biotehnos S.A., 3-5 Gorunului Street, 075100,Otopeni, county Ilfov, Romania 1

The antifungal effect of some steroidal glycoalkaloids extracted from Solanum species on the growth of the pathogen Venturia inaequalis, responsible to the appearance of apple scurf, was tested in vitro. The steroidal glycoalkaloids that we took in consideration were: solanidine, tomatine, solamargine. Of these, a certain structural group, encoded GLY, was selected, representing one of the active components of a biofungicide that is in course of patenting. The pathogen, Vi micromycetes, was isolated from the apples fruit, cv. Idared. The fungal isolate Vi17 was for the first time used in Romania. The collected biological material was placed on the agar malt medium to obtain asexual fructification. Pathogen-specific spores were taken and passed onto the potato-dextrose-agar culture medium.

From the mature culture, 0.5 mm diameter rounds were harvested with a special punch and were placed centrally in the culture vessels in which the test product was included using the food poison-technique. The concentrations of 0.1%, 0.5%, 1% were tested. Measurements of the fungus mycelium growth were performed at 3, 6, 9, 12 days. The efficacy of the product was determined after 12 days of observation. The highest efficacy values were 77.7% for the 1% variant and 55.5% for the 0.5% GLY variant, highlighting the remarkable antifungal activity of the compound.

Tomasz Wróbel1, Mariola Dreger1, Grażyna Mańkowska1, Milena Szalata1, Karolina Wielgus1, Ryszard Słomski2 1 Institute of Natural Fibres & Medicinal Plants, Department of Biotechnology, Wojska Polskiego 71b, 60-630 Poznan, Poland; 2Poznań University of Life Sciences, Department of Biochemistry and Biotechnology, Dojazd 11, 60-632 Poznan, Poland

Micropropagation is used for rapid multiplication of plant genotypes and may be an useful technique to obtain uniform plant material for the pharmaceutical and food industries. Effect of TDZ (thidiazuron) on shoot induction of chosen hemp genotypes (Epsilon 68, LKCSD, K-290 and INF 00218) was tested. Genotypes were selected due to its cannabidiol (CBD) content, adaptation to the local climate or as a positive control (INF 00218). Seeds were obtained from Fibrous Plants Gene Bank of the Institute of Natural Fibres and Medicinal Plants. Shoot tips and nodal explants were derived from three weeks – old donor plants, grown on sterile soil in the pots. Explants after surface sterilisation with commercial bleach and washing in sterile water were placed on MS medium (Murashige and Skoog 1962) containing TDZ (0.0–2.0 mg/L). The response of explants varied between genotypes. Percentage of shoot tips producing shoots was usually higher compare to nodal explants on the same medium. The best result was noted for INF 00218 genotype (from 28% to 57% of explants forming shoots, 12 shoots per explants). Unfavourable morphological changes such as: callusing, vitrification and leaf narrowing were also observed.

Acknowledgements Funded by Polish National Centre for Research and Development; project no: INNOMED/I/11/NCBR/2014

Acknowledgements The investigations were carried out within the project: PN-III-P2-2.1PTE-2016-0166.

6th Central European Congress of Life Sciences Eurobiotech 2017

82 Abstracts

P3.7

P3.8

Antioxidant activity of fruits, petioles and leaves of sweet cherry (Prunus avium L.)

High production of phenolic acids in in vitro cultures of two aronia species after addition of phenylalanine as their biosynthetic precursor

K. Dziadek*, A. Kopeć Department of Human Nutrition, Faculty of Food Technology, University of Agriculture in Krakow, Balicka 122, 30-149 Krakow, Poland, *kinga.dziadek89@gmail.com Key words: polyphenols, anthocyanins, vitamin C, sweet cherry Other authors indicate that not only fruits, but also other part of plant (fruit trees) are a good source of bioactive compounds. The objective of this study was to measure the antioxidant activity and the content of antioxidants in fruits, petioles and leaves of polish cultivars of sweet cherry (Burlat, Kordia, Regina). The research material was collected at Experimental Station of Pomology and Agriculture Department in Garlica Murowana (Poland) in 2016. The fresh material was used for preparation of methanolic extracts and to determine the content of vitamin C. In extracts, the content of total polyphenols, anthocyanins and antioxidant activity (ABTS•+, DPPH•, FRAP) were determined. The content of vitamin C was the highest in leaves. The anthocyanins was determined only in fruits. The petioles were characterized by highest content of polyphenols and antioxidant activity. Among fruits the highest antioxidant activity was found in cultivar Kordia, petioles – cultivar Burlat and leaves – cultivar Regina.

There is a need for further research of these material to be used for the production of functional foods.

Acknowledgements This study was financed by the National Science Centre, Poland (Grant no.

2015/17/N/NZ9/01136).

Agnieszka Szopa, Paweł Kubica, Aleksandra Walkowicz-Bożek, Halina Ekiert Chair and Department of Pharmaceutical Botany, Jagiellonian University-Medical College, Kraków, Poland Our earlier experiments with in vitro cultures of Aronia melanocarpa – black aronia and A. arbutifolia – red aronia documented, that they could be a rich potential source of phenolic acids. This group of plant metabolites is important in health food, pharmaceutical and also in cosmetic industry, because of their high antioxidant potential. The aim of the present studies were the investigations on the influence of egzogenic phenylalanine (Phe) as a precursor on the accumulation of phenolic acids in agitated culture system.

The cultures of two aronias were maintained on Murashige-Skoog [1] medium with 1 mg/l BAP and 1 mg/l NAA during 20 days (3 series). After 10 days different concentration (0.1–10 mmol/l) of Phe were added into the media.

In the methanolic extracts from biomasses the HPLC assays of phenolic acids (22 compounds) were performed [2].

The presence of seven compounds were confirmed. The main metabolites in both aronia cultures were depsides: chlorogenic and isochlorogenic acids. The max. total amounts of phenolic acids were respectively 2.04 and 2.84 times higher after addition of Phe in comparison to the controls and riched the amounts of 592 and 550 mg/100g DW, respectively.

The results documented that the addition of Phe into the culture media stimulate the production of investigated antioxidants.

References [1] Murashige T Skoog F (1962) Physiol Plant 15: 473–497 [2] Ellnain-Wojtaszek M Zgórka G (1999) J Liq Chrom Rel Tech 22: 1457–1471

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 83

P3.9

P3.10

The influence of egzogenic cinnamic acid on the production of phenolic acids in in vitro cultures of two aronia species

Use of nano-silver to optimize disinfection of Carex muskingumensis Schwein. rhizome explants in in vitro cultures

Agnieszka Szopa, Paweł Kubica, Aleksandra Walkowicz-Bożek, Halina Ekiert

Bairam Ismael, Danuta Kozak, Marzena Parzymies, Alicja Świstowska

Chair and Department of Pharmaceutical Botany, Jagiellonian University-Medical College, Kraków, Poland

University of Life Sciences in Lublin, Department of Ornamental Plants and Landscape Architecture, Poland

Phenolic acids (PhAs) are very valuable plant secondary metabolites with antioxidant activity. They are important in phytotherapy, cosmetology and also in health food industry.

In vitro cultures of Aronia melanocarpa and A. arbutifolia (black and red aronias) established in our Department produce high amounts of some PhAs. The aim of the present research was the investigations on influence of egzogenic cinnamic acid (CA) on the production of PhAs in in vitrocultures of both aronias. The agitated cultures were maintained for 20 days (3 series) on Murashige and Skoog medium (BAP – 1 mg/l, NAA 1 mg/l) [1] without and with addition of CA (0.1–10 mmol/l) after 10 days of growth periods.

In the methanolic extracts from biomasses the quantitative analysis of 22 PhAs was performed using HPLC method [2]. The presence of seven compounds was confirmed. The main metabolites in biomass of two aronias were depsides – chlorogenic and neochlorogenic acids, and in black aronia additionally isochlorogenic acid. The total amounts of PhAs ranged from 230 to 990 mg/100g DW and from 194 to 662 mg/100g DW in black and red aronia biomass extracts, respectively. The highest amounts of PhAs were confirmed after addition of 5 mmol/l of precursor.

The obtained results documented the great influence of egzogenic CA on production of some investigated antioxidants.

Keywords: ornamnetal grass, contaminations, AgNO3, streptomycin Carex muskingumensis is a valuable ornamental grass, characterizing with original shape. It is suitable for flowerbeds, containers and as a covering plant. It is usually propagated by division or cuttings. To meet market requirements, a more rapid propagation method is needed, such as micropropagation. Success in tissue cultures often depends on removing contaminations. The underground organs are particularly difficult to disinfect. The present study was to evaluate a potential of nano-silver (NSPs) to eliminate contaminants in Carex muskingumensis underground explants. Mother plants were pre-treated with fungicide. Plant material were sprouting buds, which were disinfected with 2.5 g dm-3 AgNO3 for 10, 15 or 20 min. Half of the explants were soaked in Streptomycin for 5 sec. The explants were placed on MS (1962) medium supplemented with BA 2.5 mg dm-3, IBA 0.1 mg dm-3 and with or without NSPs 250 mg dm-3. The addition of NSPs to the media decreased the number of contaminations. The best results were obtained when explants had been disinfected with AgNO3 for 10 min., immersed in Streptomycine and placed on the media supplemented with NSPs (25% clean regenerants) in comparison to the media without NSPs (0%).

Longer time of AgNO3 exposure caused necrosis.

References [1] Murashige T Skoog F (1962) Physiol Plant 15: 473–497 [2] Ellnain-Wojtaszek M Zgórka G (1999) J Liq Chrom Rel Tech 22: 1457–1471

6th Central European Congress of Life Sciences Eurobiotech 2017

84 Abstracts

P3.11

P3.12

Confocal laser scanning microscopy for smart and simple diagnostics of different in vitro callus cell types

LTP2 protein detection in spring barley grains exposed to multiple abiotic stresses

Ilona Mickeviča, Ilze Rubeniņa, Jeļena Kirilova, Sergejs Osipovs, Inese Kokina, Sanita Kecko, Muza Kirjušina, Inese Jahundoviča Daugavpils University, Institute of Life Science and Technology, Parādes street 1A, Daugavpils, Latvia, LV-5401 Keywords: callus, embryogenic and somatic cells, confocal laser scanning micorscopy, luminophores The recognition of embryogenic callus and somatic embryos is significant in case of proper and quick identification of embryo in in vitroplant cultures for further investigations such as propagation or plant transformation.

In in vitro conditions non-zygotic somatic embryogenesis occurs, which is a plant morphogenic response to stress, including embryos induction that’s leading promotion of regeneration of plant tissue, organs or whole plant body.

The aim of the present study is to develop a method for different callus cell diagnostic by confocal microscopy using newly synthesized luminophores – derivatives of benzanthrone. Many benzanthrone derivatives are hydrophobic luminescent dyes, which attract particular interest due to their remarkable spectral properties, such as large extinction coefficient, marked Stokes shift, negligible luminescence in anaqueous phase, high sensitivity of emission parameters to environmental polarity, etc. For this purpose, flax Linum usitatissimum, red clover Trifolium pratense and alfalfa Medicago sativa are used as model plants for in vitro callus culture formation. The newly synthesized luminophores have signifficant advantage to increases confocal image resolution. In conclusion, studied luminophores are suitable for recognition of embryogenic and somatic cells of different in vitro callus culture.

Michał Kempa1, Anetta Kuczyńska1, Krzysztof Mikołajczak1, Piotr Ogrodowicz1, Maria Surma1, Tadeusz Adamski1, Hanna Ćwiek-Kupczyńska2 1 Institute of Plant Genetics, Polish Academy of Sciences, Department of Biotechnology, Poland; 2Institute of Plant Genetics, Polish Academy of Sciences, Department of Biometry and Bioinformatics, Poland

Keywords: Hordeum vulgare, lipid transfer proteins, nsLTP2 gene, Western Blot Lipid transfer proteins have become the great of importance due to their significance in industrial applications and human health – they have been identified as a one of the main human allergens. Due to its role in lipid transfer the LTP2 may be crucial in lipidome modification in response to abiotic stress. LTP2 protein detection is the part of the project which is aimed to investigate the expression of the barley nsLTP2 gene (non-specific lipid transfer proteins) in conjunction with the lipidome (quantitative and qualitative) and phenome modification in response to various abiotic stresses (water shortage, salinity and heat stress). Studies were carried out on barley backcross and recombinant inbred lines. LTP2 protein detection was performed using the Western Blot in three biological replications. The level of protein determined by the expression of the LTP2 gene will be studied in the project to verify the hypothesis about the increased biosynthesis of this putative allergen during the drought conditions and warming climate.

Acknowledgements Project is funded by National Centre of Science (DEC-2015/17/B/NZ9/01481).

Acknowledgements The study funded by the project No. 1.1.1.1/16/A/211.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 85

P3.13

P3.14

Agrobacterium tumefaciens-mediated genetic transformation of Nigella damascena L.

A multiplex PCR assay for simultaneous detection of the most common fungal wheat pathogens

Magdalena Klimek-Chodacka, Tomasz Oleszkiewicz, Rafał Barański

Anna Kot1, Hubert Szczerba1, Agnieszka Ostrowska1, Michał Nowak 2, Marta Muszyńska1, Adam Kuzdraliński1

Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow

Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences in Lublin, Poland; 2 Institute of Plant Genetics, Breeding and Biotechnology, University of Life Sciences in Lublin, Poland

Nigella damascena L. also known as lady-in-mist or ragged lady is a herbaceous annual species belonging to Ranunculaceae family and is growing in temperate zones of Europe. N. damascena is grown as an ornamental plant due to the colorful flowers of interesting shape. Seeds are used in the kitchen, mainly due to the characteristic sweet strawberry scent. However, it is mostly appreciated for medicinal use, including the traditional medicine in Asia.

Seeds contain mainly fatty acids, proteins but also alkaloids, flavonoids and saponins. They are also essential oils present among which β-elemene, germacrene A and damascenine were mainly identified. These compounds can be derived from seeds but also produced in in vitro cultures, mainly from callus or cell suspension cultures. Many examples of in vitro cultured medicinal plants show that it is possible to control the biosynthesis of selected metabolites by using genetic transformation approaches, but no reports on N. damascena have been released till now.

In this research, for the first time, we present the Agrobacterium tumefaciens-mediated genetic transformation of N. damascena. After callus transformation with bacteria containing pCAMBIA 1105.1R plasmid most of callus clumps developed on a selection medium. Histochemical staining confirmed GUS gene activity thus the occurrence of transformation events.

1

Keywords: plant pathology, multiplex PCR, fungal detection The Septoria tritici blotch, brown/leaf rust and yellow/ stripe rust are three of the most economically important fungal foliar diseases caused by Septoria tritici (St), Puccinia triticina (Pt) and Puccinia striiformis f. sp. tritici (Pst), respectively. They are responsible for significant yield losses of cereals, especially common wheat (Triticum aestivum L.).

In this study, multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of the three pathogens. The study was based on environmental samples: leaf tissue with visual symptoms of fungal diseases collected across the Poland in the 2014/2015 growing season.

For each pathogen several sets of specific primers were developed. Their specificity was verified using naturally infected plant material with visual symptoms of target species as well as other fungal pathogens of wheat. For new PCR assay were chosen primers pairs that target different regions of fungal genome: eburicol 14 alpha-demethylase (CYP51), second largest subunits of the RNA polymerase II (rpb2) and beta-tubulin 1 (tub1) gene. Selected assays show 96–100% effectiveness in fungi detection: 69/71 of St, 55/55 of Pt and 45/45 of Pst in individual reaction.

In multiplex PCR reaction each primer sets LidSt11/12, LidPs9/10, LidPr1/2 produce single DNA fragment of 438, 240 and 144 bp, respectively. No cross-reactions were observed with tested fungal pathogens and healthy plant tissues.

6th Central European Congress of Life Sciences Eurobiotech 2017

86 Abstracts

P3.15

P3.16

Accumulation of secondary metabolites in in vitro cultures of Scutellaria subvelutina Rech. f. – preliminary results

Sulfurtransferases activity in cells of Brassica cretica ssp. botrytis cultured in vitro

Beata Kawka, Inga Kwiecień, Halina Ekiert Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, ul. Medyczna 9, 30-688 Kraków, Poland Apart from very famous in the phytotherapy world Scutellaria sp. like Scutellaria baicalensis Georgi (Baical skullcap) and Scutellaria lateriflora L. (American skullcap) [1] in the plant kingdom – is less known, native to the Mediterranean areas of Israel, Scutellaria subvelutina Rech. f. (Violet Skullcap, Velvety Skullcap).

The aim of the study was to investigate the biosynthetic potential of cells from established in vitro culture of S. subvelutina. Shoot cultures were maintained on Murashige-Skoog medium [2] supplemented with 2 mg/l BAP I 2 mg/l NAA during 4 weeks. The methanolic extracts from biomass were analyzed with HPLC method [3].

The presence of 4 flavonoids (baicalin, scutellarin, wogonin, wogonoside), 2 phenolic acids (3,4-dihydroxyphenylacetic and protocatechuic acids) and verbascoside was confirmed. The main compounds detected in biomass were wogonoside (1451,04 mg/100g DW) and verbascoside (1217,89 mg/100g DW). The total content of estimated flavonoids was 2308,12 mg/100g DW. This result shows substantial enzymatic potential of S. subvelutina cells from in vitro cultures, but it require more research.

References [1] Scutellariae baicalensis radix in: European Pharmacopoeia 7.1 Ed. (2011) Council of Europe, Strasbourg, p. 3355 [2] Murashige T, Skoog F, (1962) Physiol Plant.15: 473–497 [3] Ellnain-Wojtaszek M, Zgórka G, (1999) J Liq Chrom & Rel Tech. 22: 1457–1471

Inga Kwiecień Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, ul. Medyczna 9, 30-688 Kraków, Poland Plants can assimilate inorganic sulfur as sulfate for reduction to sulfide leading in cascade of enzymatic steps to the synthesis of sulfur-containing amino-acids. In contrast to plants, humans and animals lack the capability to reduce sulfate. As a consequence humans and animals rely on their diet for provision of reduced sulfur in cysteine and methionine. The aim of the present studies was to examine metabolism enzymes in Brassica cretica ssp. botrytis in in vitro cultures.

Brassica cretica ssp. botrytis experimental callus cultures were maintained on Murashige-Skoog (MS) medium containing different sets of plant growth regulators under constant artificial light (ca. 4 W/m2), at 24±2°C, and 4 weeks growing cycles. Medium was supplemented with sulfate ranged from 0 to 5 mM. Activities of the following enzymes involved in thiol and sulfane sulfur metabolism were assayed, rhodanese, cystationase and β-cyanoalanine synthase.

Callus in vitro cultures of Brassica cretica ssp. botrytis differed in the rate of growth. All of them can assimilate and sulfate directly from medium. However, differences in enzyme activity were observed, depending on medium variant. Sulfate concentration had an effect on rhodanese and β-cyanoalanine synthase activity. The greatest variability was observed for rhodanase. Another sulfurtransferase – cystathionase was activated using only highest sulfate concentration.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 87

P3.17

P3.18

Characteristic of the photosynthetic apparatus of Rumex tianschanicus x Rumex patientia, a high biomass yielding plant.

Acclimatization strategy of Dionaea muscipula J. Ellis to light stress in in vitro – physiological study

Magdalena Liszniańska , Halina Ślesak , Krzysztof Tokarz2 1

1

Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, Gronostajowa 9, 30-387 Cracow, Poland, e-mail: swmaga@wp.pl; 2 Institute of Botany and Plant Physiology, Department of Biotechnology and Horticulture, Agriculture University, al. 29 Listopada 54, 31-425 Cracow, Poland 1

Keywords: energy hybrid sorrel, photosynthesis, biomass Rumex tianschanicus x Rumex patientia is a cross between English spinach (R. patientia L.) (female line) and Tien Shan sorrel (R. tianschanicus A. Los.) (male line).

It belongs to energy crops, suitable for manufacturing pellets, briquettes, biogas production and is also an important medicinal plant. This hybrid significantly exceeds the original plants in terms of the yields of above-ground biomass.

We used Portable photosynthesis systems Li-6400 (LICOR Corporate) in order to compare the photosynthetic apparatus of 40-week-old maternal and paternal plants and hybrid sorrel as a high biomass yielding plant.

The highest intensity of net photosynthesis (Pn), stomatal conductance (gs) and transpiration rate (E) was noted in R. tianschanicus. In case of hybrid form leaves, only water use efficiency (WUE) was significantly higher compare to parent plants. The relation between photosynthetic pigments content and photosynthetic capacity was also studied.

Wojciech Makowski1, Barbara Piwowarczyk1, Rafał Banasiuk 2, Aleksandra Królicka2, Monika Hanula1, Krzysztof Tokarz1* Unit of Botany and Plant Physiology, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, 29 Listopada 54, 31-425, Krakow, Poland; 2Laboratory of Biologically Active Compounds, Department of Biotechnology, Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland 1

Dionaea muscipula from Droseraceae family is carnivorous plant, which is known as medical herb, because of the ability to produce derivatives of 1,4-naphtoquinones (plumbagin).

Plants exposed to high light intensity are more vulnerable to oxidative stress, which can cause oxidative damage, but also is necessary factors to improve secondary compound production.

The aim of the study was characterization of D. muscipula tissue culture acclimatization strategy to various light conditions.

D. muscipula were cultivated on 1/2 MS medium (30g×L -1 sucrose, pH=5.5), under various light conditions: fluorescence 50 [μmol×m-2×s-1] (control), fluorescence 120 [μmol×m-2×s-1] (FL) and white LED light 120 [μmol×m2 ×s-1] (WL). After 4 weeks, content of chlorophyll a+b (Chl a+b), accumulation of malondialdehyde (MDA), activity of antioxidant enzymes: catalase (CAT) and peroxidase (POD), total phenolic concentration (TPC) and plumbagin (P) accumulation were analyzed.

In comparison to control, Chl a+b content significantly decrease under WL; MDA content significantly increased under WL and FL. There was no significantly differences in POD activity, while CAT activity increased under FL. Both examined lights led to significantly higher TPC accumulation, while increased synthesis of P was observed only under FL. Results have shown, that D. muscipula acclimatization strategy to excess light is mostly based on phenolic derivatives, acting as non-enzymatic antioxidant compounds.

6th Central European Congress of Life Sciences Eurobiotech 2017

88 Abstracts

P3.19

P3.20

Plant transgenesis- new source of biofuels

Rhizobium rhizogenes transformation of monocotyledons Iris pseudacorus plants

Małgorzata Marszałek1, Joanna Zeyland1, Agnieszka Nowak1, Ryszard Słomski1,2, Daniel Lipiński1

Angelika Michalak, Aleksandra Królicka

1 Poznan University of Life Sciences, Department of Biochemistry and Biotechnology, Poland; 2Institute of Human Genetics, Polish Academy of Sciences, Poland

Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, Department of Biotechnology, Laboratory of Biologically Active Compounds, Poland

Keywords: genetic modification, transgenic plants, bioethanol Currently, the main material for bioethanol production are grains, potatoes and sugar beets. Unfortunately in Poland, bioenergy use of these plants faces discussions due to the reduction of their acreage destine for food production. Therefore, development and description of genetic construct for higher content of sucrose in sorghum and tabacco to increase the efficiency of bioethanol production seems to be very interesting.

In order to increase the sucrose content we developed and prepared genetic constructs allowing to overexpression of genes encoding enzymes in the sucrose synthesis pathway as UDP-glucose pyrophosphorylase, sucrose synthase and sucrose-phosphate phosphatase. The effect of the expression of sucrose metabolism genes may include: increase the proportion of sucrose and biomass production, higher primary and secondary plant growth.

Designing and preparation of genetic constructs contain genes encoding enzymes in the sucrose synthesis pathway affect to plant metabolism in increasing the proportion of sucrose in plant biomass. In the future it will obtain to produce a larger amount of bioethanol from plant biomass.

In many cases synthesis of bioactive phytochemicals is tissue specific. This may limit the possibility of obtaining them from in vivo source. Compounds produced in rhizomes and roots potentially may be also produced in genetically transformed roots obtained by Rhizobium rhizogenes infection. Their yield of secondary metabolites is comparable to that of the intact plants but hairy root culture is more genetically stable, hormone independent and suitable for scaling up thanks to high growth rates of the cultures.

In our research we are focused on monocotyledons Iris pseudacorus plants which produce biologically active secondary metabolites mostly in roots and rhizomes. Some of them are highly active against numerous human pathogenic bacteria, also those resistant to commercially used antibiotics. To produce bioactive phytochemicals in I. pseudacorus in laboratory conditions, we performed R. rhizogenes mediated transformation which was not previously reported in literature.

We tested 4 different strains of R. rhizogenes: 3 wild types and 1 GFP-tagged. We confirmed the transformation on the molecular level by PCR analysis of virG, rolB and rolC genes presence. To establish a stable and fast growing hairy roots culture we are testing different culture conditions (medium, supplementation, photoperiod).

Multiplication of initial hairy roots and stabilisation of hairy roots culture is in progress.

Acknowledgements This work was supported by project No. PBS1/A8/9/2012

Acknowledgements Project financially supported from Diamond Grant program. 0145/DIA/2015/44

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 89

P3.21

P3.22

Effect of beta-ketothiolase gene expression modification on benzenoid synthesis pathway in flax

Assessment of the genetic relationships among raspberry cultivars using molecular markers

Justyna Mierziak, Daria Nitarska, Kamil Kostyn, Aleksandra Boba, Jan Szopa-Skórkowski, Anna Kulma

Magdalena Simlat1, Agata Ptak1, Emilia Morańska1, Adam Kula1, Agnieszka Orzeł2

University of Wroclaw, Faculty of Biotechnology, Department of Genetic Biochemistry, Poland

1

Keywords: beta-kethiolase, benzenoids, flax, biolistic transformation, ODNs Benzenoids are significant secondary metabolites of many important functions for plants, such as growth regulation, protection from environmental stress, signal transduction. Despite relatively simple structure and its widespread occurrence, knowledge on their biosynthesis and regulation is still incomplete. One of the enzymes participating in benzenoid biosynthesis is beta-ketothiolase (bK).

It shows wide substrate specificity and can catalyze many reactions of both primary and secondary metabolism pathway. Acetyl-CoA, the main substrate for the thiolase reaction driven by this enzyme, is used for biosynthesis of such compounds as oils, carbohydrates, waxes, terpenoids and many other secondary metabolites. We studied the function of the bK gene by its silencing using two approaches: a non-vector ODN (oligodeoxynucleotide) technology and vector-based biolistic transformation. We analyzed the expression of the genes involved in benzenoid biosynthesis and measured contents of the corresponding metabolites.

The main benzenoids in flax are benzoic acid, benzoic aldehyde, vanillin and their derivatives. Their content was changed in the studied flax plants with bK gene silenced.

The plants were also characterized by changed expression profile of other genes involved in the benzenoid pathway, particularly isochorismate synthase (ICS) and benzoic acid/salicylic acid methyltransferase (BSMT). We suggest that bK is one of the key genes involved in the regulation of benzenoid synthesis in flax.

University of Agriculture in Krakow, Department of Plant Breeding and Seed Science, Krakow, Poland; 2 NIWA Berry Breeding Ltd., Brzezna, Poland Keywords: raspberry; genetic similarity; RAPD; SSR Raspberries belong to the genus Rubus which is one of the most diverse genera in the plant kingdom due to the number of species and ploidy level. However, domestication of red raspberry (R. idaeus L.) and black raspberry (R. occidentalis L.) has reduced the morphological and genetic diversity of these species. In consequence, the narrow pool of the ancestral cultivars which has been used for breeding has resulted in high similarity of modern cultivars. To improve the efficiency of crossing, breeders need detailed information on the genetic relationships among germplasms.

In this study, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) loci were used to investigate the genetic relationships within a group of 22 raspberry cultivars of different geographical origin.

The genetic similarity index (SI) calculated on the basis of RAPD data ranged from 0.14 to 1.0 and SI calculated on the basis of SSR data ranged from 0.16 to 0.68, indicating a large range of genetic variability among the analyzed cultivars. Cluster analysis by UPGMA and PCA clearly delineated the genetic relationships among all the accessions. The obtained results confirmed the usefulness of RAPD and SSR markers for discriminating among closely related raspberries and for determining the genetic variability among cultivars. This information may be helpful for breeders to plan strategically the breeding program.

6th Central European Congress of Life Sciences Eurobiotech 2017

90 Abstracts

P3.23

P3.24

Oxidative stress in Leucojum aestivum L. in vitro cultures

Roundup utilization by chemical methods

Emilia Morańska , Magdalena Simlat , Edyta Skrzypek 2, Marzena Warchoł2, Agata Ptak1 1

1

University of Agriculture in Krakow, Department of Plant Breeding and Seed Science, Krakow, Poland; 2The F. Górski Institute of Plant Physiology, Polish Academy of Sciences, Krakow, Poland 1

Key words: Leucojum aestivum, liquid cultures, sugars, antioxidant enzymes Leucojum aestivum L. is a source of pharmacologically active alkaloids – galanthamine and lycorine (Ptak et al., 2017). Biosynthesis of alkaloids in in vitro cultures may be connected with plant self-defense reaction to stress factors. Moreover, oxidative stress can modify growth and development of propagated plants. In the case of in vitro cultures several methods for inducing stress conditions are used, for example by the addition to the medium of osmotic stress agents, such as sugars.

In the present study, the effects of sugars: sucrose, glucose, fructose, maltose (3, 6, 9%), used as a source of carbon in liquid medium, on the growth of L. aestivum plants and induction of oxidative stress were examined.

The plants cultivated on the medium enriched with 3% fructose were characterized by the highest biomass increments (1.48 g fresh weight) and highest activity of catalase (0.306 U·µg protein-1). On the other hand, the osmotic treatment with fructose at a high concentration (9%) resulted in the highest activity of superoxide dismutase (3.49 U·µg protein-1). However, the highest activity of ascorbate peroxidase was found in plant material grown on the medium with the addition of 9% maltose (1.335 U µg protein-1).

Marcin H. Kudzin1, Renata Żyłła1, Zdzisława Mrozińska1, Mateusz Niedzwiecki1, Paweł Urbaniak 2, Jadwiga Sójka-Ledakowicz1 Textile Research Institute, Brzezinska 5/15, Lodz 92-103, Poland; 2Faculty of Chemistry, University of Lodz, Tamka 12, Lodz 90-136, Poland, *kudzin@iw.lodz.pl

1

Keywords: Roundup, glyphosate, PMG, pesticide, herbicide, Fenton’s reaction, hydrogen peroxide Pesticides containing glyphosate (N-phosphonomethyl-glycine) are nowadays the most used herbicides worldwide. Glyphosate (PMG) is a broad-spectrum systemic herbicide, brought to market in 1974 by Monsanto under the trade name Roundup. [1] During the last decades there has been a rising number of data revealing the possible toxicity of glyphosate and its commercial products – Roundup.

[2] Therefore several studies on the chemical degradation of glyphosate have been undertaken. [3-6] In this communication we present our results on the chemical degradation of aqueous solutions of the herbicide formulation Roundup, including Fenton’s reaction.

References [1] PANAP, Pesticide Action Network Asia & the Pacific Monograph on Glyphosate (2009).

[2] Mesnage R, Defarge N, de Vendômois J S, Séralini G E (2015) Food Chem.

Toxicol. 84: 133–153 [3] Assalin M R, De Moraes S G, Queiroz S, et al. (2010) J. Environ. Sci. & Health B 45: 89–94 [4] Manassero A, Passalia C, Negro A C, Cassano A, et al. (2010) Water Res. 44: 3875–3882 [5] Jaisi D P, Li H, Wallace A F, Paudel P, et al. (2016) J. Agric. Food Chem. 64: 8474–8482 [6] Wang M, Zhang G, Qiu G, Cai D, Wu Z (2016) Chem. Eng. J. 306: 693–703

References Ptak A et al (2017) Plant Cell Tiss Organ Cult 128: 335–345 Acknowledgements This research was supported by the Polish Ministry of Science and Higher Education (contract number DS 3129/KHRiN).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 91

P3.25

P3.26

The impact of exogenous plant growth regulators on carotenoid composition and carotenoid pathway gene expression in carrot cells in vitro

Elimination of contaminating bacteria from plant tissue culture

Tomasz Oleszkiewicz1, Magdalena Klimek-Chodacka1, Anna Kostyn2, Aleksandra Boba3, Jan Szopa3, Rafał Barański1 1 Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Kraków, Poland; 2Institute of Genetics and Microbiology, Faculty of Biological Sciences, University of Wrocław, Poland; 3Department of Genetic Biochemistry, Faculty of Biotechnology, University of Wrocław, Poland

Carotenoid accumulation depends on a plant developmental stage and plastid biogenesis that may be affected by signal molecules. Plant hormones like abscisic acid, auxins or giberellins may function as a factor regulating carotenoid biosynthesis and accumulation. Callus cultures in vitro are considered as model systems for research on genetic control of biosynthetic pathways.

The aim of our research was to determine the effect of exogenous plant hormones (GA3, 2,4-D and ABA) on carrot callus cells in their ability to accumulate carotenoids. For this purpose we used three carrot cell suspension cultures of two varieties, Koral and Amsterdamska, and a double haploid line. Growth regulators were supplemented to the solid culture medium, on which cell suspension was evenly spread. After two months, part of callus mass was directed to UPLC analysis. Samples with changes in carotenoid content were directed to gene expression analysis.

UPLC analysis showed different amounts of individual carotenoids in 2,4-D and ABA treated callus in compare to control. Gene expression analysis of 2,4-D and ABA treated callus revealed differences in some of key enzymes in carotenoid pathway. Results of these experiments indicate optimal culture conditions for the assessment of genetic determinants in carrot carotenoid biosynthesis.

Teresa Orlikowska, Tadeusz Malinowski, Lucyna Ogórek Research Institute of Horticulture, Skierniewice, Poland, Teresa.Orlikowska@inhort.pl Keywords: bacterial contamination, inner sterilization, vacuum Plant-associated bacteria form both epiphytic and endophytic populations through plants and their complete elimination from plant tissues is not possible despite of superficial sterilization of initial explants. Microorganisms, even in cryptic stage, may have detrimental effects (called vitropathy) on the cultures concerning multiplication, rooting, acclimatization or phenotypic stability. They can also influence the results of experiments affecting metabolism of explants either directly or through changing the media and the atmosphere composition in the vessels.

Sanitation protocol should eliminate as much as possible bacteria from initial explants, including: cultivating donor plants at high the phytosanitary condition, isolating the smallest possible shoot tips for culture initiation, using effective sterilization. To eliminate endophytic bacteria from long term raspberry shoot cultures, we applied an inner sterilization by infiltration of plantlets with different biocides, also at lowered pressure atmosphere. All treatments, especially those cyclically repeated were able to decrease bacteria population to some extent. The most effective approach was the use of 0.05% water solution of HgCl2, with two periods of 15 min incubation under 300 mbar pressure.

The study was conducted under the Multiannual Program of the Ministry of Agriculture and Rural Development

Acknowledgementss The financial support of the National Science Centre, Poland is acknowledged (decision No. DEC-2013/09/B/NZ9/02379).

6th Central European Congress of Life Sciences Eurobiotech 2017

92 Abstracts

P3.27

P3.28

Piriformospora indica as growth stimulator and bioprotector of rhododendron plants for Phytophthora cinnamomi

Use of tissue culture for ex situ conservation of the endangered plant species -Salix lapponum Marzena Parzymies1, Magdalena Pogorzelec2, Alicja Świstowska1

Aleksandra Trzewik , Lucyna Ogórek , Teresa Orlikowska1, Evelyn Klocke2 1

1

University of Life Sciences in Lublin, Department of Ornamental Plants and Landscape Architecture, Poland; 2University of Life Sciences in Lublin, Department of Hydrobiology, Poland 1

Research Institute of Horticulture, Skierniewice, Poland; Julius Kühn Institut, Institute for Breeding Research on Horticultural Crops, Quedlinburg, Germany, orlikowska.edytor@inhort.pl 1

2

Piriformospora indica, fungal endophyte, colonizes root cells and intercellular spaces. Several authors found its beneficial influence on different colonized plant: increase of biomass and seed yield, and enhancement of tolerance for biotic and abiotic stresses. The goal of our study was to determine if P. indica will colonize rhododendron plants and protect them before Phytophthora cinnamomi. In our experiments, P. indica colonized young rhododendron ‘Nova Zembla’ and ‘Alfred’ plants, which was observed microscopically as chlamydospores in roots. Colonized plants ‘Nova Zembla’ were higher by 50% after 3 months and by 63% after 7 months and ‘Alfred’ by 50% and 40% respectively comparing to non-inoculated control. In protection test, plants propagated in vitro were inoculated with P. indica at acclimatization in the greenhouse. After 3, 4 or 5 weeks, the growth substrate was infected with P. cinnamomi. All control plants (unprotected with endophyte) died within 6 months. Of the inoculated and infected died 11 to 14 plants from 27, and in the another experiment survived 10 plants out of 25 in comparison to total mortality in control treatment. We can conclude that this model of bioprotection can be usable in rhododendron nurseries.

Keywords: active protection, micropropagation, in vitro preservation A possibility to use micropropagation for a protection of the rare species from the Red List of Polish Flora, Salix lapponum, was studied. It is a valuable glacial relict species in Polesie Lubelskie, threatened with extinction.

A decrease in the number of stands and populations was noted. Therefore, a project concerning an active protection of the species was undertaken. The aim was to develop a micropropagation protocol for S. lapponum, to produce plants that would be reintroduced into natural habitats.

Because of few mother plants and bad quality or no seeds available, micropropagation was chosen as the best way to obtain new plants. The explants were nodes collected from plants growing in natural stands. They were disinfected with NaOCl (1%, 30 min.), AgNO3 (0.5%, 15 min.) or HgCl2(0.1%, 10 sec.), and placed on MS media [1962] with (in mg·dm-3): BA 0.1 + IBA 0.01, BA 0.1 + IBA 0.01 + AA 20, BA 1 + IBA 0.1 + AC 2 g·dm-3. Initiation of cultures was difficult, as only few shoots were free from contaminants.

Good results were obtained with NaOCl and HgCl2. There were no differences between the media used. The obtained shoots were transplanted into a fresh media. The shoots rooted spontaneously. Good quality, rooted microplants were planted into acid peat : sand mixture (2:1) and acclimatized. Around 80% of plants survived and after a year of cultivation they were ready to be planted in natural stand.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 93

P3.29

P3.30

Comparative genomics of homozygous B10 cucumber line individuals shows dynamics within functional loci

Genomic comparison of cucumber growth type mutants

Paweł Osipowski, Michał Wojcieszek, Magdalena Pawełkowicz, Agnieszka Skarzyńska, Zbigniew Przybecki, Wojciech Pląder

Michał Wojcieszek, Magdalena Pawełkowicz, Zbigniew Przybecki, Wojciech Pląder

Warsaw University of Life Sciences, Department of Plant Genetics, Breeding, and Biotechnology, Nowoursynowska 159, Warsaw, Poland Keywords: comparative genomics, structural variants, cucumber Comparative genomics of eukaryotic genomes can be facilitated by proper bioinformatics analyses. Approaching new genome draft assembly from single molecule, real-time PacBio 68.9x reads and using Illumina reads 30x data from 3 individuals we uncover some aspects of B10 cucumber inbred line genome dynamics. In its present state, newly assembled genome shows both best contiguity and quality characteristics among cucumber genome drafts (GenBank: GCA_001483825.2). Within B10 line genomes we predicted single nucleotide variants (SNVs) and structural variants (SVs) unique to certain plant individuals. Some of the predictions span within functional loci (promoters, genes) as well as overspread with annotated transposable element (TEs) sequences of the genome indicating that highly inbreed homozygous plants remain genetically dynamic. Relatively low polymorphism frequency within the B10 line and TEs’ might serve as a trace to better recognition of TE dependant phenotypic changes.

Warsaw University of Life Sciences,Department of Plant Genetics, Breeding, and Biotechnology, Nowoursynowska 159, Warsaw, Poland Keywords: cucumber, resequencing, mutants, variant calling Being one of the most important crop C. sativus is target of intensive genomic study focused on determination mechanism involved in processes significant for new variety development. In this research we try to elucidate pathways responsible for different type of growth in three cucumber mutants: W-SK, W19 and Sh. We conducted resequencing of genomes with Illumina techniques and commenced variant calling against reference genome. Results showed high number of changes due to invasive mutagenesis in coding and non-coding regions. Identified changes in both promotor and exon structures are responsible for growth type difference and will be subject for more detailed research in the future.

Acknowledgements This work was supported by grant from National Science Centre 2013/11/B/ NZ9/00814

Acknowledgements This work was supported by grant from National Science Centre 2013/11/B/ NZ9/00814

6th Central European Congress of Life Sciences Eurobiotech 2017

94 Abstracts

P3.31

P3.32

The relationship between the silencing of the GMPase gene and the sugar composition of flax

Silver thiosulfate enhance accumulation of phenolics in hairy roots of Lactuca virosa

Katarzyna Pelc, Marta Preisner, Alicja Metryka, Jan Szopa, Anna Kulma

Katarzyna Pieron, Janusz Malarz

Faculty of Biotechnology, Department of Genetic Biochemistry, University of Wroclaw, Poland Flax (Linum usitatissimus L.) is an broadly studied industrial plant originating from Middle East, presently occurs in a Mediterranean and temperate climate zone.

Most of this research is focusing on understanding of the genetic mechanisms and production of plants with new, useful features. GMPase is an enzyme catalyzing production of compounds like GDP-mannose pyrophosphorylase (GMPase) responsible for synthesis of GDP-mannose. GDP-mannose is a substrate for the synthesis of GDP-fructose, GDP-rhamnose and GDP-galactose. GMPase influences many important processes necessary for the proper functioning of the plant, like synthesis of cell wall polymers, ascorbic acid, and in the process of glycosylation of proteins. Previous studies showed that the silencing of GMPase gene had pleiotropic effects. Thus, the aim of this study was to generate flax plant with silenced gene encoding GMPase and determining how the modification affected composition and structure of the cell wall and sugar metabolism in plants.

Institute of Pharmacology, Polish Academy of Sciences, Department of Phytochemistry, 31-343 Kraków, Smętna street 12, Poland Keywords: Caffeic acid derivatives, Hairy roots, Lactuca, Sesquiterpene lactones, Silver thiosulfate Sesquiterpene lactones and phenolic compounds – derivatives of caffeic acid are two groups of secondary metabolites which are effectively produced by hairy roots of Lactuca virosa. However, little attention has been paid to regulatory aspects of this production. Individual sesquiterpene lactones demonstrated different patterns of accumulation during the growth cycle whereas maximum content of caffeic acid conjugates in the roots was detected at the day 10th of the culture cycle. Ageing of the tissue resulted in slightly reduced accumulation of the phenolic compounds. We used silver thiosulfate, an inhibitor of the ethylene action in plant cell cultures, to study an effect of ethylene inhibition on the secondary metabolite accumulation in the examined roots.

The accumulation of sesquiterpene lactones and caffeic acid derivatives in hairy roots of L. virosa, which were treated with silver thiosulfate (15–100 µM) at the logarithmic phase of growth, was studied using HPLC/PDA method. The ethylene inhibition had little effect on the sesquiterpene lactone content 24 h and 96 h after silver thiosulfate addition. Accumulation of caffeic acid derivatives in the roots was significantly higher, after 24 h and 96 h of the treatment (30–100 µM). The most affected were cichoric and 3,5-dicaffeoylquinic acids contents (about 150% of the control value).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 95

P3.33

P3.34

In vitro regeneration and wogonin production in genetically transformed culture of Scutellaria bornmuelleriana

The effect of Methylomethionine Sulfonate (Vitamins U) on physicochemical changes of rapeseed oil under accelerated oxidation conditions

Zahra Gharari1, Khadijeh Bagheri1, Ali Sharafi2, Hossein Danafar2

Małgorzata Kucia, Elżbieta Kondratowicz-Pietruszka

University of Zanjan, Zanjan, Iran; 2 School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran 1

Keywords: In vitro regeneration, genetically transformed culture, Scutellaria bornmuelleriana Introduction: Scutellaria bornmuelleriana has several important medicinal compounds such as Baicalein, Wogonin, Apigenin, Baicalin and Scutellarin. The present study evaluated the in vitro regeneration and genetically transformed root cultures. ½ MS medium supplemented different concentrations of TDZ, NAA, TDZ and BA was used for shoot organogenesis. The obtained induced shoots were transferred to root induction medium containing 0.1 mg/l IBA. We used the leaves of one month sterile plants as explants in inoculation medium of Agrobacterium rhizogenes strains A4, ATCC15834, MSU440 and A13. The explants transferred to a modified co-cultivation MS mediums for 48 h. The explants were placed on MS medium supplemented with 400 mg/l cefotaxime.

The genomic DNA was extracted from transformed roots using CTAB method. Molecular analysis of transformed root lines was confirmed by PCR using specific primers of the rolB gene. It was revealed that combination of 0.5 mg/l TDZ and 1 mg/l BA was the most effective for callus formation and shoot induction. A significant increase in transformation frequency occurred when the strain MSU440 was used. Wogonin content in genetically transformed root cultures was detected by HPLC analysis. The present protocol introduced a simple and rapid regeneration system for S. bornmulleria in short period via adventitious shoot induction. An efficient hairy root induction system was developed through A. rhizogenes-mediated transformation as an alternative approach for wogonin production.

Department of General Chemistry, Faculty of Commodity Sciences, Cracow University of Economics, Sienkiewicza 5, 31-510 Krakow, Poland Keywords: Methylomethionine Sulfonate, oxidative stability, thermal treatment, photooxidation, rapeseed oil Methylmethionine (vitamin U) was obtained for the first time from raw cabbage leaves in 1966 [1]. Studies from the previous years have shown the effect of drinking cabbage juice on gastric ulcers treatment [2]. Vitamin U is characterized by its antioxidant, anti-inflammatory and UVA and UVB-protective properties [1]. It can be a potential antioxidant and gastrointestinal additive preventing gastric and duodenal ulcers and it may relieve symptoms of gastroesophageal reflux.

Edible oils, due to the presence of unsaturated bonds, are characterized by significantly short duration and oxidative susceptibility [3]. Despite the wide range of vegetable oils, rapeseed oil is of greatest interest.

The aim of the study was to determine the usefulness of Methylmethionine sulfonate as an antioxidant as well as to evaluate the oxidative stability of pressed rapeseed oil. The study included the determination of an appropriate dose of antioxidant in the range of 50–200 mg/kg and determination of its efficacy during heating at 40 0C and 1000C and UV irradiation of oil tests under conditions simulating its storage on a store shelf. The oil quality before and after thermal treatment was assessed by measuring anisidine, acid, peroxide, viscosity, color, fatty acid content and oxidation stability.

The results showed that the addition of Methylmethionine sulfonate slowed down the oxidation of the oil.

References: [1]. Won-Serk K at al (2015) Int J Mol Sci 16(8): 17088–17100.

[2]. Cheney G at al (1956) Calif Med 84(1): 39–42.

[3]. Bartkowiak-Fludra E at al. (2006) Bromatologia i Chemia Toksykologiczna 39(2): 127–133.

6th Central European Congress of Life Sciences Eurobiotech 2017

96 Abstracts

P3.35

P3.36

Different approaches of NGS reads correction for structural variant calling of cucumber somaclonal lines

Is transgenesis significantly altering the cucumber genome?

Agnieszka Skarzyńska, Paweł Osipowski, Magdalena Pawełkowicz, Wojciech Pląder Warsaw University of Life Sciences, Department of Plant Genetics Breeding and Biotechnology, Poland Keywords: NGS, cucumber, read correction, variant calling The development of next egeneration sequencing techniques allows to use of huge data sets and bioinformatics analysis for various genomic studies. Many bioinformatics tools are currently available, but it is essential to evaluate and select the best approach for a given data set. Herein we compare two commonly available programs for correction of sequencing reads – ecc and bfc. We studied the predicted structural variants in cucumber somaclonal lines in comparison to the reference genome B10 in three parallel approaches: 1 – reference genome and mapped reads corrected with bfc, 2 – reference genome and mapped reads corrected with ecc, and 3 – reference genome corrected with ecc and mapped reads corrected with bfc. Results showed differences between read correction approaches.

Selection of proper tool for NGS read correction allows for reduction of false-posed polymorphisms.

Acknowledgements This work was supported by grant from National Science Centre 2013/11/B/ NZ9/00814

Agnieszka Skarzyńska, Magdalena Pawełkowicz, Maria Szwacka, Wojciech Pląder Warsaw University of Life Sciences, Department of Plant Genetics Breeding and Biotechnology, Poland Keywords: cucumber, transgenesis, thaumatin, resequencing, variant calling A common allegation against the use of genetically modified plants is the occurrence of unintended effects associated with the integration of the transgene into the genome. In this project we tested how the introduction of the thaumatin gene into the cucumber genome affects the whole genome. For this purpose we conducted resequencing of three transgenic lines with Illumina HiSeq 2000 and performed variant calling in comparison to the reference genome B10. Results showed high number of insertion-deletion type polymorphism and SNP variants, and the number of selected polymorphisms was similar for all analyzed lines. Identified changes in both coding and non-coding regions could be the effect of transgene integration. Genes and promoters within which changes have been identified will undergo further analysis.

Acknowledgements This work was supported by grant from National Science Centre 2013/11/B/ NZ9/00814.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 97

P3.37

P3.38

Determination of cannabinoids content in water extracts prepared from fibre type hemp (Cannabis sativa L.)

Novel primer sets for identification of Tapesia yallundae, T. acuformis and Rhizoctonia sp. based on PCR.

Milena Szalata, Karolina Wielgus

Hubert Szczerba1, Anna Kot1, Agnieszka Ostrowska1, Michał Nowak 2, Marta Muszyńska1, Adam Kuzdraliński1

Institute of Natural Fibres & Medicinal Plants, Department of Biotechnology, Wojska Polskiego 71b, 60-630 Poznan, Poland Hemp (Cannabis sativa L.) has been cultivated since ancient times for fibres, oil and medicines. The most known compounds responsible for pharmacological properties are Δ9-tetrahydrocannabinol (THC) responsible for psychoactive properties and non-psychoactive cannabidiol (CBD).

Among the most popular preparations made from narcotic type of C. sativa were water extracts prepared by infusion in cold and hot water. The aim of the work was preparation of water extracts from fibre type hemp and evaluation if water extracts contained cannabinoids. For extraction were used three hemp monoecious varieties allowed to cultivation in Europe. The hemp panicles containing 2.42–3.61% CBD and 0.6–0.13% THC in dried plant were extracted with hot water (boiling, 3 min.) and cold water (shaking, 100 rpm, 24 h). Generally extraction with cold water showed significantly lower content of CBD and THC (0.50 μg/ml and 0.05 μg/ml respectively) in obtained extract then in hot water extract (4.09 μg/ml and 1.43 μg/ ml respectively). There was not observed significant influence of variety on cannabinoids content. The experiment showed that extraction of CBD and THC with hot water is more efficient although cold water extracts also contained cannabinoids. Prepared water extracts potentially have pharmacological properties.

Acknowledgements Presented work was financially supported by The National Centre for Research and Development (grant number INNOMED/I/11/NCBR/2014).

University of Life Sciences in Lublin, Faculty of Food Science and Biotechnology, Skromna 8 Street, 20-704 Lublin, adam.kuzdralinski@up.lublin.pl; 2University of Life Sciences in Lublin, Institute of Plant Genetics, Breeding and Biotechnology, Akademicka 13 Street, 20-950 Lublin 1

Fungi from the genus Rhizoctonia are responsible for brown patch, root rot and many other plant diseases causing especially significant wheat production loss, including Poland. However, Tapesia yallundae and T. acuformis are responsible for eyespot disease, also called as strawbreaker. Large losses caused by these fungi and problems with their morphological identification make that designing new molecular tests for its early identification based on DNA is necessary.

The aim of this work was to develop new PCR-based methods for Tapesia yallundae, T. acuformis and Rhizoctonia sp. identification. The study was conducted on environmental samples – stem bases of common wheat (Triticum aestivum L.) with visual symptoms of diseases caused by these fungi from across the Poland, collected in the 2014/2015 growing season.

The first step in designing species-specific oligonucleotides was the analysis of genetic resources deposited in NCBI GenBank. On this basis, we selected genes suitable to design primers for the tested species. Subsequently, we analyzed the intraspecific genetic variability of selected genes. This analysis allowed us to choose regions in selected genes that were suitable for the identification using oligonucleotides. We designed ten primer sets for Rhizoctonia sp., thirteen and four primer sets for Tapesia yallundae and T. acuformis, respectively. The initial screening test showed that four primer sets for Rhizoctonia sp., eight for T. yallundae and four primer sets for T. acuformis were species-specific. The amplification conducted on environmental samples with the use of chosen primer sets indicated the presence of pathogen in 100% of them. In the second step, we will perform sequencing of selected amplicons. Subsequently, we will chose the most specific primer sets and validate them on more environmental samples.

New molecular tests will allow to identify the pathogens and determine their presence at an early stage of infection. In consequence, it will promote the preventive use of pesticides.

6th Central European Congress of Life Sciences Eurobiotech 2017

98 Abstracts

P3.39 Insights into unequal distribution of norflurazon through the wheat seedlings Kamil F. Trzebuniak, Beata Myśliwa-Kurdziel Department of Plant Physiology and Biochemistry; The Faculty of Biochemistry, Biophysics and Biotechnology; Jagiellonian University, Poland Keywords: Wheat, norflurazon, oxidative stress Norflurazon is a herbicide widely used to control weed.

It is also applied in research concerning plant pigments or oxidative stress. It works by inhibiting the phytoene desaturase. As a result the carotenoid biosynthesis pathway is arrested. When the plant is exposed to sunlight it is unable to dissipate the excess energy and photobleaching occurs. There are multiple studies on norflurazon effect on plants but researchers have focused on whole tissues like roots or leafs. In our research we study the regulation of photosynthetic pigment biosynthesis which include the influence of norflurazon on plant greening.

We used fluorescence spectroscopy to measure difference in the greening process of wheat in the leaf base, middle and tip. Additionally we acquired fluorescence photographs of the seedlings and determined their maximum photosystem quantum yield. We have also determined the actual concentration of norflurazon in different leaf parts using chromatographic methods.

Our findings show that the effects and distribution of norflurazon vary greatly through the leaf. The base is mostly influenced, while the tip remains almost unaffected. We have shown that when using norflurazon in research more information should be provided about exact parts of the affected tissue and actual norflurazon concentration should be measured.

Acknowledgements This work was financially supported by grant UMO-2013/10/E/NZ3/00748 from the Polish National Science Centre

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 99

Microbial elimination of deleterious compounds Keynote lecture

Lectures

L4.1

L4.2

Legacy and emerging persistent organic pollutants of foods and ambient environment Jerzy Falandysz

Diversity of endophytic bacteria from Miscanthus exposed to diclofenac and sulfamethoxazole mixed pollution. A cultivation dependent vs a cultivation independent approach

Laboratory of Environmental Chemistry & Ecotoxicology, University of Gdańsk

Anna Węgrzyn1, Viviane Radl2, Andrés Sauvêtre2 and Peter Schröder2

Perfluorinated and polyfluorinated aliphatic substances (PFASs), flame retardants and especially brominated flame retardants (BFRs), polybrominated dibenzo-p-dioxins (PBDDs)/polybrominated dibenzofurans (PBDFs) and Bisphenol A (BPA) and its substitutes are classified amongst persistent organic pollutants (POPs) of foods and environment that became actually a challenging problem for environmental sciences, analytical chemistry, toxicology, epidemiology, environmental policy and industry. Some of those classes of compounds has been manufactured from a several decades while their occurrence in the environment and foods as toxic contaminants as well accumulation in human and animals has been discovered relatively late, while some new substitutes with similar chemical, physical and biological properties has been introduced recently.

Highly toxic PBDDs and PBDFs (also polybrominated biphenyls, PBBs) can be found as by side impurities in brominated flame retardants, e.g. polybrominated diphenyl ethers (PBDEs) while can be spontaneously formed and released in high temperature processes related to fires, combustion, waste incineration and metals reclamation in presence of bromine donors such as brominated flame retardants and materials and products contaminated with BFRs. Bisphenol A and large number of substitutes for this compound (e.g. Bisphenol F, Bisphenol S, Bisphenol Z and other compounds) has been manufactured in large quantity and used in plastics, epoxy resins and many other materials and consumer products. Perfluorinated- and polyfluorinated substances including perfluorinated carboxylic acids (PFCAs), perfluorinated sulphonic acids (PFSAs) and a vast number of other highly fluorinated moieties has been widely used from several decades in large quantity and found a vast number of industrial appliances and in consumer products. An example is perfluorooctanoic acid (PFOA) and perfluorosulphonic acid (PFOS) and their sodium salts which recently become internationally prohibited, while many new such compounds were manufactured. Many individual compounds from the mentioned above classes of POPs become nowadays a common contaminates of foods and environment that are noxious for human and animal life. Nevertheless, still is deficit of toxicological, environmental, epidemiological and other data enabling better understanding the environmental fate and biological impacts of the those widespread contaminants and many information comes far late from the time of their first commercial use. One of key questions is how is possible a safe elimination of those legacy and emerging persistent organic pollutants (largely halogenated compounds) in waste water after sewage treatment, utilization of a municipal and industrial wastes or in preventing drinking water, food and environment pollution.

Silesian University of Technology, Environmental Biotechnology Department, Poland; 2Helmholtz Zentrum München German Research Center for Environmental Health, Research Unit Comparative Microbiome Analysis, Germany 1

The consumption of drugs has resulted in a significant contamination of environment with pharmaceuticals, including diclofenac (DCF) and sulfamethoxazole (SMX).

Miscanthus can be successfully used in constructed wetlands and the endophytic bacteria may play an important role in the wastewater treatment processes. Therefore, this research focuses on the effect of DCF and SMX on the diversity of endophytic bacteria from Miscanthus. The cultivable community was identified using cultivation-dependent isolation, while the total bacterial community was investigated using Illumina sequencing technology. For both procedures, differences in endobacterial diversity in samples obtained from plants non-exposed and exposed to DCF and SMX were revealed. In non-exposed roots, the class of Alphaproteobacteria was represented by the higher number of isolates. In samples exposed to DCF and SMX, the most abundant phylum was Actinobacteria and the enrichment of genera Streptomyces, Microbacterium and Glycomyces was noticed, while the abundance of the Proteobacteria was reduced. Some of that results were coherent with the data from pyrosequencing. The lowest representation of Alphaproteobacteria was observed in samples exposed to DCF and SMX, while an increasing presence of Streptomyces was noticed. The abilities of isolates to DCF transformation were also examined and the highest transformation rate was observed for Streptomyces curacoi (99.9%) and Microbacterium flavescens (64%).

6th Central European Congress of Life Sciences Eurobiotech 2017

100 Abstracts

L4.3

L4.4

Lipidomic adaptations in the filamentous fungus, auxin herbicides degrader

Environmental impact of herbicidal ionic liquids – from synthesis to advanced field studies

Przemysław Bernat*, Justyna Nykiel-Szymańska, Paulina Siewiera, Julia Dackowa, Monika Pietrzak

Ł. Chrzanowski

Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha Street 12/16, 90-237, Lodz, Poland Auxinic herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) are widely used in agriculture to selectively control broadleaf weeds in cereal crops. These pesticides have been the most extensively used herbicides worldwide for more than 70 yr. Many bacterial degradation studies dealing with 2,4-D have been reported in the literature. However, only a few of these investigations have involved fungi.

In our study, we observed that the filamentous fungus, Umbelopsis isabellina,eliminated 2,4-D and MCPA added at 25 mg L−1. Because 2,4-D and MCPA are highly lipophilic weak acids, to investigate the response of the fungus to the herbicides, a comparative lipidomics approach was employed using the gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry techniques. Lipids from fungal biomass were extracted using Folch procedure. Profiling of fungal lipids leading to the identification of 89 species of phospholipids, glycolipids and sterols, which were quantified through multiple reaction monitoring method. Herbicides caused a decline in the amounts of many molecular species of phosphatidylethanolamine and an increase in the levels of phosphatidylcholine. In the presence of the pesticides, we observed that the amounts of gamma-linoleic acid and the lipids species containing this acid decreased.

Faculty of Chemical Technology, Poznan University of Technology, M. Skłodowskiej-Curie 2, 60-965 Poznan, Poland Herbicidal ionic liquids (HILs) are a novel group of organic salts which exhibit numerous advantages compared to conventional herbicides. HILs are less volatile, more soluble in water and usually more active at lower concentrations against common weed species in comparison to parental herbicides, such as 2,4-D, MCPA, dicamba or glyphosate. Although HILs exhibit excellent herbicidal activity, their potential widespread in agriculture may result in contamination of the environment. This study provides a comprehensive summary of a 5-year research conducted on novel HILs. Several HILs will be presented with respect to their efficiency against common weed species in both greenhouse and field studies. Additionally, evaluation of their biodegradability as well as toxicity towards a wide range of organisms will be discussed. To assess the actual biodegradability of the studied compounds, experiments conducted with C13 labelled HILs in both aquatic and soil systems were performed. Moreover, Next-Generation Sequencing studies were conducted in order to investigate the influence of HILs on the structure of autochthonic soil bacterial community during field studies.

Acknowledgements This study was supported by the National Centre for Science in Krakow, Poland, Poland (Project No. 2015/19/B/NZ9/00167).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 101

L4.5

Posters

The impact of xylose based surfactants on environmental microorganisms

P4.1

Amanda Pacholak1, Wojciech Smułek1, Agata Zdarta1, Zuzana Hricovíniová2, Ewa Kaczorek1 Institute of Chemical Technology and Engineering, Poznan University of Technology, Berdychowo 4, 60-965 Poznan, Poland; 2 Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia 1

Sugar based surfactants with alkyl chain belong to the group of non-ionic, synthetic surface active agents. Their synthesis is based on renewable sources, hence they are described to be the low-cost materials. Moreover, the surfactants are considered as low-toxic compounds with high level of surface activity and effective emulsification properties. Therefore, they have a range of applications in food industry, cosmetics and pharmaceuticals. The wide use of these surfactants might result in the pollution of waters and soils. Thus, their effect on environmental microorganisms should be analysed.

The aim of the study was an examination of the influence of four alkyl D-xylosides, varying in the chain length, on environmental bacteria species. The experiments performed include the determination of bacterial membrane permeability and the hydrophobicity of cell surface (CSH).

The results show that the bacteria are mostly characterized by hydrophobic properties. However, there is no direct correlation between the chain length and the modification of CSH. The lowest CSH has Achromobacter sp. strain cultivated on 1-nonyl D-xyloside, however the highest value has Aeromonas sp. grown on 1-octyl D-xyloside.

Regarding to the results obtained for membrane permeability, the values mostly increase with the increase of the hydrophobic chain length in surfactant.

Acknowledgements This study was supported by Poznan University of Technology, grant No. 03/32/ DSMK/0721 and Slovak Grant Agency VEGA, grant No. 2/0100/14

Bioremediation of model post-fermentation sludge liquors with bacteria and yeasts Małgorzata Hałat-Łaś1, Paweł Kaszycki1, Przemysław Malec P.2, Sebastian Borowski3, Jan Burczyk4 Unit of Biochemistry, Institute of Plant Biology and Biotechnology, University of Agriculture in Krakow, Poland; 2 Department od Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophisics and Biotechnology, Jagiellonian University, Poland; 3Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Poland; 4Laboratory of Biotechnology, Cieszyn, Poland; 1

Methane fermentation during biogas production generates digester supernatants (post-fermentation liquids) whose properties depend on substrates used for anaerobic bioprocess. Although sludge liquors may differ in their susceptibility to bioremediation they all cause risk of water pollution and eutrophication. They can be categorized into two types: (A) exhibiting high content of ammonia (NH4+) and low chemical oxygen demand (COD), and (B) with both high NH4+ and COD levels.

The aim of the work was to evaluate application of bacterial consortia and yeast for treatment of two model post-fermentation liquids. Both were produced with a laboratory-scale anaerobic digester system upon biomethanization of sewage sludge (to represent type A, COD = 1220 mg O2/l, NH4+ = 860 mg/l) and slaughterhouse waste (type B, COD = 15570 mg O2/l, NH4+ = 1780 mg/l). Bacterial biocenoses were either the wastewater autochthons or the ZB-01 specialized aerobic community and the yeasts were represented by four different species. The microorganisms were tested for their tolerance towards the sludge liquors by monitoring cell survival.

Both the effluent indigenous bacteria and the ZB-01 allochthons proved capable of ammonium removal (the 7-d efficiency of 84% and 54% for A and B, respectively) whereas the yeasts were found less efficient (below 50%).

It is concluded that selected bacterial consortia may be used to construct activated sludges for aerobic treatment of eutrophic digester wastewaters.

6th Central European Congress of Life Sciences Eurobiotech 2017

102 Abstracts

P4.2

P4.3

Enzyme activity in petroleum contaminated soil as an indicator of physiology state of indigenous microorganisms in the context of biodegradation

Surface charge dependent fungistatic proprieties of silver nanoparticles

Pola Lomza, Lukasz Drewniak

Ewelina Matras1, Magdalena Oćwieja2, Anna Gorczyca1 University of Agriculture in Cracow, Department of Agricultural Environment Protection, Poland; 2Polish Academy of Sciences, Jerzy Haber Institute of Catalysis and Surface Chemistry, Poland

1

Laboratory of Environmental Pollution Analysis, Department of Biology, University of Warsaw, POLAND Keywords: soil, hydrocarbons, contamination, enzyme activity, microorganisms, bioremediation Petroleum release from leaking fuel storage tanks is one of a main reason of soil contamination with hydrocarbons and serious ecological risk. Among various technologies used to removal of petroleum pollutants bioremediation seems to be an environmental friendly, effective and economic solution. The aim of this study was to examine capacity of microorganisms to produce enzymes that play a key role in metabolic pathways of hydrocarbon biodegradation as lipases, dehydrogenases, cellulases. Activity of mentioned enzymes are a proximate indicator of physiology state of autochthonous microbial community and an indirect factor of environmental pollution. The contaminated soil was tested for the most probable number (MPN) of heterotrophic bacteria and enzymes content. MPN and enzyme activity of bacteria able to degradation of different alkanes were estimated. The results showed that MPN of heterotrophic bacteria in contaminated soil samples was comparable with control (non-polluted) sample. Disparate enzyme activity in tested soil samples were higher than in control sample.

Keywords: nanoparticles, silver, fungi, toxicity Two types of silver nanoparticles (AgNPs) of diverse surface charge were synthesized in a chemical reduction method using cysteamine and trisodium citrate as stabilizing agents. The obtained AgNPs suspensions and solution of reagents were applied into Fusarium avenaceum and F. equiseti conidia solutions to assess their fungistatic proprieties. The activity of compounds in concentration 10, 5 and 2.5 ppm was checked via fungi inoculations, culture sporulation and TEM cells visualization. The fungi inoculation demonstrated that silver nitrate(V) and its mixture with trisodium citrate at 10 and 5 ppm after 24 h were fungistatic for both species. AgNPs synthesized using trisodium citrate was fungistatic at 10 ppm after 24 h but at 5 ppm only after 240 h. AgNPs synthesized using cysteamine inhibited inoculotion at 10 ppm after 240 h, but only F. equiseti species. Sporulation was strongly differentiated without significant reaction effect. TEM imaging confirmed the results of inoculations tests. The assessed AgNPs damaged the conidia membranes and penetrated into the cells but not in all cases of spore treatment.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 103

P4.4

P4.5

Rapid eradication of plant pathogenic bacteria by direct current atmospheric pressure glow discharge generated in contact with a flowing liquid cathode

Elimination of 2,4-dichlorophenoxyacetic acid by the microscopic filamentous fungus Umbelopsis isabellina

Agata Motyka1, Anna Dzimitrowicz2, Piotr Jamroz2, Ewa Lojkowska1, Pawel Pohl2, Wojciech Sledz1

Justyna Nykiel-Szymańska, Paulina Siewiera, Julia Datskowa, Przemysław Bernat

Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, Department of Biotechnology, Poland; 2 Wroclaw University of Science and Technology, Department of Analytical Chemistry and Chemical Metallurgy, Poland

Institute of Microbiology, Biotechnology and Immunology, Department of Industrial Microbiology and Biotechnology, University of Lodz, Poland

1

Plant pathogenic bacteria are responsible for significant economic losses in crop production worldwide. As the applied control methods mainly involve prevention, development of the atmospheric pressure plasma-based system for potent eradication of phytopathogenic bacteria from contaminated liquid disposals might notably contribute to limitation of their spread. A high-throughput system based on direct current atmospheric pressure glow discharge (dc-APGD) generated in contact with flowing bacterial suspension acting as a liquid cathode was designed and applied for eradication of Dickeya solani, Xanthomonas campestris pv. campestris, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium atrosepticum and Clavibacter michiganensis subsp. sepedonicus. Reduction in CFU ml-1 in a range of 99.96–100.00% was achieved in less than 1 min per 10 ml of OD600 ≈ 0.1 bacterial suspension. Besides, physicochemical parameters of the applied dc-APGD in addition to characteristics of the plasma-treated liquids, putatively crucial for the observed antimicrobial activity were defined. In conclusion, some of the proposed applications for herein described simple, universal, rapid, and cost-effective dc-APGD-based plasma-reaction system include elimination of plant pathogenic bacteria from industrial and agricultural wastewaters, wastes from 1–4 biosafety level laboratories and sterilization of any other liquid disposals putatively contaminated with phytopathogens. This sterilization method is the subject of patent application no. P.419246

2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic plant auxin that is widely employed in many crops. Penetration of the compound into surface water and groundwater poses a threat to living organisms. Microbial degradation has a crucial relevance in the elimination of 2,4-D from the environment. In the polluted areas degradation processes are carried out by selected microflora capable of surviving in the presence of toxic substances. The aim of the study was to examine the microscopic filamentous fungus Umbelopsis isabellina, which is able to degrade 2,4-D. Liquid cultures of U. isabellina with different concentrations of 2,4-D (10–100 mg/L) were incubated on a rotary shaker (160 rpm) at 28oC. The quantitative and qualitative analyses of the pesticide distribution and products resulting from its biotransformation were performed using LC/ MS/MS and GC/MS/MS chromatography techniques. It has been shown that the tested strain is characterized by a high tolerance (80–95%) for the presence of 2,4-D in the culture in the concentration range of 10–100 mg/L. The fungus U. isabellina is capable of effective removal of different concentrations of the herbicide combined with 6-hydroxy-2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenol formation. The obtained results could be considered as evidence of a reduction in the risk from 2,4-D to the natural environment.

Acknowledgements This study was supported by the Grant of the National Centre for Science in Cracow, Poland, No 2015/19/B/NZ9/00167

6th Central European Congress of Life Sciences Eurobiotech 2017

104 Abstracts

P4.6

P4.7

The effect of saponins on biodegradation of clotrimazole by activated sludge

Efficient dibutyltin (DBT) elimination by the microscopic fungus Metarhizium robertsii under conditions of intensive aeration and ascorbic acid supplementation

Amanda Pacholak1, Joanna Simlat1, Wojciech Smułek1, Agnieszka Zgoła Grześkowiak 2, Ewa Kaczorek1 Institute of Chemical Technology and Engineering, Poznan University of Technology, Berdychowo 4, 60-965 Poznan, Poland; 2 Institute of Chemistry and Technical Electrochemistry, Poznan University of Technology, Berdychowo 4, 60-965 Poznan, Poland 1

Clotrimazole is one of the most common antifungal agents used, among others, in human and veterinary medicine. The widespread usage of clotrimazole may lead to substantial amounts of its residues in the environment.

The presence of this fungicide in the environment might cause adverse impact on fauna and flora. One of the efficient and environmentally friendly methods of the azole fungicides removal from the environment is a biodegradation. The process is usually slow and ineffective, thus it should be enhanced. A promising factor might be a presence of surface active agents.

The aim of the study was the biodegradation of clotrimazole by activated sludge coming from the municipal sewage treatment plant. An important part of the experiments was to investigate the impact of two plant extracts containing saponins on the process of biodegradation. Moreover, a characteristic of activated sludge and isolation of microorganisms with the potential ability to degrade clotrimazole was performed.

The 40-days biodegradation tests clearly showed that the activated sludge has demonstrated high ability to biodegrade clotrimazole, although its total degradation has not been achieved. There was no significant influence of saponins on the process. However, the biodegradation could be an efficient method of clotrimazole removal, both in the systems with and without the addition of surfactants.

Acknowledgements This study was supported by Poznan University of Technology, grant No. 03/32/ DSPB/0600

Paulina Siewiera, Justyna Nykiel-Szymańska, Przemysław Bernat Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz; 12/16 Banacha street, 90-237 Lodz.

Keywords: dibutyltin utilization, intense aeration, hyphae morphology, ascorbic acid, malondialdehyde Dibutyltin (DBT) is a useful heat stabilizer of polyvinyl chloride. Because of its widespread use, the compound has become a global pollutant. One of the mechanisms of DBT toxicity is induction of reactive oxygen species.

In this study, an attempt was made to enhance DBT elimination by the fungal strain Metarhizium robertsii.

We observed enhanced fungal growth in the bioreactor (pO2 ≥ 20%) compared to flask cultures (μmax increased from 0.061 to 0.086 h−1). Moreover, under aerated conditions, M. robertsii mycelium with hairy morphology biodegraded DBT (20 mg l−1) 10-fold faster in the bioreactor than in the flask cultures. Monobutyltin (MBT) was detected as a by-product of dibutyltin debutylation. In order to protect fungal cells from oxidative stress caused by DBT presence, ascorbic acid (AA, 20 mg l−1) was applied. Supplementation with AA resulted in a 3-fold acceleration of MBT removal during the first 7 h of incubation. Using the HPLC-MS/ MS technique, a quantitative analysis of malondialdehyde (MDA), a marker of oxidative stress, was performed. In the AA presence, a decrease in the MDA amount (about 45%) was observed compared to the case with fungal cells exposed to DBT alone.

An additional supply of oxygen led to a hairy morphology for the M. robertsii hyphae contributing to intensive fungal growth and finally improved butyltin biodegradation. Supplementation of the growth medium with ascorbic acid protects fungal cells exposed to DBT from oxidative stress.

Acknowledgements This study was supported by the National Science Centre, Poland (Project No. 2015/19/N/NZ9/00459).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 105

P4.8

P4.9

Chromium (VI) toxicity to tolerant fungal strains under sulfates starvation

The enhanced biodegradation of brominated diphenyl ether by environmental bacteria

Mirosława Słaba, Justyna Nykiel-Szymańska, Aneta Zawierucha, Sylwia Różalska

Wojciech Smułek, Agata Zdarta, Ewa Kaczorek

University of Lodz, Faculty of Biology and Environmental Protection, Department of Industrial Microbiology and Biotechnology, Poland Chromium is a metal widely used in various industries.

It is estimated that the annual global chromium consumption in industrial processes remains at the level of 12.5 million tons. The sources of chromium emission to the environment are mainly combustion processes and industrial production. Its toxicity is closely dependent on the valence and the pH of the environment. Chromium (III) is poorly soluble and low toxic, whereas chromium (VI) is highly soluble and toxic, a hundred times more toxic than Cr(III). It also shows mutagenic and carcinogenic properties. Cr(VI) present in the form of chromates or dichromates, easily penetrates into the cells via sulfate transport channels, due to the similar structure of these anions.

The aim of the study was to investigate the toxic effect of chromium (VI) on tolerant fungal strains, isolated from metal and dye polluted environments in the conditions of sulfates starvation. Among all tested strains , 7 were able to grow in the presence of chromium (0.5 g/l) up to 50% in comparison to control cultures. Chromium uptake, catalase activity, ROS and lipid peroxidation level in chromium stressed and sulfate starved mycelia were measured.

Obtained results show that sulfate starvation can affect chromium toxicity in tolerant fungal strains.

Institute of Chemical Technology and Engineering, Poznan University of Technology, ul. Berdychowo 4, 60-965 Poznań, Poland Keywords: biodegradation, brominated diphenyl eters, cell surface hydrophobicity The brominated derivatives of diphenyl ether have found many applications in production of multi-functional composites and they are recognised as useful flame retardants. Unfortunately, one of their main disadvantages are high resistance to biodegradation processes and high bioaccumulation. Hence, there is necessity of intensive research aimed at effective biodegradation brominated diphenyl ethers.

The aim of the study was to determine the influence of addition of co-substrates on biodegradation brominated diphenyl ether by several environmental bacteria strains.

The decay of the degraded compounds in liquid bacteria culture was determined with gas chromatography. Moreover, the modifications in cell surface properties, e.g. cell surface hydrophobicity, was also measured to evaluate cell adaptation mechanisms.

The conducted research revealed the positive effect of the cultures supplementation with co-substrates in the case of all tested strains. During the 7 days the strain from the Pseudomonas genera degraded nearly 20% of 4-bromodiphenyl ether. The same strain in the cultures containing also phenol, biodegraded over 50% of the compound. Additionally, the observed changes in cell surface properties indicated the significant interaction of the co-substrate.

Acknowledgements This study was supported by the National Centre of Science (Poland) awarded by the decision number DEC-2015/19/N/NZ9/02423

6th Central European Congress of Life Sciences Eurobiotech 2017

106 Abstracts

P4.10

P4.11

Cytosolic fraction proteins of Metarhizium robertsii IM 2358 involved in the degradation of 4-n-nonylphenol

Impact of tributyltin (TBT) on Cunninghamella echinulata IM 2611 proteome

Anna Litwin, Adrian Soboń, Sylwia Różalska University of Łódź, Faculty of Biology and Environmental Protection, Institute of Microbiology, Biotechnology and Immunology, Department of Industrial Microbiology and Biotechnology, Poland Nonylphenols possess an ability to interfere with the hormonal systems of living organisms. These toxic compounds frequently occurring in the environment exhibit high resistance to degradation processes. Filamentous fungi from the genus Metarhizium are able to degrade nonylphenols, but the mechanisms and enzymes responsible for these processes are not fully understood yet.

The aim of the study was to search for proteins involved in nonylphenol degradation.

Biomass separated from the culture medium was disintegrated using glass beads before cytosolic fraction isolation. Unidirectional electrophoresis was performed, protein bands were subjected to in-gel digestion with trypsin and the resultant peptides were analysed by MALDI– TOF–MS. The performed analysis of the resultant peptides by MALDI–TOF–MS and Protein Pilot and Mascot database searches of the MS spectra, revealed the most significant expression of oxidoreductase activator proteins (myosin-cross-reactive antigen-like protein) and nitroreductase in the bands with the protein profile of 4-n-NPgrown cells. These proteins might be involved in nonylphenol degradation.

Adrian Soboń, Rafał Szewczyk, Jerzy Długoński University of Łódź, Faculty of Biology and Environmental Protection, Institute of Microbiology, Biotechnology and Immunology, Department of Industrial Microbiology and Biotechnology, Poland.

Proteomics is a dynamically growing field in systems biology and an important tool used for a better understanding of the biology of organisms and their response to environmental conditions. Tributyltin (TBT) is a widespread environmental toxicant commonly used in anti-fouling agents for boats, as well as a byproduct from several industrial processes. TBT is very toxic and several effects that have been reported include: (i) interference with biological membranes and disturbing their integrity and physiological functions (ii) TBT acts as an endocrine disruptor; and (iii) cell growth inhibition in bacteria and fungi. We previously described the filamentous fungus C. echinulata, which was capable of effective TBT (5 mg L ‑1) biodegradation (Soboń et al. 2016).

The aim of the study was the characterization of TBT impact and its removal by C. echinulata IM 2611 using proteomics tools.

The intracellular proteins were isolated and separated using 2-D SDS-PAGE technique. Protein spots were subjected to in-gel digestion with trypsin and the resultant peptides were analysed by MALDI–TOF/TOF. The conducted proteomic analysis revealed the significant expression of many proteins involved in proper cell functioning. Moreover, significant upregulation of enzymatic antioxidants were observed. The results revealed that TBT had a significant wide-ranging negative impact on C. echinulata proteome.

Acknowledgements This study was supported by the grant of the National Science Centre, Poland (Project No. UMO-2014/13/N/NZ9/00878).

References Soboń A et al (2016) Int Biodeterior Biodegr 107:92–101

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 107

P4.12

P4.13

Fungal metabolism of carbazole, quinoline and their derivatives with pharmacological activity

Hydrocarbons sorption on natural and synthetic materials

Katarzyna Zawadzka, Aleksandra Felczak, Marta Nowak, Katarzyna Lisowska

Agata Zdarta, Wojciech SmuĹ&#x201A;ek, Ewa Kaczorek

Department of Industrial Microbiology and Biotechnology, University of Lodz, Poland Keywords: N-heterocyclic compounds, microbial degradation, carbazole, quinoline Various groups of drugs, such as receptor antagonists and antibiotics contain N-heterocyclic rings like carbazole or quinoline in their structures. Pharmaceuticals are commonly qualified as emerging pollutants. Despite low levels of their residues in surface water, they can influence aquatic organisms and humans because of their constant release to the environment. Thus, microbial degradation of xenobiotics may be an efficient method for the removal of pharmaceuticals from environment.

The aim of this study was investigating the ability of Trichoderma sp. to degrade carbazole, qiunoline and their derivatives (ondansetron and danofloxacin).

Qualitative and quantitative analysis of carbazole, quinolone and their pharmacological derivatives elimination by tested filamentous was done using GC-MS and LC-MS/MS. Toxicity of tested compounds and their metabolites was assessed by P. putida growth-inhibition test.

Trichoderma sp. eliminated all tested compounds from its growth medium. In the case of carbazole and ondansetron, about 100% of the initial amounts of the xenobiotics was degraded. In samples containing quinoline and danofloxacin the elimination reached the value of 80%. Also metabolites of tested compounds were identified.

Poznan University of Technology, Department of Chemical Technology, Poland Keywords: hydrocarbons, sorption, sand, silicone o-rings Hydrocarbons are common pollution in the environment and they possess threat for humans due to their relatively high toxicity. Significant part of the hydrocarbon pollutants can migrate in the soil and adsorb on different soil fractions. In this research we examined kinetics of sorption and desorption of selected hydrocarbons on natural (sand) and synthetic (silicone o-rings) materials to compare their capacity and properties. We examined also bioavailability of the hydrocarbons adsorbed on the selected materials.

Kinetics of sorption and bioavailability were examined using GC/GC-MS andthe kinetics measurements results were fitted to Langmuir and Freundlich isotherms to describe sorption and desorption on the used materials. Comparison of this two materials, as a hydrocarbon carriers revealed high capacity of silicone o-rings and capability of both used materials to adsorb at least 40% of total loaded hydrocarbons amount. These results open the prospect of using silicon o-rings as a stable hydrocarbon delivery system in laboratory culture for bioremediation research.

Acknowledgements This study was supported by The National Science Centre awarded by decisions number DEC-2015/19/N/NZ9/02422.

6th Central European Congress of Life Sciences Eurobiotech 2017

108 Abstracts

The tools of epigenetics Keynote lecture

Lectures

L6.1

L6.2

Crop improvement by targeted genome editing

HD2C histone deacetylase binds to SWI/SNF chromatin remodeling complex and act together to regulate Arabidopsis heat stress response

Jan Szopa University of Wroclaw Today’s crops appears to be the product of centuries of improvement through breeding. Crossing and selection of native traits was further extended by different kind of mutation, and recently complemented with genetic engineering, enabling the introduction of novel gene beyond that originally present. Recently the extension of crop improvement toolbox has been achieved. CRISPR and TALEN systems have emerged as potent biotechnological tools. It should be however pointed out that both technologies are very efficient for gene “loss of function” while gene replacement for “gain of function” requires more research. Thus, the engineered CRISPR/Cas system may serve as a suitable DNA-targeting module for gene activation-induced cytidine deaminase to catalyze site-specific mutagenesis. Use of other types of nucleotide-modifying enzymes certainly will expand the genome-editing repertoire. Very recently, several studies have proposed a targeted genome editing based on the plant treatment with short oligonucleotide sequences (OLIGO) that are homologues to the gene of interest. In most cases OLIGO silences the target gene by interference but in several cases target gene activation was reported. Most probably the effect of OLIGO depends upon targeted gene’s structural part. The recent data suggests that OLIGO technology is effective in the analysis of gene function as well as in the generating new types of plants.

Daniel Buszewicz1, Rafał Archacki1,2, Antoni Palusiński2, Maciej Kotliński2, Anna Fogtman1, Roksana Iwanicka-Nowicka1,2, Katarzyna Sosnowska1, Jan Kuciński2, Piotr Pupel3, Jacek Olędzki1, Michał Dadlez1,4, Aleksandra Misicka5,6, Andrzej Jerzmanowski1,2, Marta Kamila Koblowska1,2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland; 2 Laboratory of Systems Biology, Faculty of Biology, University of Warsaw, 02-106 Warsaw, Poland; 3 Department of Plant Physiology, Genetics and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland; 4 Institute of Genetics and Biotechnology, University of Warsaw, 02-106 Warsaw, Poland; 5 Department of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 00-927 Warsaw, Poland; 6 Mossakowski Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, Poland 1

Histone acetylation is a reversible process that depends on the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Five classes of histone deacetylases exist in plants. HD2C, the most thoroughly studied member of the HD2 family, is involved in responses to salt and osmotic stresses and abscisic acid treatment. Studies in yeast and animals have revealed that HDACs often act as components of multiprotein complexes, including chromatin remodeling complexes (CRCs).

However, interactions between HDACs and CRCs in plants have yet to be demonstrated. Here, we present evidence for the interaction between Arabidopsis HD2C deacetylase and a SWI/SNF chromatin remodeling complex containing BRM ATPase as the catalytic subunit. Moreover, we reveal a novel function of HD2C as a regulator of the heat stress response. HD2C transcript levels were strongly induced in plants subjected to heat treatment and the expression of selected heat-responsive genes was up-regulated in a hd2c mutant, suggesting that HD2C acts to down-regulate heat-activated genes. In keeping with the histone deacetylase activity of HD2C, the altered expression of HD2C-regulated genes coincided in most cases with increased histone acetylation at their loci. Microarray transcriptome analysis of brm and hd2cmutants identified a subset of commonly regulated heat-responsive genes, and the effect of the double brm hd2c mutation on the expression of these genes was non-additive. Moreover, heat treated hd2c, brm and brm hd2c mutants displayed similar rates of growth retardation. Taken together, our findings suggest that HD2C and BRM act in a common genetic pathway to regulate the Arabidopsis heat stress response.

References Buszewicz D et al (2016) Plant Cell Environ Vol 39, Issue 10: 2108–2122

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 109

L6.3

L6.4

Oligodeoxynucleotides can regulate CHS gene expression in flax by changing DNA methylation in a sequence-specific manner

Significance of microRNA-199a-5p and microRNA199b-5p and their target genes in renal cancer cells

Magdalena Działo, Jan Szopa, Magdalena Żuk University of Wroclaw, Faculty of Biotechnology, Department of Genetic Biochemistry, Poland Chalcone synthase (CHS) has been recognized as an essential enzyme in the phenylpropanoid biosynthesis pathway. The CHS gene is one of the most intensively studied genes in flax. In our study, we investigated engineering of the CHS gene through genetic and epigenetic approaches. Considering the numerous restrictions concerning the application of genetically modified (GM) crops, the main purpose of this research was optimization of the plant’s modulation via epigenetics. In our study, plants modified through two methods were compared: a widely popular agrotransformation and a relatively recent oligodeoxynucleotide (ODN) strategy. In order to understand the molecular background of epigenetic variation in more detail and evaluate the use of ODNs as a tool for predictable and stable gene engineering, we concentrated on the integration of gene expression and gene-body methylation. The treatment of flax with a series of short oligonucleotides homologous to a different part of CHS gene isoforms revealed that those directed to regulatory gene regions (5′- and 3′-UTR) activated gene expression, while those homologous to a coding region may have a variable influence on its activity. Gene expression changes were accompanied by changes in its methylation status. However, only certain (CCGG) motifs along the gene sequence were affected. The obtained results suggest high specificity of ODN action and establish a potential valuable alternative for improvement of crops.

Bartlomiej E. Krazinski1, Jolanta Kiewisz1, Agnieszka Sliwinska-Jewsiewicka1, Anna E. Kowalczyk1, Janusz Godlewski1, Przemyslaw Kwiatkowski1, Zbigniew Kmiec12 1 Department of Human Histology and Embryology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn, Poland; 2Department of Histology, Medical University of Gdansk, Gdansk, Poland

MicroRNA-199a-5p (miR199a) and microRNA-199b-5p (miR199b) have been reported to be decreased in renal cell carcinoma (RCC) compared to normal kidneys. Thus, to analyze their role in the pathogenesis of RCC and to analyze their associations with the expression levels of their two target genes: discoidin domain receptor 1 (DDR1) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKBKB), in vitro culturing of RCC-derived Caki-1, Caki2 and ACHN and proximal tubule-derived HK-2 cell lines have been conducted. The levels of endogenous expression of miR199a and miR199b determined in analyzed cell lines were relatively low. Demethylation test indicated that hypermethylation of miRs loci is the most probable mechanism of their silencing. Transfections of Caki-1, Caki-2, ACHN and HK-2 cells with mimic oligonucleotides revealed that miR199a and miR199b can act as effective repressors of both DDR1 and IKBKB. Interestingly, all miR199a- and miR199b-transfected cell lines exhibited increased rate of proliferation. After invasion/migration assays, miR199a transfection was found to limit the invasive properties in Caki-1 cells while other cell lines reminded unaffected. Altogether, results of performed studies indicate that miR199a, miR199b and their putative target genes i.e. DDR1 and IKBKBcan take part in the pathogenesis of RCC but the current knowledge regarding their biological function in renal cancer cells requires further research.

6th Central European Congress of Life Sciences Eurobiotech 2017

110 Abstracts

L6.5

Posters

Methylation analysis of HLA-E transgene in the transgenic pigs

P6.1

Marlena Szalata1, Aleksandra Minkowska1, Patrycja Nycz1, Magdalena Hryhorowicz1, Agnieszka Nowak1, Joanna Zeyland1, Daniel Lipiński1, Ryszard Słomski1,2 Poznań University of Life Sciences, Department of Biochemistry and Biotechnology, Poland; 2Institute of Human Genetics, Polish Academy of Sciences, Poland 1

Epigenetics involves studies of changes in gene expression not connected with changes at DNA sequence level.

The best characterized epigenetic mechanism involved in control of gene expression is DNA methylation. Methylation of cytosines in CpG island within promoters usually causes gene silencing. In transgenic animals we are interested not only in introduction of transgene but also in its activity. We have prepared transgenic pigs with decreased recognition by human immune system by introduction of the human HLA-E gene under elongation factor 1 alpha promoter (EF-1α). Activity of HLA-E transgene was already confirmed on molecular level and flow cytometry.

The main purpose of this study was evaluation of specific methylation of HLA-E transgene. Genomic DNA was modified by bisulfite conversion and PCR products were analysed using pyrosequencing method. The most important steps were DNA isolation and DNA bisulfite conversion.

Pyrosequencing enabled quantification of the specific methylation level of transgene. The total level of methylation of the HLA-E transgene promoter was approximately 90% (range 67–100%) and the HLA-E gene 73.79% (29– 100%). This suggest, that level of methylation of transgene promoter does not affect directly its biological activity estimated by flow cytometry.

Financed by National Centre for Research and Development (no INNOMED/I/17 /NCBR/2014) within framework of INNOMED programm Development of an innovative technology using transgenic porcine tissues for biomedical purposes. Acronym MEDPIG

Deep sequencing of QTL-rich region on porcine chromosome 15 Piórkowska K.1, Żukowski K.2, Tyra M.2, Ropka-Molik K.1 Department of Animal Genomics and Molecular Biology, National Research Institute of Animal Production, 32-083 Balice, Poland; 2 Department of Animal Genetics and Breeding, National Research Institute of Animal Production, 32-083 Balice, Poland; 1

The subject of present study was to identified potential genetic markers in porcine chromosome 15 QTL-rich region for growth, meat quality and fat deposition traits by using targeted enrichment DNA sequencing method (TEDNA-seq) based on next-generation sequencing technology.

TEDNA-seq analysis was performed on blood samples collected from 16 pigs representing two breeds – Puławska and Polish Landrace (PL). These two breeds differ in term fat deposition and meat quality traits. The DNA libraries were prepared by using SureSelectXT NGS Target Enrichment (Agilent) and 1x tilling 127–135 Mbp SSC15 region (NC_010457.4) RNA probes. The analysis was performed on HiScan SQ (Illumina) in 75 pair-end cycles. GATK HaplotypeCaller with filtering: FisherStrand (FS) value>50, QualByDepth (QD) <2.0, RMSMappingQuality (MQ)40, Quality40 was use for identified gene variants in SSC15.

In the SSC15 QTL-rich region were identified over 32,000 SNPs and 6,000 INDELs in which 86% were counted into known polymorphisms. In Puławska pigs 9 unique missense variants in FN1, VIL1, CNPPD1, ENSSSCG00000027669, ANKZF1 and ENSSSCG00000016203 were observed. In turn, in Polish Landrace the unique inframe deletion in FAM134A gene was detected. The longest 54 bp, the non-microsatellite deletion was observed in PL in downstream gene region of RUFY4.

Acknowledgements This study was funded by National Science Center in Poland, Research Project No. 2013/09/D/NZ9/02452.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 111

Bacteriophages in Biotechnology Keynote lecture L7.1 Microbial response to environmental stress when starved of oxygen Jeff Cole School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK The general public take the supply of safe drinking water for granted, but immediately become alarmed by the emergence of a pathogen that can invade the human body by overcoming host defence mechanisms. Although the world’s largest biotechnology industry is waste water treatment, which is crucial for public health, it is the biopharmaceutical industry that generates more wealth. Red biotech is more lucrative than green biotech! Most laboratory research is completed with pure microbial cultures in carefully controlled incubators with excellent aeration.

Indeed, many industrial strains are grown in fermenters under conditions of severe oxidative stress. In contrast, survival in a water treatment plant or in the gastro-intestinal tract is far more challenging. In both of these natural environments, complex mixtures of microbes compete with each other for survival. Nutrients are often scarce, and the supply of oxygen is severely limited. Laboratory experiments with a rich broth in a shake flask are therefore largely irrelevant to how microbes survive in their natural environments. Remember the golden rule: rubbish in generates only rubbish out.

Both in the human body and in the outside world, bacteria must be able to respond to environmental stress, especially oxidative and nitrosative stress or oxygen starvation. The oxidative burst and the nitrosative burst are two major strategies used by the human body to protect us from invading pathogens. Successful pathogens have evolved survival mechanisms that can often be discovered by first determining how gene expression responds to each specific stress. Nitrosative stress is caused by the generation of nitric oxide either from nitrite, or by mammals from arginine catalysed by the inducible nitric oxide synthase.

Recent research has revealed how bacteria minimize nitrosative damage and reverse any damage as it occurs.

It involves a previously undiscovered pathway operating in a newly discovered biochemical cycle. They involve the hybrid cluster protein, which has a structure that is so far unique in nature, and transcription factors that regulate its synthesis. The response is similar in both the human intestine and in anaerobic soils and marine sediments.

Sulfate reducing bacteria are anaerobes that conserve energy released during sulphate reduction to sulfide, which is responsible for the unpleasant smell of black estuarine mud. They can also be isolated from human feces. Some sulfate reducing bacteria also reduce nitrate via nitrite to ammonia. Surprisingly, the ability to reduce nitrate is more common in the human body than in the outside world. Wherever they live, they are able to protect themselves against nitrosative stress. Our results show

how the expression of many genes in Desulfovibrio desulfuricans respond to the presence of nitrate or nitric oxide.

Nitrate reduction, nitrite reduction and nitrosative stress are tightly regulated in response to substrate availability.

Transcriptional responses to nitrate or nitric oxide are largely regulated independently, but are coordinated by three sets of transcription factors. Our data exposed many gaps in knowledge of the physiology and biochemistry of these anaerobic bacteria, but provide fascinating problems for future research.

Lectures L7.2 Auresine – new weapon against Staphylococcus Elżbieta Jagielska, Paweł Mitkowski, Izabela Sabała International Institute of Molecular and Cell Biology in Warsaw, Poland Keywords: autolysin, Staphylococcus, non-antibiotic treatment Autolysin called LytM is a peptidolytic enzyme cleaving cell walls of some of Gram positive bacteria, in particular Staphylococcus sp. including S. aureus leading to instant cell lysis. LytM is a member of the M23 zinc-dependent metallopeptidase family1. These enzymes are recognized primarily for their glycylglycine endopeptidase activity leading to lysis of Gram-positive bacterial cell wall through cleavage of cross-bridges in the peptidoglycan chains. Although LytM is abundant in S. aureus cell wall, its native form is inactive. Based on structural studies, the active form, called Auresine, has been engineered1. Auresine is extremely active, stable and affordable non-antibiotic weapon against Staphylococcus. The enzyme cleaves only staphylococcal cell walls, leaving natural microflora untouched. Moreover, Auresine is active in a wide range of temperatures (even on ice) and in low conductivity conditions (e.g. water)2. Auresine can serve as a selective, non-toxic, biodegradable antibacterial agent. It can be applied as additive to surface disinfectants, food biopreservatives, component of veterinary and human hygiene products for skin infection prevention and treatment, as well as a part of wound dressing*. Due to its specificity, Auresine may be also used as a part of diagnostic test.

References 1 Odintsov S et al (2004) JMB 335:775–85 2 Sabala I et al (2012) BMC Microbiology, 12:97 * data not published

6th Central European Congress of Life Sciences Eurobiotech 2017

112 Abstracts

L7.3

L7.4

Antistaphylococcal enzybiotics – structural bases of enzyme engineering

Analysis of phage tgl gene encoding a new candidate for an antistaphylococcal agent

Izabela Sabała, Elżbieta Jagielska, Paweł Mitkowski

Aleksandra Głowacka1, Magdalena L. Ulatowska1, Małgorzata B. Łobocka1,2

International Institute of Molecular and Cell Biology in Warsaw, Poland Keywords: Staphylococcus, lysin, enzybiotic “Enzybiotics” is a term used for lysins that act on bacterial cell walls degrading peptidoglycans what leads to instant cell death. Having defined specificity and exceptional efficiency, these enzymes are finding divers applications from laboratory use to antibacterial therapy.

We have focused our research on M23 metallopeptidases which cleave pentaglycine crossbridges characteristic for staphylococcal species. The members of this family are very common among phage lysins but can also be produced by bacteria as bacteriocins (lysostaphin from S. simulans) or autolysins (LytM from S. aureus).

We are using structural analysis of M23 enzymes to aid engineering of their crucial features, like activity, stability and specificity. Based on structure of latent LytM we have generated fully active enzyme1. The structure of mature lysostaphin2 guided us in designing chimeric enzymes with extended tolerance to environmental conditions3. The complex structures of LytM with substrate analogue4 and lysostaphin cell wall targeting domain with pentaglycine* provides solid bases for defining structural determinants of enzyme specificity and helps in engineering enzymes with extended range of targeted bacteria(*).

References 1 Odintsov S et al (2004) JMB 335:775–85 2 Sabala I et al (2014) FEBS J. 281(18):4112 22 3 Jagielska E et al (2016) Microb Drug Resist. 22(6):461–9 4 Grabowska, M et al (2015) Sci Rep 5:14833 *data not published

1 Institute of Biochemistry and Biophysics, PAS, Dept. Microbial Biochemistry, Poland; 2 SGGW, Autonomous Dept. of Microbial Biology, Faculty of Agriculture and Biology, Poland

Keywords: bacteriophage, endolysin, Staphylococcus aureus, bactericidal agent Endolysins are phage-encoded peptidoglycan hydrolases that are considered to be promising antibacterial agents.

Endolysin LysK encoded by polyvalent staphylococcal Twort-like phages has been well characterized and shown to have a strong antistaphylococcal activity (2). The lysis module that encodes LysK in the genomes of Twort-like phages is separated from the “host takeover module” by several genes of unknown function. One of them, designed by us as tgl, appears to encode a weak homolog of S. aureus virulence determinant (1). We cloned it in a shuttle vector under the control of an inducible promoter. Induction of tgl transcription caused the lysis of E. coli as well as S. aureus cells, indicating that Tgl protein may function as an additional lysin capable of crossing the cytoplasmic membrane in a holin-independent manner. The transport could occur with the help of host Sec system and a potential signal peptide (SP) at the N-terminus of Tgl. Thus, the overproduction of intact Tgl in bacteria appeared to be problematic. We constructed a recombinant version of Tgl, lacking SP. The recombinant Tgl was purified and characterized. Its strong bactericidal activity against S. aureus makes it a candidate for antistaphylococcal agent active against a wide range of S. aureus strains.

Acknowledgements This work was supported by funds from the Operational Program ‘Innovative Economy, 2007-2013’ (POIG01.03.01-02-003/08) and from the NSC, research grantPRELUDIUM programme (UMO-2012/07/N/NZ2/01596) References (1) Łobocka M. B. et al (2012) Adv Virus Res. 83:143–216 (2) O’Flaherty S. et al (2005) J. Bacteriol. 187:7161–4.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 113

L7.5

L7.6

Bacteriophage vb_Eco4M-7 as the Golden mean in fight against Enterohemorrhagic Escherichia coli (EHEC)

Production of phage lytic enzyme of high antienterococcal activity in Escherichia coli

Agnieszka Necel , Gracja Topka , Aleksandra Dydecka1, Sylwia Bloch1, Bożena Nejman-Faleńczyk1, Agata Jurczak-Kurek 2, Tomasz Gąsior3, Katarzyna Kwaśnicka-Kosznik1, Łukasz Nowakowski1, Łukasz Grabowski1, Alicja Węgrzyn3, Grzegorz Węgrzyn1 1

1

Department of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland; 2 Department of Molecular Evolution, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland; 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Kładki 24, 80-822 Gdańsk, Poland 1

Food poisoning may be caused by Enterohemorrhagic Escherichia coli (EHEC), in which the best known member is E. coli O157:H7. EHEC are highly dangerous group of pathogens that can cause i.a. haemolytic-uremic syndrome (HUS). The major source of these bacteria is cattle. Humans can get infected with EHEC mostly through consumption of infected food. The major virulence factors of EHEC are Shiga toxins, encoded by genes located on genomes of Shiga toxin-converting prophages (Stx phages). Effective production and release of toxins occurs only after induction of these prophages and start of a lytic life cycle. Many antibiotics used to treat bacterial infections stimulate induction of Stx prophages, causing exacerbation of the disease symptoms. Also, the use of medications that slow down intestinal peristalsis is not recommended.

At present, only symptomatic treatment is used and is important to search alternative methods.

A new hope against EHEC may be bacteriophages, which are currently used i.a. in phage therapy. In earlier biodiversity studies 83 bacteriophages were isolated from urban sewage. One of them i.e. vb_Eco4M-7 have an ability to lysis only E. coli O157:H7 strain (ATCC 700728) and clinical strain E. coli O157:H7 with ST2-8624 prophage.

Our study shows that bacteriophage vb_Eco4M-7 is characterized by short life cycle and high multiplication rate (about 1000 phage particle) which may indicate his potential in the fight against Enterohemorrhagic Escherichia coli (EHEC).

Agnieszka Gozdek1, Agnieszka Bednarek1, Jarosław Kosakowski1, Aleksandra Różycka1, Ewa Sadowy2, Beata Weber-Dąbrowska3, Andrzej Górski3, Małgorzata Łobocka1,4 Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland; National Institute of Medicines, Warsaw, Poland; 3Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, PAS, Wrocław, Poland;4Faculty of Agriculture and Biology, Warsaw University of Life Sciences-SGGW, Warsaw, Poland 1 2

Keywords: bacteriophage, endolysin, Esnterococcus, bactericidal agent Enterococcus faecalis and Enteroccoccus faecium are Gram-positive bacteria, forming a natural component of mucous membrane microflora in human and animal gastrointestinal tract. In recent years, strains of these bacteria have become causes of frequent hospital infections, due to the acquisition of drug resistance genes, including the resistance to vancomycin – a last-chance antibiotic (1).

The enterococci have thick, multilayered peptodyglycan of atypical structure. Therefore, lytic enzymes that effectively digest peptidoglycan of other bacteria, such as lysozyme or mutanolysin, are inactive or show a weak activity against enterococcal cell walls. We identified a gene encoding the endolysin of complex structure (Ef12c29-ami) in the genome of E. faecalis bacteriophage Ef12c29. Attempts to clone the intact Ef12c29-ami gene were unsuccesful due to the unexpectedly high toxicity of its product to bacteria. Modifications in the Ef12c29-ami gene allowed us to overproduce an active version of the enzyme (Ef12c29-amiv2) in E. coli cells. Purified Ef12c29-amiv2 appeared to be highly active in lysis of death enterococcal cells and its specific activity exceeded that of mutanolysin. Unlike certain known enterococcal lysins it could lyse living cells of several Enterococcus sp. strains indicating its usefulness as anti-enterococcal agent as well as as a component of lytic kits used in the acquisition of microbial community cell content for metagenomic studies.

Acknowledgements This work was supported by funds from the Operational Program ‘Innovative Economy, 2007-2013’ (Project No. POIG.01.03.01-02-003/08) and from statutory funds for the Institute of Biochemistry and Biophysics, PAS. References (1) Arias CA, Murray BE. 2012. Nat Rev Microbiol. 10: 266–78

6th Central European Congress of Life Sciences Eurobiotech 2017

114 Abstracts

Posters

P7.2

P7.1

Novel Shiga toxin-converting bacteriophages – a comparative analysis of genomic sequences

Production of phage lytic enzyme of high anti-enterococcal activity in Escherichia coli Agnieszka Gozdek1, Agnieszka Bednarek1, Jarosław Kosakowski1, Aleksandra Różycka1, Ewa Sadowy2, Beata Weber-Dąbrowska3, Andrzej Górski3, Małgorzata Łobocka1,4 Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland; National Institute of Medicines, Warsaw, Poland; 3Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, PAS, Wrocław, Poland;4Faculty of Agriculture and Biology, Warsaw University of Life Sciences-SGGW, Warsaw, Poland 1 2

Keywords: bacteriophage, endolysin, Esnterococcus, bactericidal agent Enterococcus faecalis and Enteroccoccus faecium are Gram-positive bacteria, forming a natural component of mucous membrane microflora in human and animal gastrointestinal tract. In recent years, strains of these bacteria have become causes of frequent hospital infections, due to the acquisition of drug resistance genes, including the resistance to vancomycin – a last-chance antibiotic (1).

The enterococci have thick, multilayered peptodyglycan of atypical structure. Therefore, lytic enzymes that effectively digest peptidoglycan of other bacteria, such as lysozyme or mutanolysin, are inactive or show a weak activity against enterococcal cell walls. We identified a gene encoding the endolysin of complex structure (Ef12c29-ami) in the genome of E. faecalis bacteriophage Ef12c29. Attempts to clone the intact Ef12c29-ami gene were unsuccesful due to the unexpectedly high toxicity of its product to bacteria. Modifications in the Ef12c29-ami gene allowed us to overproduce an active version of the enzyme (Ef12c29-amiv2) in E. coli cells. Purified Ef12c29-amiv2 appeared to be highly active in lysis of death enterococcal cells and its specific activity exceeded that of mutanolysin. Unlike certain known enterococcal lysins it could lyse living cells of several Enterococcus sp. strains indicating its usefulness as anti-enterococcal agent as well as as a component of lytic kits used in the acquisition of microbial community cell content for metagenomic studies.

Tomasz Gąsior1, Katarzyna Licznerska2, Aleksandra Dydecka2, Sylwia Bloch2, Gracja Topka2, Bożena Nejman-Faleńczyk 2, Grzegorz Węgrzyn2, Alicja Węgrzyn1 1 Laboratory of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warszawa, Poland; 2 Department of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland

Keywords: bacteriophage, E. coli, bacteria, Shiga toxin, pathogen Escherichia coli is a wide-spread, symbiotic bacterium.

However some of its strains can be extremely dangerous pathogens, especially to humans. One such group are the enterohemorrhagic E. coli (EHEC), which have the ability to produce Shiga toxins. Genes encoding these toxins are located in genomes of Shiga toxin-converting bacteriophages (Stx phages).

Recently, the number of new isolates of Stx phages is rapidly growing, while the knowledge on their genomic sequences, molecular mechanisms and interplays with the bacteria is limited. The aim of this work was to determine the genomic sequences of four novel Stx phages (P22, P27, P32 and ST2-8624) and compare them with the known sequences of other lambdoid phages: λ, 933W and Φ24B. The genomic sequences were determined using next-generation sequencing (NGS). The contigs assembly was analyzed and corrected with the use of BLAST and Progressive MAUVE programs. All genomic annotations were created using myRAST software and manually corrected using the UGENE program. ClustalW algorithm was used for all pairwise and multiple sequence alignments.

Despite high global similarity in genomic sequence, key regions responsible for the phage development show differences in sequences. Special attention was also paid to fragments of highly conserved sequence but still unknown function, such as the exo-xis region of lambdoid phages.

Acknowledgements This work was supported by funds from the Operational Program ‘Innovative Economy, 2007-2013’ (Project No. POIG.01.03.01-02-003/08) and from statutory funds for the Institute of Biochemistry and Biophysics, PAS. References (1) Arias CA, Murray BE. 2012. Nat Rev Microbiol. 10: 266–78

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Abstracts 115

P7.3

P7.4

Characterization and stability analysis of prophage ΦBH1 identified in the genome of probiotic Lactobacillus rhamnosus Pen

Bacteriophage vb_Eco4M-7 as the Golden mean in fight against Enterohemorrhagic Escherichia coli (EHEC)

Piotr Jarocki, Marcin Podleśny, Oleksandr Kholiavskyi, Michał Dworniczak, Elwira Komoń-Janczara, Zdzisław Targoński

Agnieszka Necel1, Gracja Topka1, Aleksandra Dydecka1, Sylwia Bloch1, Bożena Nejman-Faleńczyk1, Agata Jurczak-Kurek 2, Tomasz Gąsior3, Katarzyna Kwaśnicka-Kosznik1, Łukasz Nowakowski1, Łukasz Grabowski1, Alicja Węgrzyn3, Grzegorz Węgrzyn1

Department of Biotechnology, Human Nutrition and Food Commodities, University of Life Sciences in Lublin, Lublin, Poland

Department of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland; 2 Department of Molecular Evolution, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland; 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Kładki 24, 80-822 Gdańsk, Poland 1

Lactobacillus rhamnosus Pen is an endogenous strain with well-documented health-promoting properties which is used to produce probiotic formulations. In this study, we present a complete genome sequence of Lactobacillus rhamnosus Pen, which consists of a circular 2,884,4966 bp chromosome with GC content of 46.8%. The analysis performed using CRISPRs finder indicated one regularly interspaced short palindromic repeat locus consisting of 1092 nt region with 16 spacers (length of 30–31 nt). One intact prophage of about 40.7 kb and GC content of 44.8% was identified as well. This prophage sequence shows only 94% (query coverage 59%) and 91% (query coverage 59%) similarity with two previously described L. rhamnosus bacteriophages – Lrm1 and Lc-Nu, respectively. Finally, based on comparative analysis with L. rhamnosus bacteriophages, the attachment sites (attB, attP, attL and attR) involved in phage integration and excision were identified.

Additionally, a real-time PCR assay was developed to determine the spontaneous induction frequency of ΦBH1.

A thorough insight into the genomic information confirms probiotic properties of L. rhamnosus Pen and may indicate new biotechnological applications of this industrially-important strain.

Acknowledgements This work was financially supported by grant UMO-2013/09/N/NZ9/01617 from the National Science Centre, Poland.

Food poisoning may be caused by Enterohemorrhagic Escherichia coli (EHEC), in which the best known member is E. coli O157:H7. EHEC are highly dangerous group of pathogens that can cause i.a. haemolytic-uremic syndrome (HUS). The major source of these bacteria is cattle. Humans can get infected with EHEC mostly through consumption of infected food. The major virulence factors of EHEC are Shiga toxins, encoded by genes located on genomes of Shiga toxin-converting prophages (Stx phages). Effective production and release of toxins occurs only after induction of these prophages and start of a lytic life cycle. Many antibiotics used to treat bacterial infections stimulate induction of Stx prophages, causing exacerbation of the disease symptoms. Also, the use of medications that slow down intestinal peristalsis is not recommended.

At present, only symptomatic treatment is used and is important to search alternative methods.

A new hope against EHEC may be bacteriophages, which are currently used i.a. in phage therapy. In earlier biodiversity studies 83 bacteriophages were isolated from urban sewage. One of them i.e. vb_Eco4M-7 have an ability to lysis only E. coli O157:H7 strain (ATCC 700728) and clinical strain E. coli O157:H7 with ST2-8624 prophage.

Our study shows that bacteriophage vb_Eco4M-7 is characterized by short life cycle and high multiplication rate (about 1000 phage particle) which may indicate his potential in the fight against Enterohemorrhagic Escherichia coli (EHEC).

6th Central European Congress of Life Sciences Eurobiotech 2017

116 Abstracts

P7.5

P7.6

Identification of Fully Human Fab Fragment Antibody Targeting Protease Activated Receptor 1

”Characterization of selected bacteriophages isolated from urban sewages and their potential biotechnological importance”

Tavassoli E1, Rahimi Jamnani F2,3, Jahandar H1 Department of Biotechnology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran (IAUPS); 2 Innovation Center, Pasteur Institute of Iran, Tehran, Iran; 3 Microbiology Research Center, Department of Mycobacteriology and Pulmonary Research Pasteur Institute of Iran, Tehran, Iran 1

Keyword: Fab antibody, PAR1, Phage display Introduction:Cancer is one of the leading cause of death worldwide.Angiogenesis plays the main role in this scenario. In this way, Protease activated receptor 1 (PAR1),is a key player in angiogenesis [1]. This receptor is activated by different proteases including thrombin. The relation between over-expression of PAR1 in cancer inavasion and in metastatic patients led to develop the Fab antibodies against PAR1 [2].

Methods:A synthesized peptide which is a part of the extracellular domain of PAR1, was used for the enrichment of phages displaying Fab fragments. Multiple rounds of panning were carried out including amplification of Fab-displaying phages, binding of phage-Fab to antigen, washing, elution, and re-amplification of PAR1 specific phages in bacteria. After checking four inputs and outputs by polyclonal phage ELISA, the soluble Fab fragments derived from the selected phages were generated in bacteria and their binding were checked by ELISA. Result: After four rounds of panning, the phages of fourth round showed the best signal intensities compared with the control. Fifty clones were checked by Fab ELISA which among them, three Fabs exhibited stronger signals than other antibody fragments examined.

Conclusion:It seems that the selected Fab antibodies, have great potential to use with other chemotherapeutic agents to treatment of cancer.

References [1] Zhao P et al.(2014) Frontiers in endocrinology. May 13;5:67 [2] Ramachandran R et al (2012). Nature reviews Drug discovery. Jan 1;11(1):69–86

Gracja Topka1, Sylwia Bloch1, Bożena Nejman-Faleńczyk1, Agata Jurczak-Kurek 2, Agnieszka Necel1,Aleksandra Dydecka1, Tomasz Gąsior3, Grzegorz Węgrzyn1, Alicja Węgrzyn3 Katedra Biologii Molekularnej, Uniwersytet Gdański, Wita Stwosza 59, 80-308 Gdańsk, Polska; Katedra Ewolucji Molekularnej, Uniwersytet Gdański, Wita Stwosza 59, 80-308 Gdańsk, Polska; 3 Instytut Biochemii i Biofizyki, Polska Akademia Nauk, Kładki 24, 80-822 Gdańsk, Polska

1

Bacteriophages have played a significant role in the development of molecular biology, biotechnology and genetic engineering. As we know, bacteriophages are viruses that infect bacterial cells. These viruses serve as tools to understand molecular biology, as vectors of horizontal gene transfer and drivers of bacterial evolution, as sources of diagnostic and genetic tools and as novel therapeutic agents. Bacteriophage diversity appears to be huge as phages are the most abundant biological entities on Earth – their number is estimated to be 1031. As only a few bacteriophages have been selected as model organisms, our current knowledge on vast majority of these viruses is very limited.

During preliminary research, 83 previously unknown bacteriophages infecting various bacterial species, were isolated. In the next stage, some properties of the isolated phages have been determined (host range, virion morphology, plaque morphology, sensitivity to physical and chemical agents). On the basis of the observed unusual biological features of particular phages, 5 of them have been selected for further analyses. We determined complete nucleotide sequences of their genomes, and looked in more detail at lytic development of these phages.

Morphological and physiological analyses of these bacteriophages indicated that there is high biodiversity among phages. Some of the presented features of investigated phages, make them potentially interesting in the context of biotechnological applications.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 117

Molecular mechanisms of plant response to environmental stresses Keynote lecture

Lectures

L8.1

L8.2

Metal homeostasis and tolerance in plants: cellular and symbiotic aspects

Endoplasmic reticulum (ER) bodies consist a defense system in Brassicaceae

Przemysław Malec1 and Katarzyna Turnau2

Kenji Yamada1, Mikio Nishimura2, Ikuko Hara-Nishimura3

Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology; 2 Institute of Environmental Studies, Faculty of Biology; Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland 1

Metal pollution has drastically increased during the last centuries and it is expected to rise in the future. Plants growing in metal-polluted habitats have developed complex mechanisms to tolerate elevated concentrations of metals and to control cell homeostasis in potentially harmful environment. A regulated system of immobilization, chelation, translocation and deposition functions in plant cells to control the uptake, redistribution and detoxification of metal ions.

Also, a set of signalling pathways and enzymatic activities are involved in the redirection of cellular metabolism to the functioning under stress conditions.

In paralell, plant-associated microbes increase the capacity of plants to survive in the presence of potentially toxic metals. In particular, the association of plant roots with mycorrhizal fungi gives the mycorrhizal plants a competitive advantage over others, that are devoid of properly selected microbiota. Recently, the complexity of plant microbial associations and their role in plant tolerance to metals has been recognized. Several field and laboratory experiments have shown that for successful metal phytoremediation consortia of mycorrhizal, endophytic fungi, endophytic and rhizospheric bacteria have to be applied.

These complex inocula have better chance to stimulate plant growth, photosynthesis, pathogen resistance and abiotic stress attenuation. The inoculation with selected microbial consortia has a beneficial effect on various species of plants. The inocula can be used in phytostabilisation and phytoextraction of such elements as Ni, where the proper selection of microbes can result in improving the efficiency of phytomining.

Malpolska Centre of Biotechnology, Jagiellonian Uiniversity, Poland; 2Department of Cell Biology, National Institute for Basic Biology, Japan; 3Facurty of Science and Engineering, Konan University, Japan 1

Endoplasmic reticulum (ER) bodies are spindle-shaped structures in Brassicaceae plants. Numerous ER bodies develop in epidermal cells of seedlings that are exposed to environmental stresses in Arabidopsis thaliana, a model plant in Brassicaceae. ER bodies accumulate β-glucosidases (BGLU21 and BGLU23). The substrates of these BGLU21 and BGLU23 include defensive glucosinolates, suggesting that ER bodies provide a defense system.

A Brassicaceae specific ER body-protein NAI2 can produce ER bodies in non Brassicaceae plants when expressed together with BGLU23. Our results suggest that Brassicaceae plants have evolved the ER-body dependent defense to protect seedlings from insects and pathogens.

6th Central European Congress of Life Sciences Eurobiotech 2017

118 Abstracts

L8.3

L8.4

Roles of LON protease and autophagy in the peroxisomal quality control

Genome-wide identification of genes involved in the potato response to drought indicates functional evolutionary conservation with Arabidopsis plants

Shino Goto-Yamada1, Shoji Mano2,3, Kazusato Oikawa4, Mikio Nishimura2 1 Jagiellonian University, Malopolska Centre of Biotechnology, Poland; 2National Institute for Basic Biology, Japan; 3The Graduate University for Advanced Studies, Department of Basic Biology, Japan; 4 RIKEN Center for Sustainable Resource Science, Japan.

Plant peroxisomes are involved in various cellular processes such as β-oxidation of fatty acids and photorespiration. A notable feature of plant peroxisomes is their flexible adaptive responses to environmental conditions, e.g., peroxisomes involved in lipid catabolism are rapidly replaced with those involved in photorespiration during post-germinative stage. This is called the functional transition of peroxisomes. However, the detailed molecular mechanisms underlying the functional transition of peroxisomes have previously been unclear. Here, we show that LON protease 2 (LON2) and autophagy are responsible for the functional transition of peroxisomes. In the Arabidopsis aberrant peroxisome morphology 10 (apem10) mutant, which is defective in LON2, the peroxisome degradation was accelerated and the number of peroxisomes was reduced. We found that the enhanced peroxisome degradation in apem10/lon2 was caused by autophagy, which is suppressed by LON2 chaperone function in wild-type plants. We also found the proteolytic consequence of LON2 for the functional transition of peroxisomes; this process is balancing autophagy and chaperone functions of LON2.

In addition to the above, we also show recent our study about peroxisome and autophagy.

Pieczynski M1, Wyrzykowska A1, Milanowska K1, Boguszewska-Mankowska D2, Zagdanska B3, Karlowski W4, Jarmolowski A1 and Szweykowska-Kulinska Z1 Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland; 2 Potato Agronomy Department, Plant Breeding and Acclimatization Institute, National Research Institute, Division Jadwisin, 05-140 Jadwisin, Poland; 3 Department of Biochemistry, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; 4 Department of Computational Biology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland 1

Potato is one of the four most important food crop plants worldwide and is strongly affected by drought, which is the main climate threat causing a major decrease in tuber yield. The development of high-throughput RNA sequencing (RNA-seq) techniques has provided novel tools for qualitative and quantitative global analyses of gene expression. The following two pairs of potato cultivars, which are related in ancestry but show different drought tolerances, were chosen for comparative gene expression studies: Gwiazda/Oberon and Tajfun/Owacja. Comparative RNA-seq analyses of gene expression differences in the transcriptomes obtained from drought-tolerant versus drought-sensitive plants during water shortage conditions were performed. The twenty-three top-ranking genes were selected, 22 of which are described here as novel potato drought-responsive genes. Moreover, all but one of the potato genes selected have homologs in the Arabidopsis genome. Of the seven tested A. thaliana mutants with altered expression of the selected homologous genes, compared to the wild-type Arabidopsis plants, six showed an improved tolerance to drought. The evolutionary conservation of the functions of the selected genes in the plant response to drought confirms the importance of these identified potato genes in the ability of plants to cope with water shortage conditions. Knowledge regarding these gene functions can be used to generate potato cultivars that are resistant to unfavorable conditions. The approach used in this work and the obtained results allowed for the identification of new players in the plant response to drought.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 119

L8.5

Posters

MicroRNAs in soybean chilling stress response

P8.1

Jakub Kuczyński, Joanna Gracz, Agata Tyczewska

Identification of Pisum sativum L. genes differentially expressed in resistant reaction to Didymella pinodes in – preliminary results

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Department of Protein Biosynthesis, Poland Soybean is the major legume crop that owes its importance to high protein (40%) and oil (20%) content, as well as the ability to host the nitrogen fixating bacteria in root nodules. Two major fields of soybean use are food production and animal feed enrichment with latter being the cause of import of more than 20 million tons of soybean meal in Europe. Low temperatures in spring common for Polish climate render it not suitable for effective soybean cultivation. In order to remedy this issue and introduce soybean to Polish agriculture efforts have been made to breed cultivars resistant to chilling stress. To better understand the mechanism of abiotic stress tolerance related to low temperatures in plants we employed cultivar Fiskeby V, established in Sweden. Based on literature data survey we selected microRNAs (miRNAs) that potentially play a role in plant low temperature response. Gene ontology analysis indicated that their target genes encode various transcription factors and antioxidants. We determined expression levels of these miRNAs and therefore highlighted their significance in soybean’s stress response. Small RNAs were isolated from leaves and seedlings of plants cultivated in stress conditions. Assessment of miRNA expression levels was performed using novel technique called digital droplet PCR, which precisely defines the concentration of target sequence in each probe. We confirmed that all studied miRNAs are involved in chilling stress response in soybean cultivar Fiskeby V. Acknowledgements The work was supported by a grant no.2014/15/B/NZ9/02312 from National Science Centre, Poland and by the Polish Ministry of Science and Higher Education, under the KNOW program.

Anna Durska1, Magdalena Gawłowska1, Wojciech Święcicki1, Lech Boros2 1 Institute of Plant Genetics, Polish Academy of Sciences, Genomics Department, Poland; 2 Plant Breeding and Acclimatization Institute Radzików, Seed Science and Technology Department, Poland

Keywords: Didymella pinodes, isoflavone synthase, Pisum sativum, real time PCR Ascochyta blight, caused by Didymella pinodes, is the most destructive foliar pathogen of the peas in the temperate zones of Europe. This disease causes 10% yield losses as an average and can reach 50% under certain conditions.

Current control practices are uneconomic and inefficient.

Very little is known on the mechanisms or genes that control host plant resistance against these fungus. Flavonoids are compounds with antioxidant and antimicrobial properties. Isoflavone synthase is the key enzyme catalyzing the biosynthesis of isoflavonoids, which have demonstrated efficient antibacterial and antifungal activities. Herein, we investigated the expression change of isoflavone synthase in 8 cultivated pea accessions before inoculation and when the first symptoms of the disease appeared. Trials with controlled inoculation were performed at Radzików, Poland. Disease severity assessment was done, according to Xue et al. (1996) (the scale: 0 – resistant, 9 – susceptible).

The expression profiles of a chosen gene were analyzed by qRT-PCR in control and D. pinodes-infected plants of chosen cultivars. The results showed that the gene involved in secondary metabolism was differentially expressed in resistant versus susceptible cultivars after inoculation with D. pinodes. Futher analyses could confirm the role of the secondary metabolism genes in defense and finally provide more resistant cultivars.

Acknowledgements The study was supported by National Multi-Year Program.

6th Central European Congress of Life Sciences Eurobiotech 2017

120 Abstracts

P8.2

P8.3

Structural and biochemical response of native model membranes to ozone stress – protective effects of gallic acid

Gene onthology analysis of miRNA targets involved in soybean chilling stress response

Barbara Dyba , Elżbieta Rudolphi- Skórska , Apolonia Sieprawska1, Jolanta Biesaga- Kościelniak 2, Andrzej Kornaś1, Maria Filek 2 1

1

1 Institute of Biology, Pedagogical University, Podchorążych 2, 30- 084 Cracow, Poland; 2The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Cracow, Poland

Keywords: ozone stress, wheat calli, model membranes, gallic acid The aim of the study was to describe the effect of gallic acid – one of the hydrophilic antioxidants – on the physiochemical properties of native (wheat calli cells) and model (lipid monolayers) membranes under ozone stress conditions. The intensity of the ozone stress was determined by the lipid peroxidation and antioxidant enzymes activities.

Langmuir technique has been used to quantify the effects associated with the structural parameters of membranes such as: the area occupied by specific molecule in a monolayer, the pressure and the fluidity of membranes. It was concluded that the changes of these physiochemical parameters may serve as key factors in the precise description of early stages of both stress-induced and protective mechanisms. The presence of the gallic acid in the environment stimulates the increase the membrane fluidity, which is significantly blocked by the ozone treatment.

Joanna Gracz1, Wojciech Karłowski2, Jakub Kuczyński1, Agata Tyczewska1 1 Department of Protein Biosynthesis, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland; 2 Department of Computational Biology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland

Stress response in plants is mediated through broad spectrum of cellular mechanisms and one of them is alteration in expression of microRNAs (miRNAs) molecules. As a consequence of its action we can observe differentiation in expression levels of genes targeted by those miRNA. Most plant miRNAs perform their gene expression regulation function through target cleavages. The resulting slicing sites on the target transcripts could be mapped by sequencing of the 3’-cleavage remnants, called degradome sequencing. For selected miRNA previously described as involved in chilling stress response in plants, we performed analysis of potential target genes in soybean – economically important crop species, which is highly used in feed industry. The analyzes were performed based on data from PMRD: plant microRNA database, structural properties of [RNA: RNA] complex and data from degradome sequencing available from Phytozome database. For most probable target genes we performed also gene onthology analysis to indicate its function in chilling stress response.

Studied genes encode, among others, transcription factors and molecule of antioxidant function which are key players in sustaining status quo of cell during stress condition.

Acknowledgementss This work was supported by grant from National Science Center Poland no.

2014/15/B/NZ9/02312 and Ministry of Science and Higher Education of the Republic of Poland by KNOW program.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 121

P8.4

P8.5

MicroRNAs in soybean chilling stress response

Proteome changes under herbicidal stress in maize

Jakub Kuczyński, Joanna Gracz, Agata Tyczewska

Agata Tyczewska, Izabela Bielińska, Joanna Gracz, Marta Sikora, Tomasz Twardowski

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Department of Protein Biosynthesis, Poland Soybean is the major legume crop that owes its importance to high protein (40%) and oil (20%) content, as well as the ability to host the nitrogen fixating bacteria in root nodules. Two major fields of soybean use are food production and animal feed enrichment with latter being the cause of import of more than 20 million tons of soybean meal in Europe. Low temperatures in spring common for Polish climate render it not suitable for effective soybean cultivation. In order to remedy this issue and introduce soybean to Polish agriculture efforts have been made to breed cultivars resistant to chilling stress. To better understand the mechanism of abiotic stress tolerance related to low temperatures in plants we employed cultivar Fiskeby V, established in Sweden. Based on literature data survey we selected microRNAs (miRNAs) that potentially play a role in plant low temperature response. Gene ontology analysis indicated that their target genes encode various transcription factors and antioxidants. We determined expression levels of these miRNAs and therefore highlighted their significance in soybean’s stress response. Small RNAs were isolated from leaves and seedlings of plants cultivated in stress conditions. Assessment of miRNA expression levels was performed using novel technique called digital droplet PCR, which precisely defines the concentration of target sequence in each probe. We confirmed that all studied miRNAs are involved in chilling stress response in soybean cultivar Fiskeby V. Acknowledgements The work was supported by a grant no.2014/15/B/NZ9/02312 from National Science Centre, Poland and by the Polish Ministry of Science and Higher Education, under the KNOW program.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Department of Protein Biosynthesis, Poland To guarantee plant’s survival under adverse conditions plants have developed exquisite adjustments to stresses at all levels (anatomical, morphological, cellular, biochemical and molecular). While transcriptomic approaches are an important resource, functional gene expression profiles can only be achieved by proteomic analysis as different families of proteins are known to be associated with plant responses to stresses by being newly synthesized, accumulating or decreasing. Among other things, these proteins are involved in signalling, translation, host-defence mechanisms, carbohydrate metabolism and amino acid metabolism. Thus, quantitative analysis of gene expression at the protein level is essential for determining plant responses to stress conditions.

Few years ago it has been observed that some maize lines show higher sensitivity to herbicide spraying than others, but molecules determining such heightened resistance remain unknown to this day. Therefore our goal is to identify molecular basics of plant’s increased/decreased resistance to herbicides by analysing changes in proteomes of two maize lines showing differential resistance to Roundup®. For better characterization of proteome responses we have chosen 2 maize inbred line, one sensitive to the glyphosate (S79757) and the another one tolerant (S245).

Our approach is based on two-dimensional gel electrophoresis (2DE) since it combines separation of polypeptides according to isoelectric properties and molecular weight. The identification of proteins is performed using mass spectrometry (MALDI Tof) and MASCOT software.

Acknowledgements The work is supported by grants no. UMO-2011/01/D/NZ9/03631 and 2012/06/A/ NZ9/00125 from National Science Centre, Poland.

6th Central European Congress of Life Sciences Eurobiotech 2017

122 Abstracts

Biomaterials Engineering – New Material Technologies for Tissue Engineering Lectures

L9.2

L9.1

3D printing of chitosan and graphene oxide hydrogels for biomedical applications

Bioinspired thin films materials structured by laser interference lithography for direct blood contact Klaudia Trembecka-Wojciga1, Roman Major1, Juergen M. Lackner2 Hanna Plutecka3 and Boguslaw Major1 Institute of Metallurgy and Materials Science, Polish Academy of Sciences, Krakow, Poland; 2Joanneum Research Forschungsges mbH, Institute of Surface Technologies and Photonics, Functional Surfaces, Leobner, Austria; 3Department of Medicine, Jagiellonian University Medical College, Krakow, Poland 1

The work focused on multidirectional phenomenological description of structure and surface properties of the biomaterial forming a bio-matrix for human endothelial cells seeding and influencing the controlled formation of blood vessels and self-selective adsorption of proteins to the surface affecting the minimization of clotting in conditions of high shear forces. Modification of titanium alloys was carried out according to methods of physical vapor deposition (Pulsed Laser Deposition, PLD), and laser interference lithography techniques, and encompass both topographical variations and surface microstructure. Periodical structures in the form of 3D patterns with a specific morphology was fabricated using the direct method of the laser interference lithography. Multiscale analysis of the material was carried out. Transmission Electron Microscopy (TEM) was used to analyze microstructure of a cross-section of materials. Thin foils for TEM analysis were prepared using the focused ion beam technique of gallium on the device QUANTA 200 3D technology (Focused Ion Beam FIB). A hemocompatibility test was carried out on the basis of clinically used solutions (tester Impact-R) based on an evaluation of the interaction of the blood-material in dynamic conditions. In order to obtain full information about hemocompatibility of materials, after the test, blood was taken above the analyzed material and was undergo to cytometric analysis to determine the degree of platelet activation and the formation of its aggregates. Simultaneously the tested surfaces were subjected to analysis using Scanning Confocal Microscopy (CLSM) and dedicated monoclonal antibodies.

Patrycja Domalik-Pyzik1,2, Joseph A.M. Steele2, Joachim Kohn2, Jan Chłopek1 AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department of Biomaterials, Al. Mickiewicza 30, 30-059 Kraków, Poland; 2New Jersey Center for Biomaterials, Rutgers, The State University of New Jersey, 145 Bevier Road, Piscataway, New Jersey 08854, United States

1

Three-dimensional (3D) printing has recently gained much attention in tissue engineering and regenerative medicine, as one of the most reliable additive manufacturing fabrication methods. Many different biomaterials, including polymeric, ceramic, metallic, and composite materials, have already been successfully produced by 3D printing. Hydrogels are of great interest due their excellent biological properties. Their three-dimensional polymer networks can mimic microenvironment of a natural extracellular matrix (ECM), thus creating optimal conditions for cell culture and proliferation [1]. However, processing hydrogels into structures with highly controlled architectures is challenging. In this work pure chitosan and chitosan/graphene oxide hydrogels were used for 3D printing of tissue engineering scaffolds with different geometries.

Results of hydrogel ink printability, physicochemical properties and in vitro biocompatibility evaluations are presented, highlighting promising features of the developed chitosan/graphene oxide formulations, as well as further perspectives for hydrogels 3D printing and its applications.

Acknowledgementss This research was supported by the NJCBM (through ICF-BSE International Summer Exchange Program), and by the grants No 15.11.160.019 and 23.23.160.02020 (AGH University of Science and Technology).

References [1] M. Guvendiren, J. Molde, R. Soares, J. Kohn. ACS Biomater. Sci. Eng. 2016, 2, 1679−1693

Acknowledgementss The research was financially supported by the Project no. 2016/21/N/ST8/00186 “Functional carbon based coatings on titanium substrate, modified by laser ablation designed for the integration with cardiac tissue and ultimately inhibit the blood clotting process” of the Polish National Center of Science.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 123

L9.3

L9.4

Improving the thermal stability of nanocrystalline cellulose: The influence of hydrolysis and modification conditions

Preparation and characterization of polyoxymethylene-based nanocomposites containing functionalized hydroxyapatite for biomedical applications

Agnieszka Leszczyńska, Paulina Radzik, Katarzyna Haraźna, Krzysztof Pielichowski Department of Chemistry and Technology of Polymers, Cracow University of Technology, 24 Warszawska St., 31-155 Cracow, Poland Due to combination of low density high modulus and natural origin, cellulose nanocrystals (CNC) are interesting material for applications in polymer nanocomposites.

However, its strong hydrophilic character and low thermal stability limit its application and performance of obtained bionanocomposites. In this research we report the influence of the hydrolysis agent type and concentration on the thermal stability, morphology, and structure of CNC. With the increase of the acid concentration, regardless of the acid type, the aspect ratio of nanoparticles increased but the thermal stability of cellulose nanocrystals was diminished. The onset temperature of degradation (Tonset) of CNC prepared in phosphoric (V) acid was higher by over 70°C comparing to those obtained in sulfuric (VI) acid. Further improvement of CNC thermal stability was achieved by its modification with succinic anhydride (SA) and the optimum molar ratio of SA to cellulose hydroxyl groups was found 3:1.

Acknowledgements This research work was supported by Polish National Science Centre under the grant No. UMO/2015/19/B/ST8/01060.

Kinga Pielichowska, Klaudia Król-Morkisz, Piotr Szatkowski AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department o Biomaterials, Al. Mickiewicza 30, 30-059 Kraków, Poland, e-mail: kingapie@agh.edu.pl Polyoxymethylene (POM) is well-known thermoplastic polymer displaying excellent stability and mechanical properties, widely used in the medical field for joint replacement components, long-term bone implants and in heat valve disc occluders [1, 2].

Hydroxyapatite is the main component of animal and human bones and teeth and is widely used in biomaterials, especially composites for bone tissue regeneration and dental materials [3]. HAp has the unusual property of containing both acid and basic sites in a single crystal lattice and it can be used as a catalyst in many organic reactions [4] as well as due to presence of hydroxyl groups it is possible its functionalization with different polymers.

In our approach we functionalized HAp by grafting poly(ethylene glycol) (PEG) and poly(e-caprolactone) (PCL) on HAp nanoparticles surface using an aliphatic 1,6-hexamethylene diisocyanate (HDI) as a coupling agent to obtain new hybrid inorganic-organic system HAp-graft-polymer. In the next step, we introduced functionalized HAp to POM copolymer using melt processing methods. The effect of HAp modification on selected POM/HAp-g-PEG nanocomposites properties was investigated. Our results show that functionalized HAp, in contrast to unmodified HAp, significantly improved the POM thermal stability and cause a decrease in the amount of formaldehyde released from POM samples during incubation. This effect can be attributed to the reduction of the number of basic OH active centers in HAp which were active sites involved in catalytic reactions of POM depolymerization in POM/ HAp nanocomposites. HAp functionalization allow to incorporate large amount of bioactive compound to polymer matrix that is desirable in orthopaedic applications.

Acknowledgements Authors are grateful to the Polish National Science Centre for financial support of projects under the Contract No. DEC-2016/21/B/ST8/00449.

References [1] Lüftl, S., V.P. M, and S. Chandran, Polyoxymethylene Handbook: Structure, Properties, Applications and their Nanocomposites. Polymer Science and Plastics Engineering. 2014: Wiley.

[2] Eschbach, L.,. Injury, 2000. 31, Suppl. 4(0): p. D22-D27.

[3] Tsuchida, T., et al., Journal of Catalysis, 2008. 259(2): p. 183–189.

[4] Diallo-Garcia, S., et al., The Journal of Physical Chemistry C, 2014. 118(24): p. 12744–12757.

6th Central European Congress of Life Sciences Eurobiotech 2017

124 Abstracts

L9.5

Posters

Dynamics of content changes selected hormones (rh-GH or rh-PRL) in kidney prefunds rinse with Biolasol

P9.1

Florian Ryszka1, Barbara Dolińska2,1, Aleksandra Dziurowicz-Kochańska2, Katarzyna Czyż1 1 Pharmaceutical Research and Production Plant „Biochefa”, Sosnowiec; 2Department of Applied Pharmacy, School of Pharmacy and the Division of Laboratory Medicine, Medical University of Silesia, Poland

We examined dynamic changes of prolactin content (rhPRL) or somatotropin (rh-GH) which was aded to Biolasol during perfusion, preservation and reperfusion isolated pigs kidneys. Hormones content was marked IRMA method in prefunds in the proces of rinsing and preservation kidneys with modified Biolasol. Samples were taken for examination after 5 and 30 minutes after perfusion, and past 24 hours organs preservation, as well after 5 and 30 minutes reperfusion. Sensitivity of hormones marking method was in the limits 3–200 pg/ml. Hormone content which was aded to fluid assumed for 100%. During kidneys perfusion with modified Biolasol with rh-PRL observed that after 5 minutes hormone content in prefunds was lower from 100% to ~ 84% and after 30 minutes got higher ~ 40%. During reperfusion observed that after 5 minutes increase of content rh-PRL ~ 16%. There was no significant changes of rh-GH content during perfusion and preservation kidneys in modified fluid came out with 100%. On the other hand they observed reduction of this hormone (to ~ 24%) in reprefunds. Statistical analysis showed essential differences in dynamic content changes rh-PRL and rh_GH in prefunds during rinsing and preservation kidneys with modified Biolasol.

Selection of reaction conditions of polymeric carriers based on polyhydroxyalkanoates (PHAs) Katarzyna Bialik-Wąs1, Dagmara Malina2, Klaudia Pluta2, Agnieszka Kulawik-Pióro1, Michał Zielina3 Institute of Organic Chemistry and Technology; Institute of Inorganic Chemistry and Technology; 3 Institute of Water Supply and Environmental Protection, Cracow University of Technology, 24 Warszawska St., 31-155 Cracow, Poland, *kbialikwas@chemia.pk.edu.pl 1 2

Keywords: polymeric carriers, polyhydroxyalkanoates, drug delivery systems In the early 1990s, PHAs became candidates for medical and biomedical applications such as tissue engineering and drug carriers due to their biodegradability, biocompatibility and their degradation throughout surface erosion.

Furthermore, PHAs are characterized by immunological indifference, lack of cytotoxicity and bioresorbability, because their monomers are present in the human body as products of the metabolism. Here we report the studies focused on the selection of the most appropriate conditions of the reaction of the polymeric carriers based on aliphatic polyesters preparation. Moreover, based on the rheological properties the type of stabilizer and its concentration was chosen. The size of polymeric carriers distribution was determined by dynamic light scattering (DLS). The structure of obtained materials was confirmed using FTIR spectroscopic technique.

Acknowledgements This research was supported by Polish Ministry of Science and Higher Education — project C-2/488/2017/DS-M.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 125

P9.2

P9.3

Studies on preparation of hydrogel materials containing extract from Echinacea purpurea

Bacterial cellulose composites with CMC and HEC as potential dressings for chronic wounds

Katarzyna Bialik-Wąs

Izabela Cielecka, Przemysław Rytczak, Edyta Gendaszewska-Darmach, Stanisław Bielecki

Cracow University of Technology, Institute of Organic Chemistry and Technology, 24 Warszawska St., 31-155 Cracow, Poland, *kbialikwas@chemia.pk.edu.pl Keywords: hydrogels, Simulated Body Fluids, Echinacea purpurea Currently, hydrogel materials, due to their unique properties such as softness, hydrophilicity, superabsorbancy, viscoelasticity, biodegradability, biocompatibility and their similarity with extra cellular matrix gain more and more interest. They can be applied in medicine, pharmacy and biomedicine such as carriers for drug delivery systems.

The introduction of the Echinacea purpurea extract, as the natural active substance, into the polymeric matrix, has a beneficial effect on improving wound healing. Echinacea purpurea contains many substances, such as polysaccharides, caffeic acid derivatives (including cichoric acid), alkylamides, and glycoproteins. The obtained hydrogels were tested for their swelling behavior and biological properties in water and simulated body fluids. Moreover, the active substances release behavior of the hydrogels was evaluated in phosphate-buffered saline (PBS) at 37°C. The structure of prepared hydrogels was confirmed using FT-IR spectroscopic technique.

Acknowledgements This research was supported by Polish Ministry of Science and Higher Education — project C-2/349/2017/DS.

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences Łódź University of Technology, Poland Keywords: Bacterial cellulose, absorption properties, transparency Bacterial cellulose (BNC) is a natural polysaccharide produced by bacteria of the genus Komagataeibacter. The scientific and commercial interest in BNC, especially in biomedical applications results from its unique properties such as biocompatibility, high mechanical strength, high crystallinity degree and water holding capacity.

Due to its properties, bacterial cellulose can be successfully used a wound dressing material. The dressing made from BNC maintain the proper moisture level and constant temperature of the wound bed which can significantly accelerate wound healing process with renewal of skin cells.

The study describes the preparation and characterization of bacterial cellulose composites with 2-hydroxyethyl cellulose (HEC) and carboxymethylcellulose (CMC), additionally modified with glycerol, which can be used as a wound dressing material for treatment of chronic wound.

The obtained composites have significantly increased absorption properties (according to ISO 13726) and mechanical strength. The FTIR and SEM analysis showed changes in material structure which improve sorption properties.

Modification with glycerol of obtained BNC composites acquires several important, in relation to dressing material, attributes like: transparency, flexibility and increased hydration at elevated temperatures. Additionally, the composites were tested in vitro using skin cell lines (fibroblasts and keratinocytes).

6th Central European Congress of Life Sciences Eurobiotech 2017

126 Abstracts

P9.4

P9.5

Association between single-nucleotide polymorphisms of genes responsible for repair, replication and degradation of mitochondrial DNA, and depressive disorder

Antibacterial hybrid material for bone regeneration

Piotr Czarny1, Paulina Wigner2, Janusz Szemraj1, Tomasz Śliwiński2

AGH-UST University of Science and Technology, Faculty of Materials Science and Ceramics, Krakow, Poland,

1 Department of Medical Biochemistry, Medical University of Lodz , Poland; 2Laboratory of Medical Genetics, University of Lodz , Poland.

Calcium phosphate based biomaterials are commonly used in regenerative medicine mainly due to their excellent biocompatibility. The major drawback of CaPs-based materials is their low mechanical strength. In order to improve mechanical properties, various organic modifiers such as sodium alginate or chitosan were proposed [1,2].

Furthermore, antibacterial additives were used to prevent postoperative infections, which are one of the biggest problems in orthopaedic surgery.

In this study hybrid biomaterials on the basis of hydroxyapatite and methylcellulose were synthesized using a revised wet chemical method. To obtain antibacterial properties hybrids were modified with silver nanoparticles.

XRD and FT-IR analysis revealed that synthesized materials consisted of one crystalline phase – hydroxyapatite.

XRF analysis and SEM observations confirmed presence of silver nanoparticles in the samples. Application of methylcellulose resulted in increase of compressive strength. Obtained hybrids were chemically stable. Presence of apatite layer after incubation in SBF indicated on bioactive character of materials.

This study demonstrated that methylcellulose and silver are promising modifiers of CaPs materials and may impart them with antibacterial properties without compromising their bioactivity.

Mitochondrial dysfunction is observed in course of depressive disorders (DD). Thus, we genotyped two single nucleotide polymorphisms (SNPs) located in genes encoding polymerase γ (POLG) and endonuclease g (ENDOG).

The former is responsible for replication and repair of mitochondrial DNA (mtDNA), while the latter for degradation of damaged mtDNA molecules.

We used TaqMan probes to analyze two SNPs: c.1370T>A – POLG (rs1054875) and c.-394T>C – ENDOG (rs2977998) in 473 DNA samples (255 DD cases and 218 controls).

For c.-1370T>A – POLG we did not find any statistically significant differences between controls and cases.

However, in case of c.-394T>C – ENDOG we found that C/C genotype and C allele increased the risk of DD occurrence, whereas T/T genotype and T allele of the same SNP decreased this risk.

We showed for the first time that SNPs of genes involved in degradation of damaged mtDNA may modulate the risk of depression occurrence.

Acknowledgements Supported by the Polish National Science Centre (grant no. DEC-2014/13/N/ NZ7/00232 and no. UMO-2015/19/BNZ7/00410).

Joanna Czechowska, Aneta Zima, Dominika Siek, Anna Ślosarczyk

References [1]Dorozhkin SV (2009) J Mater Sci 44(9): 2343–2387 [2]Olkowski R, Kaszczewski P, Czechowska J, Siek D, Pijocha D, Zima A, Ślósarczyk A, Lewandowska-Szumieł M (2015) J Mater Sci Mater Med 26(12): 270

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 127

P9.6

P9.7

Permeate level of calcium to small intestine according to composition their formulation

Study of an active substance release from hydrogel polymer nanocomposites

Barbara Dolińska1,2, Marta Jelińska2, Aneta Ostróżka-Cieślik1 Florian Ryszka2

Ewelina Łęgowska1, Małgorzata Grzywińska1, Marta Miotke 1, Justyna Strankowska1,Michał Strankowski2, Łukasz Piszczyk 2,Jerzy Kwela1

1 Department of Applied Pharmacy, School of Pharmacy and the Division of Laboratory Medicine, Medical University of Silesia, Poland; 2Pharmaceutical Research and Production Plant „Biochefa”, Sosnowiec

Problem to increase effectiveness of calcium supplementation due to effects and costs is still valid. For that matter they examined composition of water formulation containing eggshell in form of calcium salt : carbonate (F-1; F-2) or citrate (F-3) relative to chelate compound as bisglycinate (F-4 reference). Additionally formulation contained F-1 (118 mg inulin; 96 mg starch); F-2 (400 mg inulin; 500 mg starch; 2,5 mg vitamin D3); F-3 (6 mg collagen; 5 mg vitamin D3), F-4 (1,2 mg vitamin D3). Formulation was prepared in the form 5 mmol calcium solution in 0,1 M solution of hydrochloric acid. They have examined calcium permeate from small intestine segment in diffusion cells by Frantz. Most of all calcium permeate from solution F-3 (~30%), then from F-2 (~25%), and F-1 (~23%) and the least from F-4 (~19%). 1,3 times more calcium permeate from solution calcium carbonate with vitamin D3 and collagen (F-3) in comparison to solution of calcium carbonate itself (F-1). They discovered that type of salt (carbonate, citrate, bisglycinate) and suplement of vitamin D3 and collagen have significant influence for permeate calcium level. Vitamin D3 and collagen increase this proces.

1 Institute of Experimental Physics, Faculty of Mathematics, Physics and Informatics, University of Gdansk Wita Stwosza 57, 80-308 Gdansk, Poland; 2 Department of Polymer Technology, Faculty of Chemistry, Gdansk University of Technology Narutowicza 11/12, 80-233 Gdansk, Poland

Keywords: nanocomposite, montmorillonite, paracetamol Hydrogel polymer nanocomposites are a new group of materials with wide application possibilities. These hybrid materials are composed of polymer matrix, in which nanofiller particles are dispersed. The nanofiller presence in the polymer matrix can significantly change the mechanical and swelling properties of the system [1, 2].

In our studies, the layered structured montmorillonite nanoparticles were dispersed in polyurethane matrix.

The aim of the study was to investigate the transport mechanism of the paracetamol in a hydrogel polymer nanocomposite matrix with different concentration of ­Cloisite®30B nanoparticles. Analysis of the swelling and release curves allowed to determine the diffusion mechanism in studied systems using known theoretical models [3].

The swelling measurements were done for samples containing 0%, 0.5% and 1% wt. of nanofiller in 1% concentration of paracetamol in ethanol solution in water. These studies have shown that transport of the active substance strongly depends on the amount of nanofiller. When concentration of nanofiller increases, value of swelling parameter also increases.

In case of release studies,the lowest absorbances were registered for the matrix containing 1% Cloisite 30 ® B. These results show that, as concentration of nanofiller increases, the barrier effect for paracetamol molecules is stronger.

The results of release and swelling measurements allow to conclude that hydrogel nanocomposite PU/PEG 4000 with 1% concentration of nanofiller can be used as sustained release drug delivery wound dressing.

References [1] S.S. Ray, M. Okamoto, Elsevier, Prog. Polym. Sci. 28 (2003) 1539–164.

[2] J. Strankowska, Ł. Piszczyk, M. Strankowski, M. Danowska, K. Szutkowski, S. Jurga, J. Kwela, The European Physical Journal Special Topic (2013), 222, 2179–2186.

[3] P. R. Pepas, Journal of Controllled Release (1987), 23–26.

6th Central European Congress of Life Sciences Eurobiotech 2017

128 Abstracts

P9.8

P9.9

Diagnostic of parasites using novel luminescent dyes and confocal laser scanning microscopy

Bioactivity tests of calcium phosphates with variant molar ratios of main components

Muza Kirjušina, Aleksandrs Pučkins, Ilona Mickeviča, Jeļena Kirilova, Sergejs Osipovs, Ilze Rubeniņa, Inese Jahundoviča

Klaudia Pluta1, Agnieszka Sobczak-Kupiec1, Olga Półtorak1, Dagmara Malina1, Bożena Tyliszczak 2

Daugavpils University, Institute of Life Science and Technology, Parādes street 1A, Daugavpils, Latvia, LV-5401 Keywords: diagnostic, parasite, CLSM The importance of microscopic techniques with the use of various fixatives and dyes for high quality resolution data and diagnosis of the parasites is significant. Three systematic groups of parasites monogenea and flukes and protozoa with morphologically and physiologically complex structure were selected for investigation of parasite morphology by luminescent dyes. Our new synthesized highly luminescent lipophilic benzanthrone derivatives with emission near 600–650 nm dyes are the analogs of cell membrane hydrophobic probe – 3-methoxybenzanthrone, these derivatives are polarity-sensitive fluorescent, which have large quantum yield, high photostability, and low cytotoxicity. The samples were examined with Nikon ECLIPSE Ti-E A1+ confocal microscope system, where fluorescence was initiated by high-intensity lasers (561 nm and 638 nm wavelength). Fluorescence emission was detected in the range 525–750 nm. In conclusion, the detailed visualization of Dactylogyrus by P10 and ABM luminescent dyes provided an image with a more detailed shape of attachment apparatus and chitinous part of copulatory organ. Parasite internal morphological features image provided by dyeing with AM20 and P6 dyes. The external morphological structure of Diplostomum metacercaria and Sarcocystis cyst internal structure and cysts wall clearly visualized with P8 luminescent dye, which is essential for diagnostic.

1 Institute of Inorganic Chemistry and Technology, Cracow University of Technology, Poland; 2Department of Chemistry and Technology of Polymers, Cracow University of Technology, Poland

The first clinical trial of calcium phosphate (CPs) application as a bone substitute was made in 1920 – Albee discovered that tricalcium phosphate (TCP) positively affects the healing of bone fractures in rabbits. Since then, interest in CPs exhibiting a chemical and mineralogical similarity to inorganic phase of bones, significantly increases. Biomaterial engineering is mainly focused on two compounds – TCP being a compound in CaO-P2O5 oxide system with Ca/P molar ratio of 1.50, and hydroxyapatite (HAp) in CaO-P2O5-H2O oxide system with a molar ratio of 1.67.

In this paper, the initial bioactivity tests in simulated body fluids of natural and synthetic hydroxyapate (HAp) and tricalcium phosphate (TCP) with variable Ca/P molar ratios (1.0; 1.5; and 2.0) were conducted. The behavior of fittings before and after immersion in mentioned fluids were determined and the influence of the Ca/P values was exemplified.

Acknowledgements This research was financially supported by Gant LIDER 037/481/L-5/13/ NCBR/2014.

Acknowledgements The study funded by the project No. 1.1.1.1/16/A/211.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 129

P9.10

P9.11

The influence of cellulose nanocrystals (CNC) on the structure and dynamic mechanical properties of biopolyamide 10.10

Analysis of downregulation of α-gal epitope expression in the skin and liver of transgenic pigs as a model for xenotransplantation

Paulina Radzik, Agnieszka Leszczyńska, Agnieszka Niedziela, Katarzyna Leszko, Krzysztof Pielichowski

Jerzy Wiater1, Zdzisław Smorąg 2, Ryszard Słomski3, Janusz Karasiński1, Marek Romek 1

Department of Chemistry and Technology of Polymers, Cracow University of Technology, 24 Warszawska St., 31-155 Cracow, Poland Keywords: cellulose nanocrystals, biopolyamides, nanocomposites The development of fully biobased nanocomposites for engineering applications is currently a challenging task.

In this work we report the production of bio-polyamide 10.10/cellulose nanocrystals (CNC) composite materials with improved dynamic mechanical properties. CNC with sufficient thermal stability for melt blending with bioPA 10.10 were produced by hydrolysis in phosphoric acid and successive reaction with the succinic acid anhydride (SA).

The tested molar ratios of SA to cellulose hydroxyl groups were 2:1, 3:1, 5:1 and 10:1 and showed optimum value in terms of thermal stability of CNC at 3:1. The crystalline structure and mechanical properties of PA10.10 and its nanocomposites were investigated by WAXD, FTIR, DSC and DMTA methods. Bionanocomposites had higher crystallinity degrees than pristine PA 10.10. Additionally, the effect of CNC on the activation energy (Ea) of the α relaxation of bioPA was also investigated. Values of the Ea for composite materials were lower in comparison with unmodified PA 10.10.

References LEITE L et al (2016) Journal of Applied Polymer Science 133: 1–10.

Acknowledgements This research work was supported by Polish National Science Centre under the grant No. UMO/2015/19/B/ST8/01060.

1 Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków, Gronostajowa 9, 30-387 Kraków, Poland; 2Department of Animal Reproduction Biotechnology, National Research Institute of Animal Production in Balice near Kraków, Krakowska 1, 32-083 Balice, Poland; 3Institute of Human Genetics Polish Academy of Sciences, Strzeszyńska 32, 60-479 Poznań, Poland

Xenotransplantation from pig-to-human may overcome the worldwide organ donor shortage. There are several strategies to reduce the amount of α-gal epitope on the surface of pig cells. This epitope is involved in hyperacute rejection of xenograft.

The aim of this study was to compare the efficiency of four transgenesis methods for the reducing of α-gal epitope expression in the skin and liver of pigs.

For experiments we used pigs, which show the expression of human genes: FUT2, GLA, both FUT2×GLA, and the α1,3-galactosyltransferase knock-out (GTKO). Experiments were performed on tissue sections and cells cultured in vitro by using immunofluorescence, Western blot and qRT-PCR. Our results show that the level of α-gal epitope expression in the skin and liver of transgenic pigs is lower compared to control group (non-transgenic). The lowest expression was recorded in tissues and cells from double transgenic pigs (FUT2×GLA). Interestingly, the level of α-gal epitope expression in the skin and liver of GTKO pigs was lower than control group but higher than in other groups.

Summarizing, each of the used transgenesis methods led to decreasing of the α-gal epitope expression. The best way to reduce the amount of α-gal on the surface of skin and liver cells seems to be a transgenesis method based on the introduction of both genes encoding human α-galactosidase and α1,2-fucosyltransferase enzymes.

Acknowledgements Supported by grant of The National Centre for Research and Development INNOMED/I/17/NCBR/2014

6th Central European Congress of Life Sciences Eurobiotech 2017

130 Abstracts

P9.12

P9.13

Association between single nucleotide polymorphisms of TPH1 and TPH2 genes and depressive disorders

Growth of adipose mesenchymal stromal cells on different scaffolds – polymers, bacterial cellulose and acellular swine pericardium

Paulina Wigner1, Piotr Czarny2, Tomasz Sliwinski1

Ewa Wiśniewska1, Klaudia Kulik1, Agnieszka Langrzyk1, Marcin Szustak 2, Stanisław Bielecki2, Michał Sobota3, Jakub Włodarczyk3, Janusz Kasperczyk3, Paulina Gach4, Karolina Jendryczko4, Martyna Faber4, Piotr Wilczek4, Józef Dulak1,5

Laboratory of Medical Genetics University of Lodz, Lodz, Poland; 2 Department of Medical Biochemistry, Medical University of Lodz, Lodz, Poland 1

Keywords: depression, tryptophan hydroxylase, single nucleotide polymorphism Background: Disorder of tryptophan catabolites pathway is observed in patients with depression. Moreover, single nucleotide polymorphisms of tryptophan hydroxylase genes (TPH) may modulate the risk of occurrence of the disease. The aim of our study was confirm of the association between presence of polymorphic variants of TPH1 and TPH2 genes and development of depressive disorders.

Methods: We selected two polymorphisms: c.803+221C>A (rs1800532) of TPH1, and c.-844G>T (rs4570625) of TPH2. A total of 510 DNA samples (230 controls and 280 patients) were genotyped using TaqMan probes.

Result: Among the studied SNPs the T/T homozygote of c.803+221C>A – TPH1, the G/G homozygote and G allele of c.-844G>T – TPH2 were associated with the depression occurrence. However, the C/A heterozygote of c.803+221C>A – TPH1 decreased the risk of development of the depressive disorders.

Conclusion: Each of the studied polymorphisms modulated risk of depression for selected genotypes and alleles.

These results support the hypothesis on the involvement of TRYCATs pathway in pathogenesis of depressive disorders.

Acknowledgements Source of support: This study was supported with funding from scientific research grant from the Polish National Science Centre (no. UMO-2015/19/BNZ7/00410)

1 Regenerative Medicine and Isolated Tissue and Organ Laboratory, Kardio-Med Silesia Sp. z o.o., Science and Technology Park, 10C M. Skłodowskiej- Curie St., 41-800 Zabrze, Poland; 2Institute of Technical Biochemistry – Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 4/10 Stefanowskiego St., 90-924 Łódź, Poland; 3Centre of Polymer and Carbon Materials, Polish Academy of Sciences, 34 M. Curie-Skłodowska St., 41-819 Zabrze, Poland; 4Heart Prosthesis Institute, Bioengineering Laboratory, Wolności 345a St., 41-800, Zabrze, Poland; 5Department of Medical Biotechnology, Jagiellonian University, 7 Gronostajowa St., 30-387 Krakow, Poland

There is an increased interest in application of biomaterials as scaffolds for MSCs (Mesenchymal Stromal Cells) in regenerative medicine. The most accessible source of MSCs is adipose tissue, because of minimally invasive collecting procedure and possibility to obtain an abundant quantities.

The aim of the study was to determine the viability of cells cultured on tested biomaterials, determine proliferation capacity (immunostaining of antigen-Ki67) and degree of adhesion of MSCs to the tested biomaterials.

MSCs using in this study were isolated from human adipose tissue, collected during liposuction from anterior abdominal wall. MSCs expressed surface antigens: CD105, CD73, CD29, CD44, CD90 and were devoid of hematopoietic (CD45, CD34), endothelial (CD31) cells antigens. Cells was seeded on the tested biomaterials: PLGA 85/15 (poly(lactic-co-glycolic acid)), PLGA 70/30, LP 309 (poly(ester-carbonate)), bacterial cellulose (produced by K. xylinus E25 and K. hansenii) and acellular swine pericardium. These materials have ability to biodegrade after implantation or are adapted by the human body or/and are undergoing reconstruction.

MSCs demonstrate affinity to all of tested biomaterials. Double staining using Calcein-AM and Propidium Iodide visualize good viability of MSCs seeded on all tested carriers, but immunofluorescent staining with anti-Ki67 antibody shows that MSCs proliferate only on selected biomaterials.

Acknowledgements Financed by grant No STRATEGMED2/269415/11/NCBR/2015.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 131

Microbial Cell Factories Keynote lecture

Lectures

L10.1

L10.2

Bacteriophages – cell factories for metal and metal oxide nanoparticle synthesis

The perspectives of biodegradation of chloroaromatic pollutants enhanced with natural surfactants

Joanna Karczewska-Golec1, Kamila Żelechowska2, Piotr Golec3, Andrzej M. Kłonkowski4, and Grzegorz Węgrzyn1

Wojciech Smułek, Amanda Pacholak, Ewa Kaczorek

Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland; 2 Faculty of Applied Physics and Mathematics, Solid State Physics Department, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland; 3 Laboratory of Molecular Biology (affiliated with the University of Gdansk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland; 4 Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland

1

Keywords: bacteriophage M13, phages, phage display, nanoparticles, metal nanoparticles, metal oxide nanoparticles With their unique physical and chemical properties, metal and metal oxide nanoparticles (NPs) have been of great interest both in industrial and scientific settings over the last decade, offering potential for breakthrough (bio)technological applications, but also presenting challenges to scientists. In terms of value, the global metal nanoparticle market is expected to reach nearly USD 51 billion by 2026, while the market size for Au nanoparticles alone is anticipated to cross USD 8 billion by 2022. One important challenge in nanoparticle research and development is that the conventional physico-chemical methods for the synthesis of metal and metal oxide nanoparticles are not environmentally friendly, efficient enough or cost-effective. This presentation will focus on the use of novel biological systems as an attractive green alternative for nanoparticle synthesis, and will explore the application of bacteriophages exposing selected peptides on virions for an efficient synthesis of ZnO, Eu2O3 and Au nanoparticles, which we have recently – for the first time – demonstrated.

The successful formation of such NPs was confirmed, and characterization of their detailed properties was performed, using atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-vis absorption spectroscopy, luminescence spectroscopy, energy-dispersive X-ray (EDX) spectroscopy and X-ray diffraction (XRD) analyses, among other approaches.

Institute of Chemical Technology and Engineering, Poznan University of Technology, ul. Berdychowo 4, 60-965 Poznań, Poland Keywords: biodegradation, chloroaromatics, natural surfactants, saponins Chlorinated aromatic compounds are widely used as solvents and reagents. What is important and many of them are recognized as the persistent organic pollutants. One of the factors limiting their biodegradation is low solubility in water and high hydrophobicity. Among the methods increasing bioavailability of the chlorinated aromatic compounds the surfactant addition is used.

The presented research focused on biodegradation of chlorinated solvents, like chlorotoluene isomers, by environmental bacteria in the presence of natural surfactants – saponins, isolated from Sapindus mukorossi and Hedera helix. Apart from biodegradation test, the changes in permeability of the cell membrane was evaluated using colorimetric methods. Additionally, the toxicity of the saponins used were also measured.

As it could be noticed, in the presence of saponins biodegradation of the chloroaromatics increased, what can be connected with the modifications in cell membrane permeability. Moreover, the saponins at concentration below their critical micelle concentration was practically no toxic to tested environmental bacteria. To summarize, the collected data indicate, that the saponins could be valuable supplement increasing biodegradability of the chlorinated aromatic compounds.

Acknowledgements This study was supported by the National Centre of Science (Poland) awarded by the decision number DEC-2015/19/N/NZ9/02423

6th Central European Congress of Life Sciences Eurobiotech 2017

132 Abstracts

L10.3

L10.4

The influence of growth conditions on the rhamnolipids production by 20 different Pseudomons aeruginosa species

Effect of phthalocyanines on S. epidermidis cells. In vitro studies

Marta Wozniak-Karczewska1, Kamila Myszka2, Łukasz Ławniczak1, Agnieszka Zgoła-Grześkowiak3, Dorota Formanowicz4, Łukasz Chrzanowski1 Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2 Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznan, Poznan, Poland; 3 Institute of Chemistry, Poznan University of Technology, Poznan, Poland; 4 Department of Clinical Biochemistry and Laboratory Medicine, Poznan University of Medical Sciences, Poland

Karolina Jakubczyk, Katarzyna Barchiewicz, Gabriela Dyrda Department of General Chemistry, Faculty of Chemistry, Opole University

1

Rhamnolipids have an important role in bacterial life and activity. Their ability to modify the surface hydrophobicity of bacterial cells is broadly studied with respect to their cell motility. Still the actual role of rhamnolipids in the environment remains unexplained.

Thus the aim of this study was to determine the qualitative and quantitate changes of rhamnolipids, originating from P. aeruginosa with respect to variable growth conditions – different carbon sources and concentrations of nitrogen. Additionally, the biodegradation of diesel and FAMEs were evaluated in relation to nitrogen conditions.

Twenty Pseudomonas aeruginosa species (16S rRNA) were isolated from the internal organs of deceased chickens (i.e. lungs and air sacs). The cause of birds’ death, which have been previously diagnosed as inflammatory changes in the respiratory tract (chronic respiratory disease CRD), was a secondary bacterial infection. The ability of rhamnolipids’ synthesis by P. aeruginosa species was also determined.

It was observed that the secretion of rhamnolipids by the same Pseudomonas aeruginosa species was medium-dependent. Low concentration of nitrogen and presence of glucose in growth medium creates favorable conditions for dirhamnolipids secretion. The biodegradation of hydrocarbon fractions is also medium-dependent e.g. the same strains utilize FAMEs as sole carbon source in low N-condition, while no degradation was observed in the presence of NH4Cl.

Keywords: phthalocyanines, PDT, Staphylococcus epidermidis In the age of rapid progress in medical sciences and biotechnology, new ways are being sought to effectively combat antibiotic-resistant microorganisms. The drug resistance is a major problem in the treatment of infections caused by bacteria from the Staphylococcus strain. The possibility of intercellular transfer of drug resistant genes and biofilm formation by bacteria render difficult a classical antibiotic therapy.

Recently, the potential application of phthalocyanines (Pc) in photodynamic therapy has raised interest, both against tumors and microorganisms.These porphyrin derivatives have interesting spectroscopic properties and may be involved in photodynamic reactions. Since they may generate reactive oxygen species (ROS) in a photochemical process, they possess the ability to destroy cells by apoptosis or necrosis.

The effect of selected metallophthalocyanines, including MgPc, SnPc and CrPc, on the cells of Staphylococcus epidermidis was investigated in vitro. The viable count of cells exposed to the particular phthalocyanines was determined using typical microbiological methods, while their inclusion within the cells was checked by spectroscopic methods. The results indicated that the viability of cells depends on the kind of phthalocyanine present in the medium, however only MgPc was found in the cell lysates.

These preliminary results are considered the basis for further investigations.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 133

L10.5

L10.6

How do bacteria adapt to long-term hydrocarbons exposure?

The role of open reading frame 63 (orf63) in the development of phage λ and Shiga toxin- converting Φ24B bacterophage

Agata Zdarta, Ewa Kaczorek Poznan University of Technology, Department of Chemical Technology, Poland Keywords: bacteria, adaptation, hydrocarbons, longterm exposure Bacteria can alter their properties to adapt to the changes in their environment, but only certain are able to metabolize toxic substrates such as hydrocarbons. The main objective of this research was to determine the impact of microbial exposure on hydrocarbon impurities on the adaptability of bacteria to biodegradation of aliphatic and aromatic compounds.For this purposes, microorganisms were isolated, and those capable of growth in the presence of hydrocarbon impurities were subjected to a 12-months exposure to aliphatic and aromatic hydrocarbons. At the end of the exposure period, changes in membrane compositions of fatty acids and protein profiles were analyzed and compared to those obtained for control strains. The results of the study showed significant changes in the properties of cells exposed to hydrocarbon contaminants compared to the control samples. In addition, the proteomic analyzes of the hydrocarbon-exposed strains showed that the cells significantly altered the activity of the intracellular protein apparatus, allowing them to degrade more effectively the available metabolic substrates.

Acknowledgements This study was supported by The National Science Centre awarded by decisions number DEC-2015/19/N/NZ9/02422.

Aleksandra Dydecka1, Sylwia Bloch1, Gracja Topka1, Bożena Nejman-Faleńczyk 1, Agnieszka Necel1, Katarzyna Licznerska 1, Tomasz Gąsior2, Grzegorz Węgrzyn 1, Alicja Węgrzyn 2 Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, Gdansk, Poland; 2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland 1

High pathogenicity of enterohemorrhagic Escherichia coli (EHEC) strains depends on production of Shiga toxins, encoded by stx genes, which are located in genomes of Shiga toxins-converting bacteriophages (Stx phages). Effective production of these toxins occur only upon prophage induction and its further lytic development.

Products encoded in the exo-xis region of genome of bacteriophage λ and stx-carrying Φ24B phage, may be the potential factors involved in the regulation of lambdoid phage development. This region contains highly conserved open reading frames (ORFs) and two genes of largely unknown functions. This genetic elements could be involved in the regulation of lysogenization and prophage induction processes. The most recent results of our research indicate that the deletion of the exo-xis region negatively influences the expression of regulatory genes of Φ24B. I decided to determine the role of the deletions of particular elements from the exo-xis region in the development of recombinant lambdoid phages. Based on the preliminary results of my experiments, I can conclude that orf63 from analyzed region has a special role in the control of phage development, especially at the stage of the lysis vs. lysogenization decision. This elements may influence: the changes of the time of prophage induction with use hydrogen peroxide, survival of the E. coli bacteria, intracellular lambdoid phage lytic development and lysogenization of host bacteria.

6th Central European Congress of Life Sciences Eurobiotech 2017

134 Abstracts

Posters P10.1 NADH-dependent erythrose reductase from Yarrowia lipolytica: production and biochemical characterization of the enzyme Tomasz Janek1, Adam Dobrowolski2, Anna Biegalska2, Aleksandra M. Mirończuk 2 1 Wroclaw Medical University, Department of Inorganic Chemistry, Wrocław, Poland; 2Wroclaw University of Environmental and Life Sciences, Department of Biotechnology and Food Microbiology, Wrocław, Poland

Erythritol is a natural sweetener that is produced as an osmoprotectant by microorganisms. Glycerol is a low-cost, main waste product from biodiesel industry. Despite many studies on erythritol synthesis by Yarrowia lipolytica [1], the enzymes involved in its metabolic pathway have never been described. In other microorganisms it has been shown that erythrose reductase (ER), which catalyzes the final step, plays a crucial role in erythritol synthesis. In this study, we chose the yeast Yarrowia lipolytica as a source for an ER gene. For the first time, we identified the ER gene from Y. lipolytica (YlER) that encodes a protein with a predicted molecular weight of ~37 kDa. Moreover, the biochemical properties of YlER were investigated. The enzyme showed strict NADH dependence and broad substrate specificity, with the highest specific activity against D-erythrose. The optimal pH for ER activity is 3.0, at higher pH, its activity significantly decreases, and only about 15% of maximal activity was noted at pH above 4.0. Due to the obtained data the process optimization was performed.

The addition of Zn2+ ions had a positive effect on the activity of YlER, as well as on the erythritol production. In conclusion, this is the first report describing an efficient production of erythritol by genetically modified microorganisms by overexpression of the native ER. References [1]. Mirończuk AM et al (2017) Biotechnology for Biofuels 10: 77 Acknowledgements This work was financed by the Polish National Center for Research and Development under project LIDER/010/207/L-5/13/NCBR/2014.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 135

Plants for the European Bioeconomy Keynote lecture

Lectures

L11.1

L11.2

Microbiome in the context of FOOD2030 and Food systems”

Registration EMEA

Marios Markakis Directorate-General for Research and Innovation European Commission; Brussels – Belgium The recent past has brought us obesity, malnutrition, emergence of food borne hazards, greenhouse gases, waste, and resource depletion. A radical rethink of how we produce, obtain and prepare our food, our nutritional wellbeing, and our water and energy usage is needed.

Today, complete human genomes can be sequenced in a matter of days due to the increasingly rapid technological advances that have taken place since the first Human Genome Project. These ground-breaking research and innovation advances have opened an entirely new chapter in genomics and have also enabled the genomic characterization of other species or systems such as microbial ecosystems. Everybody and everything is surrounded by a microbial community called a microbiome, from the metro station to the office, to the field of wheat, or the fisherman and his catch. Understanding what microbiomes do, what they are, and how they interact is a new scientific frontier made easier by rapid advances in genomic mapping, robotics, and chemical analysis. What we know and understand so far is that the microbiome has an essential impact on our health and food we produced, on plants and animals and on ecosystems in general. Unravelling their complexity offers huge potential for innovation and will be a major game changer in the way we manage our planet, obtain our food, and remain healthy.

In a similar vein the “food system” approach advocated and described under the FOOD 2030[1] initiative would benefit hugely from knowledge of the plant, marine, animal, environmental and human microbiomes. The impact will be in strengthening and improving the overall sustainability and innovation capacity of food systems and in bringing to market new and cost-effective commercial applications. Such an approach will be in full support of the Sustainable Development Goals and climate change commitments.

Overall, we aim at structuring, connecting and eventually co-ordinating world-wide microbiome R&I, with a common policy objective goal and concrete outcomes.

Co-ordination in the planning and in the implementation of the R&I activities would ensure coherence, synergy and leverage for the delivery, without imposing any constraint on each respective programme.

Camilla Liput Bayer: Science For A Better Life Title: EU legal framework for plants developed through biotechnology: present and future Currently in the EU the commercialization of GM plants is following a process-based approach resulting in extensive regulatory requirements for plants developed through certain techniques. In light of the regulatory framework no new products since 1998 have been approved for cultivation purposes in the EU. Recent scientific advancements provide the chance for re-evaluating the current legal framework to regulate the development of modern biotechnology, specifically of GMOs. Genome editing techniques such as the CRISPR-Cas technology challenge current process-based regulations and raise the question if evaluating the final product instead of the process is more suitable.

Therefore, plants developed with recent plant breeding techniques should not fall under the current EU GM regulation (Directive 2001/18) if the final plant product contains solely the stable insertion of inherited genetic material from sexually compatible plant species or if the genetic variation is the result of spontaneous or induced mutagenesis.

References [1] https://publications.europa.eu/en/publication-detail/-/publication/709af455 -c03d-11e6-a6db-01aa75ed71a1

6th Central European Congress of Life Sciences Eurobiotech 2017

136 Abstracts

L11.3

L11.4

Leaf vegetables as important source of precursor omega–3 long chain polyunsaturated fatty acids essential in the human diet

The use of biodiversity of species of the Poaceae family in wheat cultivar T. aestivum L development

Monika Bojko , Monika Olchawa-Pajor , Marek Chyc2, Dariusz Latowski1 1

1

Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland; 2Department of Environmental Protection, State Higher Vocational School in Tarnów, ul. Mickiewicza 8, 33-100 Tarnów, Poland 1

In the last decade it was widely reported that omega–3 long chain polyunsaturated fatty acids (n-3 LC-PUFA) are beneficial for human health. Currently, the main source of n-3 LC-PUFA for human are marine fishes. The precursor of other long chains in omega-3 family is α-linoleic acid (ALA, 18:3n-3 n) which is synthetized from linoleic acid (LA, 18:2n-6) and exists in plant chloroplast membranes.

In dietary intake LA and ALA are mainly delivered from vegetable oils. Moreover, it has been estimated that present human diet is deficient in omega-3 fatty acids.

The aim of these studies was to prove that leaf vegetables are the source of ALA in the human diet. Using the gas chromatography, fatty acids composition in leaves of arugula (rucola), kohlrabi, several lettuces and spinach varieties were analyzed. Additionally, the effect of low temperature storage on fatty acids profile of leaf vegetables were considered and the analysis of fatty acids composition of the leaves of lettuces were carried out separately in outer (dark green) and inner (light green) leaves.

We showed that ALA is the most dominant fatty acid in leaf vegetables. ALA content in outer dark green leaves were up to 50% higher than in inner light green ones. The lowest content of ALA as well as the lowest ratio of ALA to LA was observed in kohlrabi leaves. Short time storage of dark green leaves under low temperature caused increase of ALA level together with decrease of LA and saturated fatty acids content.

Andrzej Z. Czaplicki1, Józef Pilch1, Jacek Żebrowski2, Katarzyna Makowska1, Janusz Zimny1 1 Plant Breeding and Acclimatization Institute – NRI, Radzików, Poland; 2University of Rzeszów, Rzeszów, Poland

Wheat and its relatives have evolved through divergence at the diploid level, followed by evolutionary convergence via polyploidy. This type of reticulate evolution has led to immense variability in the wheat tribe. According to the hypothesis of pivotal‑differential evolution, one genome of an allopolyploid such as wheat remains stable during evolution, and serves as the “pivotal” genome. The other genomes become modified (i.e., differential), partially due to crossing with other sympatric polyploid species which have a common pivotal genome. Acceptance of this hypothesis has not only academic implications, but also influences the way plant breeders design and implement their crossing programs.

These species contain both homeological chromosomes and rDNA sequences very similar to T. aestivum L. Such a system allows the introgression of alien genes and their incorporation into the genomes A, B and D of wheat, where they can function permanently. Many of them have already been transferred to the varieties of T. aestivum L. But not all the species could be used in the alien-gene-introgressions because of the anatomical, physiological and genetical reasons. The obtained lines were developed using: (1) interspecific and intergeneric generative hybridization, (2) the genetic systems of wheat (T. aestivum L) including the crossability genes (Kr), and the Ph genes – homeology/homology pairing system (5B-chromosome system), and (3) positive selection directed on both spike and grain characters.

In total, 543 plants was regenerated by androgenesis process. The obtained lines showed improved connections of 7–11 features in 15 different combinations, giving breeders the ability to choose the line with the desired parameters.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 137

L11.5

Posters

Extension of oil biosynthesis during the mid-phase of seed maturation increases seed oil in Arabidopsis thaliana

P11.1

Masatake Kanai1, Shoji Mano1,2, Maki Kondo1, Makoto Hayashi3, Mikio Nishimura1 1 National Institute for Basic Biology, Japan; 2SOKENDAI (The Graduate University for Advanced Studies), japan; 3 Nagahama Institute of Bio-Science and Technology, Japan

Regulation of oil biosynthesis in plant seeds has been studied, and transgenic approaches have been designed to enhance seed oil content. Oil and protein synthesis is negatively correlated in seeds, but the mechanisms controlling interactions between these two pathways are unknown. To understand the molecular mechanism controlling oil and protein content in seeds, we utilized transgenic Arabidopsis plants overexpressing WRINKLED1 (WRI1), a master transcription factor controlling seed oil biosynthesis, and knockout mutants of major seed storage proteins. Oil and protein synthesis in wild-type plants was sequentially activated during early and late seed maturation, respectively.

The negative correlation between oil and protein contents in seeds arises from competition between the pathways.

Extension of WRI1 expression during mid-phase of seed maturation significantly increased seed oil content. This study demonstrates that temporal activation of genes involved in oil biosynthesis enhances the seed oil content in Arabidopsis thaliana. These results provide novel insights into potential breeding strategies to generate crops with high oil contents in seeds.

References Kanai M et al (2016) Plant Biotechnol J. 14: 1241–1250

Factors affecting seed yield in selected Festuca species Grzegorz Żurek1, Kamil Prokopiuk1, Danuta Martyniak1, Eugeniusz Paszkowski2, Urszula Woźna-Pawlak3, Maciej Jurkowski4 Instytut Hodowli i Aklimatyzacji Roślin, Państwowy Instytut Badawczy, Radzików, 05-870 Błonie; 2 DANKO Hodowla Roślin sp. z o.o., 64-000 Kościan; 3 Poznańska Hodowla Roślin, sp. z o.o., 63-004 Tulce; 4 Małopolska Hodowla Roślin, sp. z o.o., 30-002 Kraków 1

An experiment has been performed in four locations in Poland (Radzików, Leszno, Szelejewo and Nieznanice).

Fifteen genotypes from three species (Festuca arundinacea, F. pratensis and F. rubra) were measured and observed during two consequtive years. Despite of phenological observations (heading and flowering start dates), biometrical measurements (plant height, leaf dimension, number of generative stems etc.) and some physiological traits(chlorophyll contents index, nitrogen contents) seed yield of single panicle, seed yield of plant and seed yield per plot (i.e. 50 plants) were determined.Temperature and percipitation averges specific for each location were also used for calculations.

Multiple linear regression analysis with a forward stepwise variable selection procedure was used to estimate major factors affecting seed yield. It resulted in different predictors for different species.

For meadow fescue almost all traits (18 from all 19) used were selected and 16 were statistically significant.

For tall fescue it was 12 with 4 statistically significant, but for red fescue only 6 with 3 statistically significant.

Significant predictors had different weights in different species. Specific genotype features were significant only in meadow fescue, also with percipitation averges and many other traits. For tall fescue, location termal characteristic (normal value for growing season) were negatively related to seed yield. Conclusion is that for each species different measures shuld applied to predict seed yield.

6th Central European Congress of Life Sciences Eurobiotech 2017

138 Abstracts

Modern instrumental analysis in biotechnology and biomedicine Keynote lecture

Lectures

L12.1

L12.2

Improving workflows for MS- based proteomics by spectral libraries

Childhood tuberculosis – in search of diagnostic biomarkers

Sabryna Juncker, Christian Hentschker, Kristina Plate, Andreas Otto, Sandra Maass and Dörte Becher

Rafał Szewczyk1, Michał Seweryn2, Magdalena Druszczyńska3

Institute for Microbiology, Department for Microbial Proteomics, University of Greifswald, Germany

1

Mass spectrometry based proteome analyses are extensively used in basic sciences for elucidation of biological processes as well as for control of production steps in biotechnology industry. Here, discovery experiments involve sample preparation, LC-MS measurements and data analysis by database search algorithms. The last years, besides the vast range of database search algorithms the concept of spectral library searching using previously acquired data has been introduced into proteomics workflows.

Although especially helpful in differential analyses in peptide centred shotgun proteomics, assays relying on MSbased proteomics looking at post translational modifications at a large scale are still challenging especially for bacteria. This challenge may be overcome by improvements in sample preparation as well as in mass spectrometry instrumentation but compared to PTM analyses in eukaryotes the bacterial field is still lagging behind in terms of numbers of PTM identified. Therefore we have pursued the idea of using spectral libraries for improved analysis of PTM in complex samples. In doing so, we were able to not only to increase the numbers of e.g phosphopeptides identified but also to show that with regular search workflows, a large number of false positive identifications are being found in the results. We prove this by using synthetic peptides reflecting both anticipated positive and negative hits.

We went even a step further with using spectral libraries in comparison of different isolates of a pathogenic bacterium. Here, we first acquired large scale spectral data for laboratory strains that were used then for data analysis of data independent acquisition approaches based on SWATH. Relying on this, we are able to analyse clinical isolates without having the genomic information that would be necessary in regular data dependent shot gun approaches.

Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Biotechnology and Immunology, Faculty of Biology and Environmental Protection, University of Łódź, ul. Banacha 12/16, 90-237 Łódź; 2 Center for Medical Genomics OMICRON, Jagiellonian University Medical College, Kopernika 7c, 31-034 Kraków; 3 Department of Immunology and Infectious Biology, Institute of Microbiology, Biotechnology and Immunology, Faculty of Biology and Environmental Protection, University of Łódź, ul. Banacha 12/16, 90-237 Łódź

Currently available microbiological, serological or genetic diagnostic methods are not good enough to recognize tuberculosis (TB) in children. The lack of rapid diagnosis of TB in children has serious consequences because disease progression can occur more rapidly than in adults. Presented in this work data are focused on the low molecular weight compounds searching and profiling in the plasma samples collected from children. After QuantiFERON® TB Gold test the whole blood stimulated samples panel were prepared to LC-MS/MS analysis including target analysis of amino acids and their derivatives, metabolites and cofactors of glycolysis and TCA cycle and selected steroids and lipids and non-targeted analysis where the only criterion applied was a 100–1000 m/z MS scan range.

Obtained results in targeted and non-targeted workflows revealed significant differences both in the number and presence of certain molecules. The statistical tests resulted in an identification of major markers responsible for clear separation of the samples from children with early infection, developed disease, under medical treatment and healthy. Further analysis will allow the identification of biomarkers for active and latent M.tb infection, what may assess the risk of progression to active tuberculosis in children. Metabolomics data may also be used for the development of rapid diagnostic tests, new immunosuppressive vaccines as well as antituberculous drugs.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 139

L12.3

L12.4

Spectrophotometry in quantitative determination of platinum complexes on titania nanomaterials

Production, characterisation and mass spectrometry identification of novel multicopper oxidases from Myrothecium roridum

Adrian Topolski, Aleksandra Radtke, Piotr Piszczek Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń Development of modern materials is in close relation to instrumental methods which can be used for their characterization. A good evidence for that is a birth of modern nanotechnology and construction of Scanning Tunnelling Microscope by Gerd Binnig and Heinrich Rohrer in 1981.

Modern science offers many advanced techniques which let us analyze a lot of specific problems, e.g. surface coordination by adsorbed gas. These presentation discuss the use of old and well known spectrophotometry in quantitative determination of platinum complexes (K 2PtCl4 and some bimetallic platinum complexes) implemented on titania nanomaterials. A general procedure for that materials involves IR, Raman, and SEM analysis which can be enriched with TEM, XRD, or EDX techniques. However, the knowledge about molar absorption of platinum complexes in UV and relatively high molar absorption coefficients let us apply UV spectrophotometry as useful technique for qualitative and quantitative determination of platinum complexes on nanomaterial surface. The results show that there is a possibility to determine even nanomoles of the complexes on ca. 50 mm2 of titanium foil covered with titania nanoforms. Position of bands on spectrum informs about stability of the complexes. Additionally, kinetic tests on the complexes release from the surface in solution have been performed and analyzed.

Anna Jasińska, Aleksandra Góralczyk, Adrian Soboń, Jerzy Długoński University of Lodz, Faculty of Biology and Environmental Protection, Department of Industrial Microbiology and Biotechnology, Poland Keywords: Myrothecium roridum, multicopper oxidases, mass spectrometry Laccases are the most commonly investigated multicopper oxidases. This report describes the first attempt to improve their production, identify proteins using mass spectrometry and characterise M. roridum laccase-like multicopper oxidases (LMCOs). LMCOs yield was the highest (2184 U l-1) in the following conditions: 0.75% sucrose, 0.30% yeast extract, pH 6.50, and with copper supplementation. An SDS-PAGE and a native-PAGE revealed the presence of two Cu-inducible proteins with molecular weights of 67 and 50 kDa. Peptides analysis using the AB Sciex 5800 TOF/TOF system and Delta-BLAST searching allowed identifying proteins as multicopper oxidases.

The enzymes were relatively resistant to SDS and EDTA. LMCOs exhibited an activity toward ABTS, guaiacol and 2,6-DMP. They were also found to oxidize caffeic acid, catechol, pirogallol and bilirubin. These properties make LMCOs attractive candidates for specific environmental applications.

Acknowledgements This study was supported by the National Science Centre of Poland, project no.

UMO 2013/11/D/NZ9/02776.

6th Central European Congress of Life Sciences Eurobiotech 2017

140 Abstracts

L12.5

L12.6

DNA fragment analysis for sex identification of bird using capillary electrophoresis on 3100xl Genetic Analyzer

Evaluation of the Effects of S-Allyl Cysteine on Autophagy and Apoptosis in Human Leukemia Cell Line

Radko A., Podbielska A., Szumiec A., Bugno-Poniewierska M.

Neslihan Tekin1, Kamile Öztürk 2, Barış Kerimoğlu2, Mehtap Tarhan2

National Research Institute of Animal Production, Department of Animal Genomics and Molecular Biology, Krakowska 1, 32-083 Balice, Poland

1 Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey; 2Department of Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey

To identify the sex of birds, techniques of molecular biology are used increasingly, because nearly 50% of birds are sexually monomorphic. The CHD gene has been widely used to determine the sex of birds, due to the high degree of conservatism and the presence of polymorphic variants of the Z and W chromosomes. PCR products are usually visualized after electrophoresis on agarose gel, where PCR amplification of the CHD gene produces a double (ZW) and single (ZZ) bands in females and males, respectively. In our laboratory we have implemented method of capillary electrophoresis for identifications the bands. 69 individuals were tested from genera Columba and 45 individuals from genera Aprosmictus, Cacatua, Myiopsitta, Platycercus, Polytelis and Psittacula. Genomic DNAs were extracted from feathers and buccal swabs. Sex-specific products of CHD gene was amplified using the QIAGEN Multiplex PCR and specific pair primer. One of pair primers were labelled with fluorescent dye 6-FAM, the amplified products were separated on 3100xl Genetic Analyzer and genotyped using GeneMapper software (Applied Biosystems).

Variation in the intronic sizes of CHD-W and CHD-Z gene was detected by capillary electrophoresis. Two different picks of PCR products was produced from CHD-W and CHD-Z genes in females and one pick of PCR products from CHD-Z gene in males. The tested material had a high genetic diversity, were identified 5 gene variants in the range of 268–320 bp.

Natural compounds derived from food have been investigated as new sources of chemotherapy drugs. These agents act in a manner similar to the conventional chemotherapy drugs by inducing apoptosis and autophagy. S-allyl cysteine (SAC), a derivative of garlic, has low toxicity and stable oral bioavailability and acts as an antioxidant through various mechanisms. The major anticancer effects of SAC were to suppress cancer proliferation, invasion, and metastasis but their mechanisms in relation to the apoptosis and autophagy signaling pathway were not investigated. The aim of this study was to evaluate the anticancer effects of SAC on human leukemia cell line HL-60 and to elucidate the possible mechanisms. Bax, Bcl-2, caspase-3, mTOR, AKT, and PI3K expression levels were estimated using Real-time quantitative reverse transcriptasepolymerase chain reaction (RT-qPCR). HL60 cells were incubated with SAC at three different concentrations. mTOR, AKT, and PI3K expression levels were altered after 24 h treatment with SAC in HL-60 cells at some selected dose. SAC significantly induced apoptosis of HL-60 cells through suppressing expression of Bcl-2 as well as inducing expression of bax and caspase-3. Our results demonstrate that SAC induces inhibition of cell progression in HL-60 cells via a mechanism that involves modulation of apoptotic gene expression.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 141

Posters

P12.2

P12.1

Extracellular proteome of Myrothecium roridum – a valuable tool for dyes degradation

Expression of human α1,4-galactosyltransferase in CHO Lec-2 cell line Anna Bereźnicka1, Małgorzata Wasik1, Radosław Kaczmarek1, Krzysztof Mikołajczyk1, Maria Duk1, Marcin Czerwiński1,2 1 Laboratory of Glycobiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland; 2 Faculty of Physiotherapy and Physical Education, Opole University of Technology, Opole, Poland

Human P1PK blood group system includes three antigens: Pk and P1 with terminal Galα1→4Gal and NOR with terminal Galα1→4GalNAc. Pk and P1 antigens occur on erythrocytes as well as on epithelial and endothelial cells, except that P1 is found only in some individuals. Depending on the presence of P1 antigen, we have a blood group P1 (P1 antigen is present) or P2 (absence of P1 antigen). Pk and P1 antigens are synthesized with the α1,4-galactosyltransferase (Gb3/CD77 synthase) encoded by the A4GALT gene. As a result of the point mutation c.631C> G (p.Q221E), the enzyme extends acceptor specificity, obtaining an ability to attach the galactose residue to terminal N-acetylgalactosamine. This is the first case of a change in the acceptor specificity of a glycosyltransferase due to a point mutation. Pk antigen is the main receptor for Shiga toxins that cause haemorrhagic colitis and haemolytic-uraemic syndrome (HUS). TLC analysis of glycosphingolipids from NOR-positive erythrocytes showed that Shiga toxin was bound to unidentified glycosphingolipid. To determine the structure of this glycosphingolipid, CHO-Lec2 cell line produce glycosphingolipids with lower amount of sialic acid has been transfected with vector encoding consensus α1,4-galactosyltransferase and one with 211Q>E substitution. It has been shown that Shiga toxin has an elevated binding to transfected cells, and the major glycolipid recognized by Shiga toxin is Pk antigen.

Aleksandra Góralczyk, Anna Jasińska, Adrian Soboń, Jerzy Długoński Faculty of Biology and Environmental Protection, University of Lodz, Department of Industrial Microbiology and Biotechnology, Poland Keywords: decolorization, dyes, extracellular proteome, Myrothecium roridum The use of microorganisms and their enzymes is an attractive alternative for methods applied for synthetic dyes elimination from polluted water. In this work for the first time extracellular protein cocktail of fungus Myrothecium roridum was used for dyes biodegradation. The dye-decolorizing potential of crude extracellular proteins was demonstrated for different groups of dyes (anthraquinone, azo, phthalocyanine) with various applications. After 24 hours of incubation with protein cocktail Direct blue 86 (DB86) and Acid blue 74 (AB74) were decolorized by over 40%. The use of mediators such as vanillin and TEMPO allowed for almost complete elimination of AB74. In the presence of vanillin it was possible to remove the dye concentration as high as 200 mg/l within 17 hours. The native-PAGE followed by peptides analysis using the AB Sciex 5800 TOF/TOF system allowed identifying the protein responsible for dyes decolorization as an enzyme belonging to the cupredoxin superfamily.

Acknowledgements This study was supported by the National Science Centre of Poland, project no.

UMO 2013/11/D/NZ9/02776.

References: Kaczmarek R et al (2014) Transfusion Medicine Reviews 28:126–136 Kaczmarek R et al (2016) BBRC 470: 168–174

6th Central European Congress of Life Sciences Eurobiotech 2017

142 Abstracts

P12.3

P12.4

Metal-amphisin conjugates: bioactivity of metal-containing compounds

Low-frequency EPR spectrometry as a tool to monitor chromate bioremediation in living systems

Tomasz Janek1, Lígia R. Rodrigues2, Żaneta Czyżnikowska1

Anna Kostecka-Gugała1, Aleksandra Dubicka-Lisowska1, Halina Gabryś2, Tadeusz Walczak 2, Magdalena Chochlińska1, Paweł Kaszycki1

1 Wroclaw Medical University, Department of Inorganic Chemistry, Wrocław, Poland; 2University of Minho, Centre of Biological Engineering, Braga, Portugal

Amphisin is a lipoundecapeptide with an N-terminal β-hydroxydecanoyl fatty acid side chain and a nonapeptide lactone core resulting from cyclization of the Thr-3 hydroxyl group onto the C-terminal carboxylate. As far as we know, this is the first report about the molecular interaction between divalent metal ions and amphisin.

The effect of metal ions on the secondary structure of the amphipathic lipopeptide was examined by circular dichroism spectroscopy. Moreover, experimental data were supported with the results of semi-empirical calculations to determine the possible structures of formed Me2+-amphisin systems. In the present study, the antimicrobial activities of amphisin and metal-amphisin conjugates against pathogenic gastrointestinal and urinary tract microorganism such as Escherichia coli, Enterococcus faecalis, Staphylococcus epidermidis, Proteus mirabilis, and Candida albicans were investigated. The results demonstrated that amphisin ± metal ions had a broad spectrum of action, including antimicrobial activity against microorganisms with multidrug-resistant profiles. Our results suggest the possible use of this biosurfactant as an alternative antimicrobial agent for applications against microorganisms responsible for diseases and infections, thus making it a suitable alternative to conventional antibiotics.

Acknowledgements This work was supported by Polish-Portugal Executive Program for years 2017– 2018 sponsored by the Polish Ministry of Science and Higher Education and by Portuguese Fundação para a Ciência e a Tecnologia.

Unit of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, al. 29 Listopada 54, 31-425 Kraków, Poland; 2Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, ul. Gronostajowa 7, 30-387 Kraków, Poland 1

Biological reduction of highly toxic chromium(VI), an anthropogenic environmental contaminant, to a more stable and less toxic Cr(III), is one of the most effective mechanisms of its remediation. Paramagnetic character of the Cr(V) radical intermediate makes it possible to follow the reaction directly with the EPR (electron paramagnetic resonance) spectrometry. The application of the L-band microwave frequency (1,1 GHz) enables to test biological specimens in vivo in their natural aqueous environments.

The reaction kinetics determined upon the time course of EPR signal amplitude helps to explain the mechanisms of biological transformation.

We were the first to report chromate bioreduction kinetics in yeast employing a custom-built low frequency (L-band) EPR spectrometer equipped with an extended surface coil-type high-frequency resonator. However, the quantitative signal analysis was found difficult due to the lack of easily available Cr(V) standard for calibration. Recently, we have elaborated an improved measurement technique based on normalization with semi-stable free radical TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl). This enabled to directly compare Cr(VI) reduction-based processes between different pro- and eukaryotic organisms. The work presents examples of the in vivo studies carried out for selected biological systems treated with 1 mM chromate, exhibiting divergent bioremediation mechanisms: bacteria, yeast, microalgae and macrophyte phytoremediators.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 143

P12.5

P12.6

Determination of a universal animal DNA marker

Evaluation of the suitability of mitochondrial DNA for the species identification of animal origin traces

Małgorzata Natonek-Wiśniewska, Piotr Krzyścin National Research Institute of Animal Production, Department of Animal Genomics and Molecular Biology, Poland Keywords: animal DNA marker The objective of this study was to choose a universal marker for determination of animal DNA in food products.

The study used DNA from twenty animal species (mammals, poultry, fish) isolated from the meat and hair fragments without the root, as well as from more than twelve plant species (vegetables, fruit, herbs). DNA concentration was measured using the Nanodrop system, and next the concentration was balanced to the same value with nuclease-free water. DNA was amplified using primers flanking a myostatin gene fragment (Laube et al., 2007). For DNA originating from the meat, a PCR product of around 90 bp was obtained in all cases, whereas for plant DNA and DNA from hair fragments without the root no reaction product was obtained. The limit of detection (LOD) for the presented method revealed that 0.1% animal DNA was added to the plant material.The present study demonstrated that the animal tissue identification method using the myostatin gene is specific to the representatives of the animal world, while not amplifying the DNA of plants.

The amplified fragment is short enough to allow its identification in both raw and cooked foods. This method can only be used for the samples from which genomic DNA can be extracted.

Małgorzata Natonek-Wiśniewska, Piotr Krzyścin National Research Institute of Animal Production, Department of Genomics and Animal Molecular Biology, Poland Keywords: species identification of trace The aim of the study was to present the possibility of using mitochondrial DNA (mtDNA) to determine the species origin of animal traces. The study included: single dog and cat hair (without hair bulbs), 4 x 4 mm cooked chicken bone piece, goose down (0.028 g), pork swab (5 x 5 mm).

Of all these samples, DNA was isolated using the method specific to the DNA matrix. The resulting DNA extracts were characterized by concentrations above 30 ng/μl with a purity of A 260/280 from 1.7 to 2.2. Subsequently, the samples were subjected to PCR with species-specific and complement-specific primers to different mtDNA fragments for each species. In addition, a positive control based on replication of the eukaryotic-specific fragment (18S rRNA) was performed. For all DNA matrices, reaction products were obtained.

Poor quality of the DNA obtained did not disrupted the PCR reaction. The results show the suitability of mitochondrial DNA for the analysis of small samples in which genomic DNA is often not available.

6th Central European Congress of Life Sciences Eurobiotech 2017

144 Abstracts

P12.7

P12.8

Use of cytochrome b polymorphism for species identification of spots of unknown origin

Quantitative and qualitative determination of ducks and geese DNA from different matrices

Małgorzata Natonek-Wiśniewska , Anna Radko , Dominika Rubiś , Piotr Krzyścin , Angelika Podbielska

Małgorzata Natonek-Wiśniewska, Piotr Krzyścin

National Research Institute of Animal Production, Department of Animal Genomics and Molecular Biology, Poland Keywords: individual identification, species identification The aim of the study was to perform comparative analysis of DNA from trace evidence collected from blood spots revealed on the car bonnet of a person suspected of running down a red deer with the tissue of the killed red deer (Cervus elaphus), in order to determine the match with the dead animal. From the DNA of the collected meat, DNA profile was determined from 12 microsatellite loci, whereas no profile specific to the red deer species was obtained from the blood spots, which challenged the origin of the blood spots from the red deer and ruled out that the traces come from the same animal. Because of these doubts, the material obtained from the blood smear was analysed with regard to species identification. The applied method allowed distinguishing cattle, red deer and roe deer based on restriction analysis (Tsp509I) of the PCR product (195 bp) obtained through amplification of the fragment of the gene coding for cytochrome b. Because the obtained restriction profile ruled out that the traces originated from the above species, the PCR product was sequenced to receive a product of 194 bp that was 96% homologous with the gene coding for the human ryanodine receptor (NG_008799.2).

It is concluded from the present study that the experimental procedures made it possible to determine the biological traces and excluded the involvement of the dead red deer in the car accident.

National Research Institute of Animal Production, Department of Genomics and Animal Molecular Biology, Poland Keywords: determination of DNA goose, determination of DNA duck The purpose of this work was to develop a method to identify the species of DNA from ducks and geese extracted from various matrices. The study included: meat, down, whole feathers and blood. The analysis was performed using real-time PCR using MGB probes and primers that bound the cytochrome B (duck) and 12SrRNA (goose) sequences.For all matrices the reactions were species specific. Cross reactions either didn’t occur or occurred after 36 cycles, corresponding to amplification for the species content of 10–5%, i.e. several times below the detection limit (LOQ = 0.1%). The standard curves obtained by consecutive dilutions were linear (R2> 0.99), the slope ranged from -3.34 to 3.42, and the shift coefficient was 23–26.

The amplitude threshold analysis (ct) showed significant differences between successive matrices make it impossible to select one reference material to produce a standard curve effective for analyzing each type of matrix. With reference to the reference material and the test material, the accuracy of the method (the match between the actual and the mark) is above 78%.Validation methods indicate that they can be used to denote DNA ducks and geese in the analyzed matrices.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 145

P12.9

P12.10

Bacilli biosurfactants production in mixed (bacterialfungal) cultures determined by mass spectrometry

Monitoring over time cell culturing medium metabolites changes in breast cancer cell line

Anna Kuśmierska, Przemysław Bernat, Jerzy Długoński, Katarzyna Paraszkiewicz

Wojciech Wojtowicz, A. Ząbek, P. Młynarz

Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lódź, Poland; corresponding author: katarzyna.paraszkiewicz@biol.uni.lodz.pl Keywords: Bacillus, Fusarium, lipopeptide biosurfactants, mass spectrometry Some Bacillus strains are known to be producers of lipopeptide (LP) biosurfactants demonstrating both surface and antimicrobial activity. The aim of the study was to evaluate the impact of Fusarium oxysporum IM 6449 on LPs production by three Bacillus sp. strains (IM 13, IM 14 and KP 7). Luria-Bertani medium (30 ml) was inoculated with bacterial culture (OD600 adjusted to 0.25) and fungal spore suspension (5 x 106 spores ml-1) and cultivated for 96 h, on a rotatory shaker (120 rpm), at 28°C. LPs were extracted by modified QuEChERS procedure and analyzed by LC-MS/MS. The highest (about twofold) increase of LPs (surfactin and iturin) production and the strongest negative impact on mycelium morphology was estimated in mixed (bacterial-fungal) culture containing cells of Bacillus IM 13. The obtained data suggested a possibility of commercial application of Bacillus IM 13 as an antifungal agent.

Wroclaw University of Technology, Department of Bioorganic Chemistry, Wroclaw, Poland Cell culture is one of the basic methods used in biotechnology laboratories. Cell culturing processes must be strictly regulated to obtain reproducible and reliable results. In addition, the results are influenced by the timing of the experiment, associated with changes in biochemical pathways during cancer cells proliferation. Knowing the course of biochemical changes over time could add important information to systematize the results and narrow the field for potential molecular targets. In our study, we used the MDA-MB-468 cell line to determine changes in extracellular metabolites. The medium composition was determined by NMR spectroscopy, where the culture media material was collected in 4 or 8 hour intervals. The analysis was performed by statistical and chemometric methods. The observed changes were not only connected the relative concentrations of metabolites that are typical for cellular development such as pyruvic acid, glucose or glutamic acid but also related to extracellular low molecular weight compounds which are not found in the standard medium composition – pyroglutamic acid, lactic acid, ketoleucine, ketoisoleucine or in significantly increased amounts – alanine. It is also important that the majority of the compounds identified on the proton NMR spectra changed significantly their trend according to the time of cell culturing which allows to utilize this method for monitoring cell cultures.

6th Central European Congress of Life Sciences Eurobiotech 2017

146 Abstracts

P12.11 Electrochemical stem-loop biosensor for fast and sensitive detection of Bacillus anthracis DNA Robert Ziółkowski1*, Katarzyna Zacharczuk 2, Aleksandra A. Zasada3, Elżbieta Malinowska1,4 1 Institute of Biotechnology, Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, 00-664 Warsaw, Poland; 2 National Institute of Public Health – National Institute of Hygiene, Department of Bacteriology, Chocimska 24, 00-791 Warsaw, Poland; 3 National Institute of Public Health – National Institute of Hygiene, Department of Sera and Vaccines Evaluation, Chocimska 24, 00-791 Warsaw, Poland; 4 CEZAMAT PW, Poleczki 19, 02-822 Warsaw, Poland

Bacillus anthracis is a gram positive, rod shaped and spore forming highly pathogenic bacterium. The anthrax infectious disease affects animals and it can cause potentially lethal infection in humans. Its spores are extremely resistant to natural conditions. According to Centers for Disease Control and Prevention anthrax is classified as a category A agent due to its easily dissemination, high mortality rates, epidemic potential and possible high public health impact. The symptoms in anthrax can appear from 1 day to more than 2 months. The rapid and accurate identification of virulent B. anthracis strains is essential for limitation of negative effects of its release. Traditional identification is time-consuming and can last from 24 h to a few days. On the other hand, the molecular assays could provide results within a few hours, but highly specialized equipment and skilled staff are needed. Nonetheless, due to the high lethality and the potential of anthrax as a biological warfare agent, there is a pressing need in development of fully independent, rapid and field-ready monitoring systems for B. anthracis.

The presented study is focused on the elaboration of electrochemical biosensor dedicated to the detection of B. anthracis DNA in unpurified mix of asymmetric PCR. The biosensor is based on stem-loop probe modified with methylene blue as redox marker. The three different probes were investigated and the optimal construction was chosen. As was found such an approach allowed to detect as low as 4 ng ul-1of DNA (82 nucleotides) in 5 minutes with good selectivity.

Acknowledgementss This work was supported by grant from National Science Centre under the decision no. UMO-2014/15/B/NZ6/01771.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 147

Biotechnology for beauty Keynote lecture

Lectures

L13.1

L13.2

The science of healthy old age Suresh Rattan

Finding the relationship between the chemical structure and anti-microbial properties of rhamnose-based glycoconjugates

Laboratory of Cellular Ageing, Department of Molecular Biology and Genetics; Aarhus University, Denmark

Ł. Ławniczak1, G. Framski2, M. Woźniak1, M. Sydow1, A. Borkowski3, Ł. Chrzanowski1

Scientific approaches to achieving healthy old age are based in serious biogerontological research. There is a change of paradigm from “anti-ageing” to health maintenance. Ageing is not a disease; there are no ageing causing gerontogenes; and there is no “enemy within” our cells.

Ageing occurs in spite of the presence of complex pathways of maintenance, repair and defence. An evidence-based scientific strategy towards healthy ageing is that of mild stress-induced hormesis, which stimulates body’s own defence systems and its property of homeodynamics. Novel hormetins based in the principles of hormesis are in development for cosmeceutical, nutriceutical and other applications for maintaining and strengthening the self-perception of health and beauty. A wholistic understanding of ageing at the biological, sociological and psychological level is essential for maintaining health and for enhancing our well being.

Faculty of Chemical Technology, Poznan University of Technology, M. Skłodowskiej-Curie 2, 60-965 Poznan, Poland; 2 Institute of Bioorganic ChemistryPolish Academy of SciencesPoznańPoland; 3 Faculty of Geology, University of Warsaw, Żwirki i Wigury 93, 02-089 Warsaw, Poland 1

The recent focus on balanced economy and sustainable development resulted in an increased popularity of renewable products based on compounds of natural origin.

Glycoconjugates, which comprise a carbohydrate unit covalently linked with other chemical species, are a group of products which have gained increasing popularity due to their wide spectrum of promising properties. Several types glycoconjugates have already found application in various sectors of the industry, such as medicine, molecular biology or the production of pharmaceuticals.

Glycoconjugates incorporating rhamnose exhibit particularly interesting properties, which may be associated with the fact that this sugar is a rare example of L -monosaccharides. Rhamnolipids are a prime and well-studied example of rhamnose-based glycoconjugates. They can be obtained via bioconversion of waste materials and exhibit potent antimicrobial and surface activity, which makes them a valuable alternative to conventional chemical surfactants. Despite numerous reports regarding the effects of rhamnose-based glycoconjugates, there is still much inconsistency in this regard. Due to this reason, there is a need to investigate this topic further.

The results of studies focused on the characterization of antimicrobial activity of specific rhamnoconjugates and analysis of structural inclinations by means of molecular modelling confirm a relationship between the chemical structure and properties of this group of compounds.

6th Central European Congress of Life Sciences Eurobiotech 2017

148 Abstracts

L13.3

Posters

Nano-emulsions as vehicles for topical delivery of forskolin

P13.1

Małgorzata Miastkowska 1, Elżbieta Sikora1, Elwira Lasoń1, Maria Jose Garcia-Celma2, Elvira Escribano-Ferrer2, Conxita Solans3, Meritxell Llinas3 Institute of Organic Chemistry and Technology, Cracow University of Technology, Warszawska 24, 31-155 Krakow, Poland; 2 Pharmacy and Pharmaceutical Technology Department, Faculty of Pharmacy, University of Barcelona, Av Joan XXIII, s/n, 08028-Barcelona, Spain; 3 Institute of Advanced Chemistry of Catalonia, Consejo Superior de Investigaciones Científicas (IQAC-CSIC) and CIBER en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Jordi Girona 18-26, 08034-Barcelona, Spain 1

Forskolin, a diterpene produced by the Indian Coleus plant (Coleus forskohlii), activates the enzyme adenylyl cyclase and therefore increases the intracellular levels of cAMP [1]. Because of its properties forskolin have been used as: antiallergic agent, effective agent in some cardiovascular disorders, or as an agent promotes loss of weight (fat burning) [2]. Recently, forskolin has appeared in the literature as a natural substance to obtain a healthy tan [3].

Two O/W nano-emulsions based on medium chain triglycerides (MCT) and stabilized by a nonionic surfactant (Polysorbate 80 or Polysorbate 40) were studied as forskolin delivery systems. The nano-emulsions were prepared by the PIC method. The mean droplet size of the nano-emulsions with Polysorbate 80 and Polysorbate 40 with oil/surfactant (O/S) ratios of 20/80 and 80% water concentration, measured by Dynamic Light Scattering (DLS), was of 118 nm and 111 nm, respectively. Stability of the formulations assessed by light backscattering for 24 h, showed that both nano-emulsions were stable at 25ºC. Studies of forskolin in vitro skin permeation from the nano-emulsions and from a triglyceride solution were carried out at 32ºC, using Franz-type diffusion cells. A mixture of PBS pH 7.4 / ethanol (60:40) was used as a receptor solution. The obtained results have shown that the nano-emulsions developed in this study could be used as effective carriers for topical administration of forskolin.

Catechins isolation from green tea: Effect of extraction time and temperature Spyridon Achinasa, Vasileios Achinasb a Faculty of Science and Engineering, University of Groningen, Nijenborgh 4, 9747 AG Groningen, Netherlands; bUnion of Agricultural Co-operatives of Monofatsi, Asimi 700 16 Crete, Greece

Green tea is a rich source of catechins, antioxidants which account for about 30% of its dry weight. As numerous studies have linked green tea catechins with improving human health and extending the shelf-life of food products, the extraction of catechins from green tea has received considerable interest. An efficient and safe extraction system is needed for the accurate quantification of the catechins in teas and tea products and as an efficient first step for the preparation of catechin extracts and for the isolation of the individual catechins. In this study we investigated the influence of two individual factors (process time and temperature) on the extraction yield of catechins from green tea using water.

References [1] Burlando B et al (2010) Herbal Principles in Cosmetics: Properties and Mechanisms of Action, CRS Press Taylor & Francis Group: 212–216 [2] Ciotonea C et al (2010) Buletinul Institutulvi politehnic DIN IASI 4: 95–106 [3] Spry M et al (2009) Pigment Cell Melanoma Res 22: 219–229.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 149

P13.2

P13.3

Phenolic compounds extraction for Olea europaea leaves using water-based solvent

The effect of cosmetic base on the release of Methylmethionine Sulfonate

Spyridon Achinas1,Vasileios Achinas2

Małgorzata Kucia1, Agnieszka Leśniak1, Elżbieta Sikora2

1 Faculty of Science and Engineering, University of Groningen, Groningen, Netherlands; 2Union of Agricultural Co-operatives of Monofatsi,Crete, Greece

1

Phenolic compounds are becoming increasingly popular because of their potential role in contributing to human health. Experimental evidence obtained from human and animal studies demonstrate that phenolic compounds from Olea europaea leaves have biological activities which may be important in the reduction in risk and severity of certain chronic diseases. Therefore, an accurate profiling of phenolics is a crucial issue. Water was chosen instead of organic solvents as it is considered safer and environment-friendly solvent.

Department of General Chemistry, Faculty of Comodity Science, Cracow University of Economics, Sienkiewicza 5, 31-510 Krakow, Poland; 2 Institute of Organic Chemistry and Technology, Cracow University of Technology, Warszawska 24, 31-155 Krakow, Poland

Methylmethionine Sulfonate (Methylmethionine Sulfonium), called vitamin U was first isolated from leaves of raw cabbage in 1966 [1]. This substance shows antihistamine, anti-inflammatory, radioprotective and anti-irritant activity [2]. In addition, vitamin U affects the regeneration and restoration of the hydrolipid mantle. Because of its wide spectrum of biological activity Methylmethionine can be used in remedial and anti-aging cosmetics.

It is well known, that there are a few factors influencing the skin permeation and, as a consequence, efficiency of cosmetics or pharmacological products. Apart from the active substance properties, also a character of the used carrier are important. The aim of the study was to examine the influence of the cosmetic bases properties (the type of emulsion and the internal phase droplets size) on the release of vitamin U. Two types of O/W and W/O Methylmethionine-loaded emulsions, varying in internal phase droplets size were prepared. Physicochemical properties of the prepared formulations, such as the emulsions type, their stability, viscosity, pH and droplets size of the dispersed phase were evaluated. The kinetic of the vitamin U release was performed in a thermostatic diffusion chambers, at the temperature of T=32oC, with the use of dialysis membranes made from regenerated cellulose. Phosphate buffer (PBS) of pH 7.4 was used as the acceptor solution. The concentration of the released amino acid derivative was analyzed by use of UV-Vis spectrophotometer. The detection and the quantitative estimation of the amino acids has been accomplished by their reaction with ninhydrin [3].

The obtained results showed that the type of the used emulsion affects the release of Methylmethionine sulfonate.

References: [1]. Sakamoto K at al (1996) Biosci Biotechnol Biochem 60(90): 1486–1487.

[2]. Won-Serk K at al (2015) J Mol Sci 16(8): 17088–17100.

[3]. Dubin A (2003) Wydział Biotechnologii UJ, Kraków

6th Central European Congress of Life Sciences Eurobiotech 2017

150 Abstracts

P13.4

P13.5

NLC as a potential carrier system for transdermal delivery of forskolin

Safety evaluation and total antioxidant activity of the new semisynthetic lupeol derivatives

Elwira Lasoń1, Elżbieta Sikora1, Małgorzata Miastkowska1, Elvira Escribano2, Maria Jose Garcia-Celma2, Conxita Solans3, Meritxell Llinas3, Jan Ogonowski1

Magdalena Malinowska1, Monika Pasikowska2, J. Ogonowski1, E. Sikora1, I. Eris2

1 Faculty of Chemical Engineering and Technology, Institute of Organic Chemistry and Technology, Cracow University of Technology, Poland; 2Dept. of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Barcelona, Spain; 3 Institute for Advanced Chemistry of Catalonia, Consejo Superior de Investigaciones Científicas (IQAC-CSIC), Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain

Keywords: forskolin, NLC, nanostructured lipid carrier, skin permeation During the several last years, lipid nanoparticle formulations with solid matrix have shown a great potential as carriers for topical administration of poorly soluble active ingredients. In our work as an active molecule forskolin was applied. Forskolin is a diterpene produced by the Indian Coleus plant (Coleus forskohlii). It activates the enzyme adenylyl cyclase and therefore increases the intracellular levels of cAMP [1]. Moreover, forskolin has appeared in the literature as a natural substance to obtain a healthy tan [2]. Nanostructured lipid carriers (NLC), based on beeswax and caprilic/capric trigliceride mixture, were obtained by using hot high preassure homogenization technique. The influence of number of homogenization cycles on NLC properties was analyzed. Additionaly an effect of surfactant concentration (decyl glucoside) was studied. The systems stability was assessed by macroscopic observation, light backscattering and zeta potential measurements.

NLC particle size was measured by means of dynamic light scattering (DLS). The kinetically stable forskolin-loaded formulations were selected for in vitro drug permeation study (Franz cell method). The concentration of forskolin in the receptor solution (i.e. Ethanol/PBS mixture) was analyzed by high performance liquid chromatography (HPLC) with UV detection. The obtained results have shown that NLC formulations could be used as effective carriers for forskolin delivery to the skin.

1 Institute of Organic Chemistry and Technology, Department of Chemical Engineering and Technology , Cracow University of Technology, Poland; 2Dr Irena Eris Centre for Science and Research, Poland

Lupeol is a pentacyclic triterpene alcohol which shows various biological activity: antimicrobial, anticancer and antivirus. Moreover, the compound acts as antiswelling and antiinflammatory agent [1]. The rich source of lupeol is white birch bark. The dry extract of birch bark contains up to 80% of triterpene compounds [2] and lupeol is second substance after betulin which can be extracted from this source in high concentrations [3]. Relationship between compound properties and its ability to skin absorption and, as a consequence, their biological activity is well known.

The lipophilicity is the most significant factor influencing the skin permeation [4]. The aim of our work was the modification of lupeol molecule structure to enhance its biological activity.

Four lupeol esters (acetate, propionate, succinate, acetylsalicylate) were tested on full thickness skin model Epiderm as an alternative method to animal skin testing to evaluate the potential irritancy and cytotoxity to human cells. The results showed that the tested compounds increased skin cells proliferation process with no negative effect on RhE (Reconstructed Human Epidermis) model.

Moreover, antioxidant activity for ethanolic solutions of lupeol and its esters was evaluated. The inhibition of the oxidation process was measured using modified Brandt-Williams method. The most effective antioxidant was lupeol acetylsalicylate which inhibited the process of oxidation in over 5%. The obtained results showed that the lupeol esters could be applied as potential biological active substances in pharmaceutical and cosmetic products.

References [1] Siddique H R et al (2011) Life Sci 88: 285–293 [2] Abyshev A Z et al (2007) Pharm. Chem. J 41: 22–26 [3] Ekman R (1983) Holzforschung 37: 205–211 [4] Moser K et al (2001) Eur J Pharm Biopharm 52: 103–112

References [1] Burlando B et al Herbal Principles in Cosmetics: Properties and Mechanisms of Action (2010) CRS Press Taylor & Francis Group 212–216 [2] Spry M et al (2009) Pigment Cell Melanoma Res 22: 219–229

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 151

P13.6 Ultrasonic assistance micelle-mediated extraction of Calendula Anthodium Karolina Śliwa1, Paweł Śliwa1, Elżbieta Sikora1, Jan Ogonowski1, Paulina Nowicka2, Jan Oszmiański2 1 Faculty of Chemical Engineering and Technology, Cracow University of Technology, Poland; 2Department of Fruit, Vegetable and Grain Technology, Wrocław University of Environmental and Life Sciences, Poland

The UAMME extracts composition and antioxidant properties of active substances derived from Calendula Anthodium were studied. Marigold flowers extracts have a moisturizing and strengthening effect on the epidermal lipid barrier by content phytosterols and waxes [1]. Triterpene alcohols and esters present in them show antimicrobial and anti-inflammatory effects [2]. This extract has an antioxidant activity by the carotenoids and polyphenols presence.

The UAMME method using several surfactants was applied. The results were compared with those obtained for extraction with water and ethanol. The bioflavonoids compositions with UPLC-DAD-MS method were determined.

For antioxidants properties reaction with DPPH reagent and Follin’s method were measured. The structure of the selected flavonoids solubilized in micelle solution was theoretically simulated using MD method.

The results confirmed that the UAMME method might be a convenient method for the preparation of rich in natural antioxidants plant extracts. The selection of a suitable surfactant may thus provide the expected composition of extract. The extracts can be used as raw materials in the cosmetic and pharmaceutical industries.

References [1] Khalid K A et al (2012) Med. Arom. Plant Sci. Biotech. 6: 12–27 [2] Della L R et al (1994) TPlanta Medica 60: 516–520

6th Central European Congress of Life Sciences Eurobiotech 2017

152 Abstracts

Microorganisms – from environment to biotechnology Keynote lecture

Lectures

L14.1

L14.2

Plant microbe interaction and the role of endophytes in phytoremediation of pharmaceuticals

Pangenome studies of plant pathogenic bacterium Pectobacterium parmentieri

Peter Schröder, Bernadett Bartha, Feiran Chen, Hao Cui, Christine Götz, Christian Huber, Tina Riedel and Andres Sauvetre

Sabina Zoledowska1, Wojciech Sledz1, Alessio Mengoni2, Ewa Lojkowska1

Helmholtz Zentrum Muenchen, German Center for Environment and Health, GmbH, Research Unit Comparative Microbiome Analyses, 85764 Neuherberg, DE; peter.schroeder@helmholtz-muenchen.de Pharmaceuticals may pose serious threats to our water resources when released into the environment. Although modern technologies in waste water treatment have proven elimination of organic pollutants, older WWTPs are still not capable to remove pharmaca completely. Constructed wetlands might represent a rather cost and labor saving alternative to clean effluents, before they enter water bodies or soils. In order to test phytoremediation as a tool for the removal of pharmaca, we chose the UV-Filter Oxybenzone as well as the intensively used NSAID drugs Acetaminophen (paracetamol) and Diclofenac, the antidiabetic drug Metformin, and the antiepilepticum Carbamazepine, all causing massive problems due to poor elimination in WWTPs, for their possible fate in plants. Among plant species to be tested for their capabilities of uptake and metabolism of pharmaca were indian mustard (Brassica juncea), the common reed (Phragmites communis) cattail (Typha latifolia) umbrella sedge (Cyperus alternifolius) and a hairy root culture of horse radish (Armoracia rusticana).

All compounds were rapidly absorbed by plants, and already after 3h of exposure the drugs and their metabolites could be detected in plant tissues. In all cases we found phase I and II metabolites, some of them plant specific.

Since it has been proven that plants live together with a plethora of microbes on and in roots, stems and leaves, plant microbe interactions were studied. It could be shown that the enzymes responsible for xenobiotic metabolism may be induced by bacterial quorum sensing molecules.

The role of microbes assisting the remediation process was more detailed studied with Carbamazepine. Endophytic bacteria were isolated from Phragmites plants grown in a wetland in Eschede and exposed afterwards to Carbamazepine in greenhouse conditions. An enrichment culture allowed the isolation of potentially degrading bacteria. In total, about 40 strains were able to remove carbamazepine from liquid cultures while 20 were able to grow using carbamazepine as sole carbon source. Phylogenetic analysis based on 16S rDNA sequences reveals that the majority of these isolates belong to Proteobacteria, Actinobacteria and Bacteroidetes.

The significance of phytoremediation in the presence and absence of endophytic bacteria is discussed and the significance of the process observed is evaluated. Alone or in combination, the isolates found could be used as inoculates to enhance the phytoremediation of pharmaceuticals in constructed wetlands.

Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, 58 Abrahama Street, 80 307, Gdansk, Poland; 2 Department of Biology, University of Florence, Florence, Italy 1

Pectobacterium parmentieri is a newly established species within plant pathogenic Pectobacteriaceae (Khayi et al. 2016; Adeolu et al. 2016). Microorganisms belonging to this species are causative agents of diseases in economically important plants (e.g. potato, maize) (Toth et al. 2003).

Severe disease symptoms are caused mainly by activity of P. parmentieri virulence factors like Plant Cell Wall Degrading Enzymes (PCWDE). Interestingly, we observe significant phenotypic differences among P. parmentieri isolates in terms of virulence factors production or ability to macerate potato tissue. In order to establish the source of these differences, we sequenced 6 genomes of P. parmentieri (4 isolated in Poland, 2 in Belgium) with the use of Ilumina and PacBio approaches. De novo genome assembly was performed with the use of Ilumina reads and SPAdes software; later on genome polishing was done with the use of PacBio reads and Quiver software. A Pangenome study was performed on 8 genomes (6 de novo assembled, 2 references: P. parmentieri SCC3193, P. parmentieri WPP163).

P. parmentieri pangenome includes: 3788 core genes, 1201 accessory genes and 1625 unique genes. We were able to determine presence of all well-known virulence factors in core genome despite one – pnl gene encoding pectin lyase was absent in P. parmentieri IFB5486, isolated in Belgium. Moreover we confirmed presence of putative plasmid in one strain of P. parmentieri isolated in Poland

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 153

L14.3

L14.4

Inhibitory effects of quercus pubescens willd. Galls on nosocomial acinetobacter baumannii biofilm formation and quorum quenching of gram negative bacteria

Cleavage of poly(cis-1,4-isoprene) rubber particles by cultures of Gordonia polyisoprenivorans

Kübra Erkan Türkmen1, Demet Erdönmez2, Nihal Kenar2 Department of Biology, Division of Biotechnology, Hacettepe University, 06800, Ankara, Turkey; 2Department of Biology, Aksaray University, 68100, Aksaray, Turkey 1

Keywords: Oak Galls, Quorum Quenching, Nosocomial, Acinetobacter baumannii Galls are abnormal plant growth or swellings comprised of plant tissue due to the wound of tissue, infections of microorganisms, and egg-laying of insects. Galls are usually found on foliage or twigs. These are formed by plant hormones and enzymes which are interfere with stimuli produced by insects or microorganisms. Although many plants have been used for the source of various medicines, the studies about galls are not enough.

The bacterial virulence and the biofilm formation ability of pathogens are arranged by the system of Quorum-Sensing (QS). Thus, the Quorum Quenching is an important issue to fight bacterial infections and to break the resistance of pathogens against antibiotics. Therefore, we thought that Quercus pubescens Willd. galls may be effective in Acinetobacter baumannii biofilm formation and quorum quenching activity of gram-negative bacteria. The method as follows; dichloromethane-methanol extracts of Quercus pubescens Willd. galls were tested by agar well and disc diffusion assay for Quorum Quenching (anti-quorum Sensing) activity using Chromobacterium violaceum (CV12472, CVO26) reporter strains. The anti-biofilm activity of extracts was investigated on 20 different nosocomial Acinetobacter baumannii strains. The development of biofilm was determined by the crystal violet assay. The majority of extracts were tested prevented cell adhesion on polypropylene microtiter plates well.

In conclusion, effect of the extract of oak galls on anti-biofilm of Acinetobacter baumannii and Quorum Quenching activity is very effective. This study proposes that these extracts are encouraging materials for pharmaceutical research and developments in the future.

Rodrigo Andler1, Sebastian Hiessl1, Onur Yücel1, Matthias Tesch2, Alexander Steinbüchel1 1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany; 2Institut für Organische Chemie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany

Potential biotechnological recycling processes for rubber-made products include the bacterial degradation of poly(cis-1,4-isoprene) in order to achieve its total biodegradation or its biotransformation into useful products. The actinomycete Gordonia polyisoprenivoransstrain VH2 catalyzes the degradation of poly(cis-1,4-isoprene) and enables its use as a sole carbon source via β-oxidation. For the first time poly(cis-1,4-isoprene) was used as solid substrate in 2-l fermenters. Two different particle size fractions (63–500 and 500–1000 μm) and three stirring rates (300, 400 and 500 rpm) were evaluated in the process. In order to enhance the production of oligo(cis-1,4-isoprene) molecules, a transposon insertion mutant was used, which has lost the ability to transport the partial degradation products into the cells, supporting the accumulation of the degradation products in the supernatants of the cultures.

Propionic acid, glucose or glycerol were fed as additional carbon sources beside polyisoprene for cell growth of the mutant. In 2-l bioreactors with controlled pH, different feeding regimes during cultivation were performed by the addition of the carbon source every 24 or 48 h for 16 days.

After liquid-liquid extraction and a derivatization with Girard’s T reagent, the oligo(cis-1,4-isoprene) molecules were detected by ESI-MS, obtaining different ranges of molecular weights depending on the carbon source feeding regime, the cultivation time and the extraction solvent.

6th Central European Congress of Life Sciences Eurobiotech 2017

154 Abstracts

L14.5

L14.6

Capability of polyhydroxyalkanoates accumulation enhances stress resistance and cell robustness of bacteria

Screening of feeding strategies based on Trigger events in a 24-micro bioreactor platform

Stanislav Obruča1, Dan Kučera1, Iva Pernicová1, Petr Sedláček1, Filip Mravec1, Ota Samek 2, Vladislav Krzyzanek 2, Jana Nebesářová3, Lucie Mullerová1, Aneta Chytilová1, Eva Slaninová1, Ivana Márová1 1 Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic; 2 Institute of Scientific Instruments, Academy of Sciences of The Czech Republic, Vvi, Kralovopolska 147, 612 64 Brno, Czech Republic; 3 Biology Centre, The Czech Academy of Sciences, v.v.i., Branisovska 31, 37005 Ceske Budejovice, Czech Republic

Polyhydroxyalkanoates (PHA) are microbial polyesters accumulated in form of intracellular granules by numerous bacteria primarily as storage compounds. Nevertheless, recent research data indicate that their biological role is more complex and presence of PHA in bacterial cells substantially improves their stress resistance. We have recently demonstrated that protective effect can be partially attributed to PHA monomers which are very potent chemical chaperone capable of protecting biological molecules. Furthermore, presence of PHA considerably changes biophysical properties of bacterial cells which, for instance, enhance their resistance to repeated freezing.

Moreover, PHA granules demonstrate unique liquid-like properties and prevented membrane damage caused by massive plasmolysis when bacterial cells are exposed to osmotic up-shock. These fundamental research results can have numerous practical outcomes since bacteria are exposed to stress conditions in numerous biotechnological processes. Therefore, employing PHA rich bacterial cells in situ bioremediation or as agricultural inoculants is of great interest. Moreover, PHA are considered being biodegradable alternative to petrochemical plastics. The fact that PHA accumulation supports cellular robustness can be also very important for instance when bacteria are cultivated on waste-originating substrates. Therefore, PHA production is very interesting process which perfectly fits concept of biorefinery.

Bernal. C., PhD and Santos Fernandez. C, MsC Applikon Biotechnology B.V. Heertjeslaan 2, 2629 JG, Delft, The Netherlands.

Several bioprocesses exploit feeding strategies such as the minimization of acetate production in microbial cell culture (Johnson et al., 2002, Kim et al., 2004, Andersen et al., 2001). Some of these feeding strategies use values for pH or dissolved oxygen (dO2) as a trigger point. The availability of a platform able to perform this approach on a micro-scale cultivation is essential for strain optimization strategy. In the present work, a platform that holds 24 micro bioreactors (working volume 2–5 mL) with individual control of pH, dO2 and temperature. This system is able to program event-based control whereby the control strategy is based on a triggered event. In the present work, two different feeding strategies in an E.coli cultivation have been applied based on i) pH above 7.1 and ii) dO2 above 50%, using the work of Chen et al., 1997 as a reference.

The results demonstrate micro -scale can be successfully used for the screening of different feeding strategies.

Acknowledgements This work was supported by the project LO1211 and LD15031 of the Ministry Education, Youth and Sports and by the project GA15-20645S of the Czech Science Foundation (GACR).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 155

Poster

P14.2

P14.1

The influence of low-intensity extremely highfrequency electromagnetic radiation on variability and biosynthetic activity of streptomycetes

Nanomaterials as stimulating agents for bacterial metabolism Adrian Augustyniak1, Krzysztof Cendrowski2, Martyna Barylak 2, Ewa Mijowska2, Paweł Nawrotek1 Department of Immunology, Microbiology and Physiological Chemistry, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology, Szczecin, al. Piastów 45, 70-311 Szczecin, Poland; 2 Department of Nanotechnology, West Pomeranian University of Technology, Szczecin, al. Piastów 45, 70-311 Szczecin, Poland

Antonina Bratuhina T.G. Shevchenko Pridnestrovian State University, Department of Biology, Moldova

1

Nanomaterials are often used in microbiology for elimination or reduction of bacteria [1]. Nevertheless, the response to nanostructures is dependent from many factors such as their concentration, dispersion, shape, and size [2]. Recent literature indicates that addition of toxic substances to culture in sublethal doses may induce e.g. biofilm formation. This process is energetically demanding which indicates that stress caused by the substance may induce a heightened metabolic reaction which can be variable for different microorganisms [3]. Therefore, this study was to evaluate effects of selected nanocomposites on physiological reactions of bacteria and production of secondary metabolites. Bacteria from genera Pseudomonas and Streptomyces were used in the experiments. Selected nanomaterials included carbon nanotubes, silica nanotubes and nanospheres, and graphene oxide. Nanostructures were functionalised with metals (Cu, Co) or titanium dioxide. Microscopy, culture methods, qPCR, spectroscopy and flow cytometry were used. Nanocomposites caused changes in physiology of studied bacteria and their growth rate. They also stimulated tested strains to secrete secondary metabolites such as pigments. It has been found that nanoparticles may enter cells causing overexpression of genes coding efflux pumps. Based on the outcome it has been concluded that nanocomposites may trigger specific metabolic responses in pseudomonads and streptomycetes.

Keywords: Streptomyces, low-intensity EHF EMR, variability, lipids, amino acids.

Electromagnetic radiation (EMR) is known to influence the physiological and biochemical properties of microorganisms. The effect of low-intensity extremely high frequency (EHF) EMR on the variability, growth and synthesis of lipids, amino acids and antimicrobial properties of S. massasporeus strains was studied. Low-intensity EHF EMR acting on S. massasporeus CNMN-Ac-06 within 3–10 minutes reduces the heterogeneity and pigmentation of the substrate mycelium culture; within 3–15 minutes increases the content of lipids in biomass by 22.1–51.2% as well as the content of phospholipids and sterols compared to the control; within 10–30 minutes increases the total protein content, individual amino acids and the total content of essential, immunoactive, proteinogenic amino acids in the strain’s culture fluid. Low-intensity EHF EMR acting on S. massasporeus CNMN-Ac-07 within 3–15 minutes stimulates biomass accumulation by 7.9–30.8% compared to the control; within 5 minutes increases the fraction of sterols and antimicrobial activities in relation to plant pathogenic fungi. Low-intensity EHF EMR acting on S. massasporeus CNMN-Ac-08 increases the content of lipids in biomass by 11.2–177.2% and sterols by 51.5% compared to the control.

The study of the influence of low-intensity EHF EMR on S. massasporeus strains has shown that the homogeneity of the population of cultures and their biosynthetic activity are determined by both radiation exposure time and the specific strain.

References [1] Paredes D et al (2014) International Journal of Nanomedicine, 9, 1717–1729 [2] Lemire J et al (2013) Nature Reviews Microbiology, 11, 371–384 [3] Holden P et al (2014) Current Opinion in Biotechnology, 27, 73–78

6th Central European Congress of Life Sciences Eurobiotech 2017

156 Abstracts

P14.3

P14.4

Production and characterization of extracellular polysaccharide of extremely halophilic Archaea Haloferax mediterranei

Physicochemical properties and cytotoxicity of hydrogels based on Beetosan® containing sage, bee pollen and caffeine

Aneta Chytilová, Kateřina Drábková, Paulína Strečanská, Vojtěch Enev, Dan Kučera, Pavla Benešová, Stanislav Obruča

Drabczyk1, S. Kudłacik-Kramarczyk1, B. Grabowska2, M. Kędzierska3, B. Tyliszczak4

Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic Haloferax mediterranei is extremely halophilic archeon capable of production of two very interesting biopolymers.

This microorganism intracellularly accumulates polyhydroxyalkanoates (PHA) while simultaneously excreting polysaccharide. Furthermore, its demands on osmotic pressure of media substantially reduce requirements on the sterility of the biotechnological process. In this work we focused on production and characterization of its extracellular polysaccharide. To decrease cost of the cultivation media we substituted expensive yeast extract by alkaline hydrolyzed waste chicken feather which positively influenced polysaccharide yields. Further, polysaccharide was isolated purified and characterized by Raman spectroscopy, FTIR and elemental analysis. In contrast to literature, we observed very low content of sulfate groups which indicates that level of sulfonation can depend upon cultivation conditions or can be strain specific. Moreover, isolated polysaccharide was capable to provide very high protective activity for model prokaryotic (Cupriavidus necator) and eukaryotic (Saccharomyces cerevisiae) microorganisms against various stress factors such as freezing, ethanol, high temperature, osmotic pressure, oxidative pressure induced by H2O2 or SDS which imply its interesting biological properties and potential applications in biotechnology or health care.

Acknowledgements This work was supported by the project LO1211 and LD15031 of the Ministry Education, Youth and Sports and by the project GA15-20645S of the Czech Science Foundation (GACR).

1 Institute of Inorganic Chemistry and Technology, Cracow University of Technology, 24 Warszawska St., 31-155 Cracow, Poland; 2AGH University of Science and Technology, Department of Foundry Processes Engineering, Faculty of Foundry Engineering, Reymonta 23, 30-059 Cracow, Poland; 3Medical University of Lodz, Al. Kościuszki 4, 90-419 Łódź, Poland; 4Department of Chemistry and Technology of Polymers, Cracow University of Technology, 24 Warszawska St., 31-155 Cracow, Poland

Currently, more and more attention is being paid to issues related to the environmental protection, waste management as well as to the development of polymers with useful properties. Therefore, present research involved preparation of hydrogels the main component of which was Beetosan® – chitosan derived from the multi-stage processing of died bees. In presented paper series of hydrogel polymers containing in their matrices substances of natural origin have been synthesized. Apart from interesting group of additives it is worth noting that the main component of modified matrices is characterized by animal origin. Beetosan® playing a role of hydrogel matrix is a type of well-known polysaccharide – chitosan – obtained as a result of multistage chemical treatment of bees. Mentioned insects are individuals that do not survive difficult winter conditions and constitute a waste.

Therefore, proposed methodology is also interesting from the point of view of the waste management. Synthesized hydrogels were additionally modified with natural substances. Based on the research it can be stated that such combination of Beetosan® hydrogel matrix and mentioned additives resulted in a preparation of polymers characterized by negative impact on cancer cells. Furthermore, such properties as surface morphology as well as wettability of the hydrogels also were determined. Finally, it can be concluded that the use of natural-derived reagents and synthesis of polymers using these reagents (as a result of environmentally friendly photopolymerization) lead to the receipt of the materials with unusual properties, with particular emphasis on antitumor activity.

References [1] Tyliszczak B et al (2016) Przemysł Chemiczny 95(10): 2059–2062 [2] Cheba BA (2011) Global Journal of Biotechnology & Biochemistry 6(3): 149–153

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 157

P14.5

P14.6

Methanotrophs community – from environmental sample to enrichment culture

Production of ectoine and hydroksyectoine by methanotrophs isolated from Wieliczka Salt Mine

Weronika Goraj, Anna Pytlak, Anna Szafranek-Nakonieczna, Zofia Stępniewska

Weronika Goraj, Zofia Stępniewska

The John Paul II Catholic University of Lublin, Institute of Biotechnology, Department of Biochemistry and Environmental Chemistry. Poland Transfer microbiological studies under the surface of the Earth to exploration of biodiversity of subsurface biosphere is important and allows exceeding the current framework of science. Microbial life is determined by physical, geochemical, and biological factors such as the availability of liquid water, nutrients, trace elements, temperature, pH, salinity, and pressure. Recent studies have shown the presence and activity of cells in the deep biosphere.

Presented problems are particularly important because they are aimed at learning the biodiversity of microorganisms that inhabit the saline subsurface biosphere. Moreover we show biodiversity change during isolation of bacterial community.

The research material was the rocks surrounding salt deposits in Wieliczka (siltstones with veins of fibrous salt and lenses of anhydrite). Metagenomic analysis was performed on the basis of NGS by MiSeq technology (Illumina), (GENOMED S. A.). The results confirmed the presence of methanotrophic microorganisms directly in environmental sample from Wieliczka Salt Mine.

Besides the application of metagenomics for determination of biodiversity of bacterial isolates, it also has found applications in fields as diverse as bioremediation, medicine, xenobiotic metabolism, sources of new molecules and novel biocatalysts and enzymes from nature.

Acknowledgements This project was partly financed by the National Science Centre (Poland), granted on the basis of decision DEC-2014/15/N/NZ8/00315.

The John Paul II Catholic University of Lublin, Institute of Biotechnology, Department of Biochemistry and Environmental Chemistry, Poland Halotolerant and halophilic methanotrophs have been isolated from seawater, coastal lagoons, and several soda lakes. There are two fundamental strategies used by microorganisms to balance their cytoplasm osmotically with their medium. The first involves accumulation of molar concentrations of potassium and chloride. The second mechanism is the biosynthesis of organic osmotic solutes like sugars (e. g trehalose), amino acids (e.g glycine betaine, L-α-glutamate, β-glutamine, β-glutamate, L-proline, ectoine, hydroxyectoine) and polyols (e.g. glycerol).

The aim of this study is the recognition the synthesis of ectoine and hydroksyectoine by methanotrophs isolated from rocks surroundings Wieliczka salt deposits.

Methanotrophic microorganisms isolated from Wieliczka Salt Mine were identified by metagenomics analysis using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform (GENOMED S. A.). Released osmolythes was analyzed by GCMS and HPLC technique.

The bacterial isolates were found to belong to the genus: Methylomonas, Methylosinus Methylocystis. We found significant amount of extracellular and intercellular ectoine and hydroxyectoine. The concentration of ectoine range from 0.003±0.000 to 0.012±0.006 mg gDW-1 and hydroksyectoine from 0.010±0.001 to 0.018±0.005 mg gDW-1.

Acknowledgements This project was financed by the National Science Centre (Poland), granted on the basis of decision DEC-2014/15/N/NZ8/00315.

6th Central European Congress of Life Sciences Eurobiotech 2017

158 Abstracts

P14.7

P14.8

Structural determinants of Ty1 genomic RNA dimerization: the role of Gag and p22 restriction factor

Two sides of the green coin – lipid profiling of microalgal consortium selected for treatment of ammonium-rich wastewater

Julita Gumna1, Katarzyna J. Purzycka1, Agniva Saha2, David Garfinkel2 and Katarzyna Pachulska-Wieczorek1

Paweł Jedynak1, Przemysław Petryszak 2, Khongorzul Mungunkhuyag1, Jan Burczyk3, Magdalena Kędra1, Paweł Kaszycki2, Przemysław Malec1

1 Institute of Bioorganic Chemistry, Polish Academy of Sciences, RNA Structure and Function Laboratory, Poland; 2University of Georgia, Department of Biochemistry & Molecular Biology, USA

Keywords: Ty1 retrotransposon, dimerization, Gag protein Retrotransposon Ty1 is the most abundant mobile genetic element in the yeast Saccharomyces cerevisiae. Ty1 has pronounced functional similarities to retroviruses and its structure is analogous to that of retroviral proviruses [1, 2]. Gag protein is the major structural component of Ty1 viral-like particles (VLPs) and multifunctional regulator which control replication of retrotransposon. During retroviral replication Gag, via their nucleic acid chaperone (NAC) activity, facilitate genomic RNA (gRNA) dimerization and packaging into virions [3]. The gRNA in VLPs of Ty1 retrotransposon is also dimeric, but limited information is available for Ty1 gRNA dimerization [4]. Our recent studies indicate that Ty1 Gag promotes Ty1 gRNA dimerization in vitro [5]. We have also developed a secondary structure model of miniTy1 RNA in the dimeric form and indicated palindromic sequences that may be involved in the genome dimerization process.

S. cerevisiae maintain a low copy number of the Ty1 retrotransposon in their genomes trough the mechanism called copy number control. This mechanism depends on Ty1 Gag-derived restriction factor – p22 [5, 6]. The p22 also contains a region crucial for interactions with RNA but its NAC activity in dimerization assay in vitro is significantly lower. The Ty1 Gag and p22 recognize the same nucleotide sequences and thus compete for interactions with Ty1 RNA (5).

Acknowledgements This study was supported by the National Science Centre, Poland (project no 2016/22/E/NZ3/00426).

References [1] Lesage P. and Todeschini A.L. (2005) Cytogenet Genome Res 110: 70–90.

[2] Curcio M.J. et al (2015) Microbiol Spectr 3: 1–35.

[3] D’Souza V. and Summers M.F. (2005) Nat Rev Microbiol 3: 643–655.

[4] Feng Y.X. et al (2000) J Virol 74: 10819–10821.

[5] Nishida Y. et al (2015) Nucleic Acids Res 43: 7414–7431. Saha A. et al (2015) J Virol 89: 3922–3938.

1 Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology; Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland; 2 Unit of Biochemistry, Faculty of Biotechnology and Horticulture, University of Agriculture in Kraków, Poland; 3 Laboratory of Biotechnology, 43-400 Cieszyn, Poland;

The use of ammonium-rich wastewaters, containing low concentrations of organic phase, e.g. effluents from anaerobic digestion, as a medium for algal growth could combine both the biomass generation and purification of hazardous wastes. However, relatively high ammonium concentration may negatively affect lipid accumulation and thus limit the quality of biomass for subsequent biofuel production [1].

In this work we examined an effect of ammonium concentration on lipid accumulation and composition in a mix of Chlorophyta strains isolated from eutrophic habitats in Poland (ZTT-3 consortium), during photoautotrophic growth for 10 days on artificial mineral media. We have used different nitrate/ammonium ratios in concentrations comparable to technogenical contamination levels in wastewaters to investigate the responses of algae with reference to the growth rate and photosynthetic activity as measured by chlorophyll fluorescence in vivo.

Ammonium (up to 72 mg/l) had relatively weak effect on growth and photosynthetic activity of the environmental isolate ZTT-3. Moreover, ZTT-3 expressed relatively high lipid content in excess of ammonium 50% of which was C16:0. Additionally, long-chain fatty acid accumulation (C20:0; C22:0; C24:0, C26:0 and C28:0) has been detected in ZTT-3.

In summary, ZTT-3 algae provide an excellent source of strains for both the treatment of ammonium-rich wastewater and the prospective production of biofuels.

References [1] Ho SH et al (2014) Biotechnol Adv 32: 1448–1459

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 159

P14.9

P14.10

Response of environmental microalgae to anthropogenic pollutants: ammonia and chromate. Implications for bioremediation

Optimization of native S. cerevisiae yeast submerged fermentation (SmF) conditions for phytase production

Paweł Kaszycki1, Kinga Regdos1, Zbigniew Gajewski2, Cecilia Gutiérrez González1 Unit of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, Poland; 2 Unit of Botany and Plant Physiology, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, Poland

Grzegorz Kłosowski, Dawid Mikulski, Oliwia Jankowiak Department of Biotechnology, Kazimierz Wielki University, ul. Poniatowskiego 12, 85-667 Bydgoszcz, Poland

1

With growing industrialization, the waste discharged to the environment cause health hazards. Wastewaters contain numerous pollutants like toxic xenobiotics, organic matter, biogenic substances and heavy metals. Inefficient removal of these contaminants via conventional wastewater treatment brings an urgent need to search for novel remediation biotechnologies. Recently, microscopic algae have focused much interest due to their affinity for polyvalent metals and capability of removing biogenic elements from effluents.

The aim of the work was to establish sublethal concentrations of chromate and ammonia nitrogen in microalgae Chlorella sp. and Stichococcus sp. isolated from polluted niches. Toxicity was evaluated based on cell frequency microscopic counts, biomass growth inhibition (optical density measurements) and chlorophyll concentration changes (determined spectrophotometrically). Chlorella sp. was found more sensitive to Cr(VI) (sublethal concentrations of 10 µM) than Stichococcus sp. (50 µM). At the same time, the former species proved resistant to ammonia (no lethality observed for NH4+ levels up to 7.5 g/l) whereas for Stichococcus sp. all the applied concentrations (1 g/l and above) were inhibitory.

The contaminated sites are rich sources of biotechnologically important microalgal isolates. We suggest Chlorella sp. to be tested for treatment of eutrophicated wastewaters, while Stichococcus sp. seems to be suitable for bioremediation of chromate contaminated sewage.

Keywords: phytase production, S. cerevisiae, submerged fermentation After screening of microorganisms, the next step in the biosynthesis procedure is optimization of culture medium composition in order to achieve a high yield of enzymes with a specified catalytic activity. The goal of the study was to optimize the submerged fermentation of S. cerevisiae, a Finarome wine strain, aimed at the production of phytases, by the selection of process parameters, i.e .carbon and nitrogen sources, temperature and pH of the culture and the concentration of sodium phytate, which is a substrate of the enzymatic reaction. The following parameters of aerobic culture with shaking were subjected to modification: carbon source (sucrose, maltose, lactose, fructose, galactose, arabinose, mannose, glucose, xylose, inositol), nitrogen source (glycine, asparagine, arginine, yeast extract, beef extract, peptone, malt extract, ammonium sulfate, ammonium chloride, potassium nitrate), pH (3.5, 4.5, 5.5, 6.5), temperature (24, 28, 32, 37°C) and the concentration of sodium phytate (98, 144, 190, 285 μg/ml).

During the culture, after 24, 48 and 72 h, the following parameters in the media were determined: biomass concentration based on optical density (OD600), phytic acid concentration with the WADE reagent, and extracellular proteins with Bradford method. The highest degree of utilization of phytic acid after 72 h of culture was observed with mannose as a carbon source (over 50% reduction as compared to initial concentration). Asparagine as a nitrogen source enabled the reduction of phytic acid concentration by more than 80% with respect to the initial concentration after 48 hours of culture. On the other hand, pH 5.5 and 32°C made it possible to reduce phytate concentrations by more than 90%.

6th Central European Congress of Life Sciences Eurobiotech 2017

160 Abstracts

P14.11

P14.12

Strategies to improve the yield of PHAs produced from wood material

Beetosan – proecological chitosan – preparation, characterization, and in vitro cytotoxicity

Dan Kučera1,2, Pavla Benešová1,2, Markéta Kovářová2, Stanislav Obruča1,2

S. Kudłacik-Kramarczyk1, A. Drabczyk1, M. Krzan2, E. Olejnik3, B. Tyliszczak4

Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Czech Republic; 2Institute of Food Science and Biotechnology, Faculty of Chemistry, Brno University of Technology, Czech Republic

1 Institute of Inorganic Chemistry and Technology, Cracow University of Technology, 24 Warszawska St., 31-155 Cracow, Poland; 2Polish Academy of Sciences, Institute of Catalysis and Surface Chemistry, Niezapominajek 8, 30-239 Cracow, Poland; 3AGH University of Science and Technology, Department of Engineering of Cast Alloys and Composites, Faculty of Foundry Engineering, Reymonta 23, 30-059 Cracow, Poland; 4Department of Chemistry and Technology of Polymers, Cracow University of Technology, 24 Warszawska St., 31-155 Cracow, Poland

1

Polyhydroxyalkanoates (PHAs) are among the bioplastics. Chemically it is a polyester which may be produced by bacteria. Burkholderia cepacia and Burkholderia sacchari are promising candidates for the PHA production from hemicellose and cellulose hydrolysates. The wood waste biomass is generally considered as one of cheap carbon source for bacterial production. However, its use is relatively complicated due to the difficulty of converting lignocellulose to fermentable sugars.

The aim of the experiments was to compare the possibilities of pretreatment of wood sawdust and subsequently to choose the most suitable method of detoxification of wood hydrolyzate. The wood waste biomass has been subjected to chemical and enzymatic hydrolysis. The highest conversion to carbohydrates was achieved when the pre-treatment was taken by leaching in ethanol. The maximum conversion rate was 45–50%.

Nevertheless, inhibiting substances as polyphenols and furfurals was also detected. Therefore, we continued to focus on the elimination of microbial inhibitors. Over-liming and activated carbon as an adsorbent of inhibitors were employed for detoxification of wood hydrolysate. Cultivation test proved a strong positive influence pretreatment and detoxification on PHA production.

In the framework of presented research a methodology of obtaining chitosan-chitin complex derived from corpses of naturally died honeybees has been developed. Proposed methodology is very interesting from ecological point of view. The issue of the disposal of waste is quite problematic and is associated with the development of new technologies as well as with incurring additional costs. Dead honeybees that do not survive difficult weather conditions also constitute an unnecessary waste. In presented studies such dead insects were processed in multistage chemical treatment and act as a raw material in the preparation of hydrogels. Synthesized polymers were subjected to the numerous tests. On the basis of the experimental part, it can be stated unequivocally that the hydrogels obtained on the basis of Beetosan® are practically identical to those obtained using only commercially available chitosan.

The research shows that honeybees can be an alternative source of chitosan, which nowadays is mainly extracted from crustaceans on an industrial scale. An amorphous phase in the structure of Beetosan® was observed that is desirable in medical and cosmetic applications and prepared materials are considered for this particular use.

Presented materials are also characterized by an ability to release of active substance from their interior what is desirable in view of the potential application in areas such as cosmetics or pharmacy.

References Hoffman SA (2010) Advanced Drug Delivery Reviews 64: 18–23 Nemstew SV et al (2004) Applied Biochemistry and Microbiology 40:39–43

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 161

P14.13

P14.14

Unleashing the biotechnological potential of the cold-adapted Psychrobacter spp.

Waste and agricultural products as sources of carbon and nitrogen in D-lactic acid biosynthesis

Robert Lasek, Monika Krolikowski, Adrian Sówka, Dariusz Bartosik

Alicja Michalczyk, Anna Cieniecka-Rosłonkiewicz, Arkadiusz Bialek, Sylwia Garbaczewska

University of Warsaw, Faculty of Biology, Institute of Microbiology, Department of Bacterial Genetics, Poland

Institute of Industrial Organic Chemistry, Department of Synthesis, Technology and Biotechnology of Biologically Active Products, Annopol 6, 03-236 Warszawa, Poland

The study of cold-adapted bacteria is a promising branch of microbial biotechnology. Their huge applicative potential stems e.g. from the unique characteristics of their enzymes. However, the research on psychrophilic and psychrotolerant organisms is hindered by the lack of specific genetic tools for this group of prokaryotes.

Having studied in detail a pool of Arctic strains of the genus Psychrobacter, we decided to use selected genetic modules of their native plasmids in the construction of novel Psychrobacter-specific vectors. The created series of new genetic tools is functional in DAB_AL43B, a well-characterized Psychrobacter strain with a completely sequenced genome (GenBank: LT799838).

On the basis of Escherichia coli–Psychrobacter spp. shuttle vectors, we obtained and tested the following genetic tools: (i) pExPsy expression vector carrying a strong promoter PSLF, induced by sodium dodecyl sulfate and laurylaldehyde, which allows for the inducible expression of Histagged proteins (ii) pRSPsy plasmid for testing promoter activity in vivo via high throughput LacZ activity assays (iii) the two-plasmid pLOL/pROFL system for introducing markerless deletions in the DAB_AL43B genome with the use of the lacZ gene as a counterselection marker (the product of X-gal hydrolysis being deleterious for DAB_AL43B).

We believe that these novel tools will facilitate further studies of Psychrobacter spp., aimed i.a. at the construction of genetically modified bacterial strains for biotechnological application.

The modern approach to production of bulk chemical intermediates, incl. biotechnological production of lactic acid for biodegradable polylactides, requires using of waste materials, mainly from the food and agricultural sector.

The raw materials used in biotechnology should be cheap, accessible and of high purity. Using of waste materials in lactic acid biosynthesis should allow fast, efficient and selective production of desired acid enantiomers. Their preparation for fermentation (hydrolysis improving carbon and nitrogen accessibility for bacteria) has to be quick and cost-effective. The study was to investigate the possibility of using low-cost waste and agricultural raw materials as sources of nitrogen (yeast slurry, corn steep liquor, baker’s yeast, wheat middlings, soybean, medick, lupine, pea seeds) and carbon (wheat, wheat flour, beet molasses, whey, brewers grains, beet pulp) in D-lactate fermentation involving Sporolactobacillus laevolacticus DSM 442.

Screening tests in stationary cultures were focused on the maximizing amount of produced D-lactic acid (D-LA), minimizing unreacted carbohydrates and by-products in the digestion medium. Molasses and yeast slurry appeared to be the most effective substrates in D-LA biosynthesis.

Batch fermentation in BioFlo 320 bioreactor showed that the amount of D-LA obtained after 50-hour biosynthesis

by Sp. laevolacticus on the production medium, based on molasses and hydrolysed yeast slurry, was similar to the amount obtained on the medium with glucose and yeast extract.

6th Central European Congress of Life Sciences Eurobiotech 2017

162 Abstracts

P14.15

P14.16

The genotypic structure changeability and biodiversity of bacterial community in vertical flow constructed wetlands during sulfamethoxazole-rich wastewater treatment

Bacterial autofluoresce – A useful tool in biotechnological processes

Aleksandra Ziembińska-Buczyńska, Katarzyna Ślipko, Monika Nowrotek, Korneliusz Miksch The Silesian University of Technology, Environmental Biotechnology Department, Poland Constructed wetland (CW) are regarded to be cost-efficient and effective method for pharmaceutical wastewater treatment. Bactericidal substances, such as sulfamethoxazole (SMX), often present in pharmaceutical wastewater, can be the factor shaping microbial community in such biological wastewater systems influencing the wastewater treatment efficacy. That is why in this experiment we monitored bacterial community genotypic structure and biodiversity in CW columns (at the top, the middle and the bottom) during SMX-rich (5 mg/L) wastewater treatment in vertical flow constricted wetland system, partially planted with Phalaris arundinacea. The research revealed that for such type of biological material the best DNA isolation method is mechanical with 24-hour rinsing using 1 ×PBS before the extraction procedure and with no PCR inhibitors removal. PCR-DGGE (Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis) analysis revealed that in each columns there were dominant genotypes present during total length of the experiment.

There was no statistical correlation between bacterial biodiversity and plant presence or SMX dosage. The highest biodiversity was observed at the top of the columns, lower at the middle and the lowest at the bottom of the column.

Spearman correlation coefficient did not confirmed the linkage between biodiversity and the location of the community sample in the CW column.

Acknowledgements This research was partially supported by NCN with grant UMO2012/05/B/ ST8/02739 and by grant BK-217/RIE-8/16 from the Silesian University of Technology

Lucie Müllerová1,2, Filip Mravec1, Kateřina Bílková1, Stanislav Obruča1,2 Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic; 2Institute of Food Science and Biotechnology, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic 1

Cellular green autofluorescence cuased predominantly by flavins can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with the detection of low-level specific fluorescence. In our work we intend to use “green” autofluorescence to distinguish microbial cells from abiotic particles in flow cytometry analysis or, moreover, it can even serve as a marker of physiological state of the microbial culture of the interest for instance during biotechnological process. Therefore, our aim is to use the green autofluorescence of bacteria Cupriavidus necator H16 which is considered being industrial candidate strain for polyhydroxyalkanoates (PHA) production and its mutant strain Cuprivaidus necator PHB-4 unable of PHA accumulation as a viability marker in various biotechnological processes. Flow cytometry and fluorescence microscopy are used in this work as complementary techniques. By combining their strengths, i.e. the ability of flow cytometer to analyse millions of cells in a very short time, and the high resolution of advanced fluorescence microscopy when analysing single cell, the data obtained are more complex and detailed. It is shown that green autofluorescence correlates with the concentration of oxygen in production media; the higher the availability of oxygen the higher the intensity of autofluorescence was measured by either fluorescence microscopy or flow cytometry. Furthermore, time resolved fluorescence spectra of cellular autofluorescence using variety of excitation lasers are also presented, which is important factor when doing fluorescence analysis combining autofluorescence with other fluorophores.

Acknowledgements This work was supported by the project LO1211 and LD15031 of the Ministry Education, Youth and Sports and by the project GA15-20645S of the Czech Science Foundation (GACR).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 163

P14.17

P14.18

The effect of nickel and aluminum exposition on cell division under Tor1 signaling deficiency in Schizosaccharomyces pombe

Role of polyhydroxyalkanoates in biofilm formation of Burholderia cepacia and Burholderia sacchari

Miroslava Pozgajova, Alica Navratilova, Anna Trakovicka Slovak University of Agriculture in Nitra, Department of Genetics and Breeding Biology, Faculty of Agrobiology and Food Resources, Tr. A. Hlinku 2, 94976 Nitra, Slovakia Keywords: Cell cycle, Schizosccharomyces pombe, Tor1, heavy metals, nickel, aluminum Nickel is a micronutrient in trace amounts essential for many biological processes. Higher Ni concentrations exposed to the organism were associated with various toxicities such as allergies or cancer. Aluminum is a non-essential metal normally occurring in the nature. However, its exposure to the organism is connected with different toxic events as for example accumulation of amyloid-beta subsequently leading to neurodegenerative disorders.

Moreover, aluminum was categorized as an element causing cell division errors and DNA damage. In our study we investigated the effect of different nickel (NiSO4.6H2O) and aluminum (AlCl3.6H2O) concentrations on cell division of the eukaryotic model organism, fission yeast Schizosaccharomyces pombe. One of the key regulatory mechanisms of cell growth, proliferation and division under different environmental stresses is the TOR (target of rapamycin) signaling. TOR is a serine/threonine kinase formed from two distinct complexes TOR complex 1 (TORC1) and TOR complex 2 (TORC2). Two homologs of the TOR kinase, namely Tor1 and Tor2 exist in Schizosaccharomyces pombe, of which Tor1 is the catalytic subunit of TORC2 and Tor2 is the catalytic subunit of TORC1. We analyzed response of the two heavy metals in cell strains lacking the Tor1 signaling pathway in the presence or absence of the microtubule destroying drug, thiabendazole (TBZ).

Obtained results demonstrate that higher Ni and Al concentrations lead to the inhibition of the cell division, but increased TBZ sensitivity was detectable only in the presence of Ni. Furthermore, our observations suggest, that Tor1 is involved in Ni but not Al responses in eukaryotes.

Acknowledgements This work was supported by the Slovak Research and Development Agency under the Contract No. APVV-0636-11 and APVV-14-0054.

Stanislav Obruča1, Markéta Rucká1, Kateřina Mrázová1, Eva Slaninová1, Ota Samek 2, Jitka Krouska1, Vladislav Krzyzanek 2 1 Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic; 2 Institute of Scientific Instruments, Academy of Sciences of The Czech Republic, Vvi, Kralovopolska 147, 612 64 Brno, Czech Republic

Burholderia cepacia and Bukrholderia sacchari are Gram negative bacteria capable of biofilm formation and polyhydroxyalkanoates (PHA) accumulations. Their capability of PHA production is extensively utilized for PHA production especially using lignocellulose based substrates. On the contrary, biofilm formation by B. cepacia represent serious problem for immuno-deficient patients which can be infected by this bacteria. Nevertheless, there is lack of information about role of PHA in biofilm life style adapted bacteria. Generally, we observed that PHA content in biofilm bacteria is considerably lower than in planktonic cells. It seems that biofilm cells prefer production of extracellular matrix over PHA accumulation; nevertheless, PHA biosynthesis was not completely suppressed.

Surprisingly, we observed that biofilm of both bacteria strains contained crystalline PHA which indicates that some bacteria in biofilm lysed and released PHA granules which subsequently crystalized. Furthermore, cell surfaces of biofilm bacteria revealed more hydrophobic nature than PHA rich planktonic cells and stress resistance of biofilm associated cells against various stress factors was superior to PHA rich planktonic cells. But when stress resistance planktonic cells with high and low PHA content was compared, PHA rich performed much better in all the stress challenges. This indicates that PHA plays protective role predominantly in planktonic highly mobile cells and biofilm associated cells rely on protective function of extracellular matrix.

Acknowledgements This work was supported by the project LO1211 and LD15031 of the Ministry Education, Youth and Sports and by the project GA15-20645S of the Czech Science Foundation (GACR).

6th Central European Congress of Life Sciences Eurobiotech 2017

164 Abstracts

P14.19

P14.20

Newly identified ciliary proteins as a potential novel markers in PCD diagnosis

Interconnection of microbial degradation of chicken feather and polyhydroxyalkanoates production employing selected Pseudomonas strains

Paulina Urbanska, Ewa Waclawek, Rafal Bazan, Martyna Poprzeczko, Michal Niziolek, Hanan Farahat, Anna Osinka, Ewa Joachimiak, Hanna Fabczak, Dorota Wloga Laboratoty of Cytoskeleton and Cilia Biology, Dept od Cell Biology, Nencki Institute of Experimental Biology of Polish Academy of Sciences, 3 Pasteur street, 02-093 Warsaw, Poland Tetrahymena is a non-pathogenic unicellular Eukaryote of short generation time and the ability to grow to high densities in the laboratory conditions. Because of the well-developed methods enabling ultrastructural and biochemical analyses and genetic manipulations, Tetrahymena became a model organism in diverse research fields from environmental studies to gene analysis.

Tetrahymena assembles numerous motile cilia, organelles that are highly evolutionarily conserved from protist to mammals. In human cilia are assembled by sperm cells and epithelial cells lining internal tracks and their disfunction leads to primary ciliary dyskinesia (PCD), a multi-symptom disorder, resulting in male and female infertility, infection of respiratory tracks, improper leftright body asymmetry and hydrocephalus. Diagnosis of PCD is difficult. Taking advantage of the fact that ciliary proteins are highly conserved we are investigating new ciliary proteins required for cilia assembly and motility.

Identified new ciliary proteins may become new genetic markers in PCD diagnostics.

Acknowledgements This work has been supported by the National Science Centre (Harmonia 6, 2014/14/M/NZ3/00511) and the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no 665735 (Bio4Med) and by the funding from Polish Ministry of Science and Higher Education within 2016–2020 funds for the implementation of international projects (agreement no 3548/H2020/COFUND/2016/2).

Iva Pernicová1,2, Zuzana Šuráňová2, Petra Innemanova3,4, Stanislav Obruča1,2 Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic; 2Institute of Food Science and Biotechnology, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic; 3Dekonta, a.s., Dřetovice 109, 273 42 Stehelčeves, Czech Republic; 4Institute of Environmental Studies, Faculty of Science Charles University in Prague, Benátská 2, 128 01, Prague 2, Czech Republic 1

Chicken feather from poultry-processing industry represent worldwide problem, since millions of tons of feather are generated worldwide without any benefit. However, some Pseudomonas strains are able to directly degrade feather and, moreover, also accumulate polyhydroxyalkanoates (PHA). Pseudomonas putida KT2440 and two strains isolated from diesel polluted areas, Pseudomonas fulva and Pseudomonas gessardii, were used for the degradation of non-treated chicken feather and keratinase production in laboratory reactors. Total loss of feather, as well as enzymatic activities of proteinase and keratinase, were monitored during cultivation. Subsequently, bacterial biomass grown on feather was used as an inoculum for PHA productions using waste frying oil as carbon source and octanoic acid as PHA precursor. By applying this approach we reached very high intracellular PHA content (about 60% of cell dry weight), polymer consisted of 3-hydroxyoctanoate and 3-hydroxyhexanoate. Hence, microbial degradation and feather and production of PHA can be efficiently interconnected.

Acknowledgements This work was supported by the project LO1211 and LD15031 of the Ministry Education, Youth and Sports and by the project GA15-20645S of the Czech Science Foundation (GACR).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 165

P14.21

P14.22

Analysis of cellular fatty acids of selected microalgal species

In-situ monitoring of a stress-induced crystallization of bacterial polyhydroxyalkanoates

Przemysław Petryszak1, Paweł Jedynak 2, Khongorzul Mungunkhuyag2, Jan Burczyk3, Przemysław Malec2, Paweł Kaszycki1

Petr Sedláček1, Stanislav Obruča1, Eva Slaninová1, Filip Mravec1, Ota Samek 2, Vladislav Krzyžánek 2

Unit of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Kraków, Poland; 2 Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology; Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland; 3Laboratory of Biotechnology, Cieszyn, Poland 1

Oily plants are basic resources for biodiesel production.

The extensive processing of plant biocomponents affects global food supplies and prices and lead to severe environmental changes (emission of greenhouse gasses, pollution and degradation of ecologically important habitats, misbalanced biodiversity). Thus, there is an urgent need to search for alternative, efficiently growing photosynthetic biomass resistant to physiological stresses. In this regard microalgae may serve as good candidates.

In this work the effect of different nitrogen sources (various nitrate/ammonium ratios) on photoautotrophic growth (240 h) of two microalgal species: Chlamydomonas sp. and Chlorella sp. was investigated. Cell growth rates, photosynthetic activities (chlorophyll fluorescence in vivo), and lipid accumulation (GC-MS analyses of FAMEs: fatty acid methyl esters) were presented.

Chlorella sp. was more resistant to NH4+ than Chlamydomonas sp. For both strains, high ammonium levels (6 and 8 mM) negatively influenced the biomass yield, chlorophyll concentration, photosynthetic activity and total content of fatty acids. However, the FAMEs composition remained similar independently of the amount of ammonia and C16:0 and C18:1 were found dominant. We suggest that upon process optimization the tested microalgae could be used in environmental projects to remove excess nitrogen from wastewaters. Such an approach might become a source of fatty acids applicable as the third generation biofuel.

1 Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic; 2 Institute of Scientific Instruments, Academy of Sciences of The Czech Republic, Vvi, Kralovopolska 147, 612 64 Brno, Czech Republic

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized and accumulated in a form of tiny intracellular granules by numerous bacteria. Although they are considered primarily as a source of carbon and energy, according to recent findings it appears that PHAs play much more complex role in physiology of a bacterial cell. In our previous work, we have confirmed a general protective effect of PHAs in microbial cells facing adverse environmental conditions. In vivo, PHAs are present in thermodynamically unfavorable amorphous state, which is stabilized by a complex surface structure of the granules and by the presence of low-molecular plasticizers (e.g. water) inside them. When this stabilizing effect is eliminated, PHAs will crystallize to some extent.

This contribution focuses on the stress-induced phase transition of PHAs in Cupriavidus necator H16. Thermally and osmotically induced crystallization of PHAs was monitored during desiccation of the C. necator cells by various techniques of structural (FTIR and Raman spectroscopy, XRD) and morphological (TEM, cryoSEM) analysis. The In-situ monitoring was supplemented by the similar experiments performed with PHAs granules isolated from C. necator cells via chemical/enzymatical treatment. On the basis of obtained experimental results, principal role of structural changes on the surface of the granules in the phase transition of PHAs is proposed and discussed.

Acknowledgements This work was supported by the project Materials Research Centre at FCH BUT— Sustainability and Development no.LO1211 and national project GA15-20645S of the Czech Science Foundation (GACR).

6th Central European Congress of Life Sciences Eurobiotech 2017

166 Abstracts

P14.23

P14.24

Nisin-loaded magnetic nanoparticles

Accumulation of PHA granules protects bacterial cells from adverse effect of UV-irradiation

Ruta Gruskiene1, Tatjana Krivorotova2, Ramune Staneviciene3, Dalius Ratautas1,4, Elena Serviene1, 3, Jolanta Sereikaite1 1 Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Lithuania; 2 Department of Polymer Chemistry, Vilnius University, Lithuania; 3 Laboratory of Genetics, Institute of Botany, Nature Research Centre, Lithuania; 4Institute of Biochemistry, Vilnius University, Lithuania

Keywords: nisin, magnetic nanoparticles, antimicrobial activity Nisin is a known bacteriocin approved as a food additive for food preservation. It exhibits a wide spectrum antimicrobial activity against Gram-positive bacteria. Nisin is produced by Lactococcus lactis subsp. lactis. It is a small cationic peptide composed of 34 amino acid residues.

Iron oxide magnetic nanoparticles were synthesized and determined by XRD as Fe2O3 phase (Maghemite-C).

Magnetic nanoparticles were stabilized by citrate, ascorbate, gallic or glucuronic acid coating. Nisin loading on the particles was acquired by the adsorption method and confirmed by FTIR spectroscopy. The size of nisin-loaded magnetic particles was characterized by AFM and DLS. The antimicrobial activity of developed nanoparticles was evaluated on the model Gram-positive bacteria Bacillus subtilis by applying microbiological techniques.

Nisin-loaded magnetic nanoparticles could find their application in innovative and emerging technologies as antimicrobials.

Eva Slaninová, Petr Sedláček, Filip Mravec, Ondrej Hesko, Lucie Müllerová, Stanislav Obruca Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic Bacteria developed various strategies to cope with stress conditions including UV irradiation. Polyhydroxyalkanoates (PHA) are polyesters accumulated in form of intracellular granules by numerous bacteria primarily as storage compounds, nevertheless, we investigated that PHA granules are not used only as carbon and energy storage but bacteria can use this compound also as UV-protectant. This statement is based on our observation that PHA accumulating cells of Cupriavidus necator H16 survived exposure to UV irradiation challenge much better than its PHA non-accumulating mutant. Mechanisms of protective effect of PHA granules were determined by different spectroscopic approaches such as turbidity measurement, absorption spectrophotometry with integration sphere, nephelometry and continuous fluorescence microphotolysis. Generally, protective mechanisms of PHA granules is predominantly based on very effective scattering of UV radiation with wavelength of about 250 – 290 nm which protect photo-sensitive molecules, especially DNA, from their damage. Furthermore, PHA metabolism also provides reduction power to eliminate radicals induced by irradiation. Therefore, PHA accumulation capability represents very potent strategy to face UV-irradiation.

Acknowledgements This work was supported by the project LO1211 and LD15031 of the Ministry Education, Youth and Sports and by the project GA15-20645S of the Czech Science Foundation (GACR).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 167

P14.25

P14.26

Biotransformation of aliphatic and aromatic hydrocarbons with Rhodococcus sp. CUP11 isolated from brown coal deposit

Psychrophilic laccase Kabatiella bupleuri – a novel enzyme useful in biotechnology

Paulina Supel1, Przemysław Petryszak1, Piotr Kapusta2, Joanna Brzeszcz2, Paweł Kaszycki1 Unit of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, Poland; 2 Department of Microbiology, Oil and Gas Institute – National Research Institute, Krakow, Poland 1

The organic fraction of brown coal deposits is very complex and consists mostly of lignite and its derivatives. The structure, content and composition of organic substances depend on geological age and localization of a given deposit, but generally this fraction is thought to be resistant to biological degradation. Therefore it can be assumed that brown coal deposits can become sources of autochthonous strains revealing unique pathways of biodegradation of various complex organic compounds.

In this study biotransformation capabilities of a Rhodococcus sp. CUP11 isolate (R. opacus as identified with 16S rDNA) were investigated towards such xenobiotics as naphthalene, phenanthrene, pristane, squalane, squalene, hexadecane, and diesel oil. The tests were carried out for two weeks in a mineral medium containing various concentrations of xenobiotics supplemented as sole carbon sources. The changes of bacterial frequency (Koch plating method) and population density (measured turbidimetrically) were monitored along with xenobiotic concentration determination. Optimized protocols for bacterial culture cultivation and treatment as well as for obtainment of xenobiotic extracts for GC-MS analyses were developed.

The experimental results prove high bioremediation potential fo the tested strain and suggest its high application value enabling future use against environmental contaminations with organic pollutants.

Katarzyna M. Szulczewska, Aneta Białkowska, Marianna Turkiewicz The Institute of Technical Biochemistry, Technical University of Lodz, Poland Keywords: psychrophiles, cold-adapted laccase Psychrophilic yeast-like fungi, Kabatiella bupleuri IBT-G3, produces a cold-adapted extracellular laccase.

Thanks to laccases catalyse the bond formation in mild, environmentally friendly reaction conditions, they are very interesting biocatalysts for green chemistry. Laccases can be used in phenolic compounds transformation reactions, cross-coupling and decarboxylation reactions. Additionally, the psychrophilic nature of cold-adapted enzyme makes that laccases a great potential for application in reactions in organic solvents.

Laccase is characterized by high activity of 0.2 U/ml at optimal pH 3.5 with ABTS. Its optimal temperature is 30°C. Moreover, this enzyme retains up to 66% of maximal activity at 10°C and is highly thermolabile protein, it loses 80% of activity after 30 minutes at 45°C. Optimal enzyme production is conducted at 20°C in medium containing 10% of spent brewery grain (one of the main waste generated during the beer production process) in water supplemented with 0.4 mM Cu2+ and 1 mM Tween 80. These features allow to use this laccase in decolourization and detoxification processes at low temperatures and in biotransformations important for pharmaceutical and chemical industry.

6th Central European Congress of Life Sciences Eurobiotech 2017

168 Abstracts

P14.27 Myxoxanthophylls – carotenoid glycosides from cyanobacteria: isolation and intial characterization Paweł Żbik1, Paweł Kaszycki2 and Przemysław Malec1 1 Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology; Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland; 2Unit of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, Al. 29 Listopada 54, 31-425 Kraków, Poland

Carotenoid glycosides are poorly characterized group of compounds. One of them is crocin – a pigment of saffron.

Most of others occur in prokaryotes, especially cyanobacteria, including oscilloxanthin in Oscillatoria and myxoxanthophyll in Synechocystis and Anabaena species [1]. Myxoxanthophyll is a fucoside of myxol (1’,2’-dihydro-3’,4’-didehydro-3,1’,2’-trihydroxy-γ-carotene). Although the relative amounts of myxoxanthophyll were reported to increase when cyanobacteria are undergoing stress, the biological function of myxoxathophyll derivatives remains unknown [2]. The presence of this carotenoid is necessary for normal organisation of cell wall and thylakoid membrane in Synechocystis sp. PCC6803 [3]. In this work, two myxoxanthophyll derivatives were purified from Synechocystis sp. PCC6803 and Anabaena 7020. The absorption spectra in solvents of different polarity show the presence of solvatochromic effect. Furthermore, changes of chiralooptical properties in water:ethanol solutions indicate the aggregation of myxoxanthophylls. The ORAC and DPPH-based methods demonstrate the antioxidant capacity. The results suggest that these compounds may share certain properties with the expensive saffron pigments.

References [1] S. Takaichi et al (2001) Plant Cell Physiol. 42(7): 756–762 [2] K. Kłodawska et al (2015) Plant Cell Physiol. 56(3): 558–571 [3] H. E. Mohamed et al (2005) J. Bacteriol. 187(20): 6883–6892

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 169

From Molecular Machines to Molecular Medicine: Advances in Biotechnology Keynote lecture

Lectures

L15&18.1

L15&18.2

Lost in translation tRNA modifications protect the proteome

Regulatory mechanisms of tRNA modification

S. Glatt

Rościsław Krutyhołowa1,2 and Sebastian Glatt1 Max Planck Research Group hosted by the Malopolska Centre of Biotechnology of the Jagiellonian University Krakow, Poland; 2Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Poland

1

Max Planck Research Group hosted by the Malopolska Centre of Biotechnology (MCB) of the Jagiellonian University Krakow, Poland, sebastian.glatt@uj.edu.pl Two copies of each of the highly conserved six subunits of the eukaryotic Elongator complex (Elp1-6) constitute a large macromolecular complex, which in combination with additional modification pathways conducts the formation of specific post-transcriptional modifications on uridines in the wobble base position of tRNAs [1]. Its modification activity is essential for maintaining accurate translational rates, protein folding dynamics and global protein homeostasis. Neither the detailed chemistry of the reaction itself, nor the specific mechanisms by which temporarily associated regulatory factors, namely Kti11, Kti12, Kti13 and Kti14 are modulating Elongator’s activity are currently fully understood. I will present an overview of our work focusing on novel insights into the catalytic cycle of the modification reaction based on the crystal structure of a bacterial Elp3 homologue at 2.1 Å resolution. Our results reveal the unexpected arrangement of Elp3 lysine acetyl transferase (KAT) and radical S-adenosyl-methionine (SAM) domains that share a large interface to form a composite active site and tRNA binding pocket [2].

In addition, I will present the recently obtained structure of the fully assembled yeast Elongator complex solved by an integrative structure determination approach as a showcase for hybrid methods in structural biology. We were able to combine data from electron microscopy, x-ray crystallography, mass spectrometry, biochemistry, biophysics and integrative modelling to resolve the architecture and provide a pseudo-atomic model of this highly conserved and important protein complex [3]. In summary, our structural and functional analyses provide novel insight into the molecular mechanisms that allow Elongator to regulate ribosomal protein synthesis and deepen our understanding of its tRNA modification activity. In addition, our studies also provide novel ideas for clinical intervention and treatment strategies, as Elongator is known to play an important role in the onset of various neurodegenerative diseases, intellectual disabilities and cancer in humans.

Keywords: tRNA modification, Elongator, regulatory factors All known organisms use ribosomes and transfer RNA (tRNA) to form polypeptides based on sequence information provided by messenger RNA (mRNA). Decoding of this information occurs via interaction of triplets in mRNA with the appropriate anticodon loop of tRNA. The recognition rate differs between various tRNA species not only because of unequal cytoplasmic availability but also due to presence of tRNA modifications in the anticodon loop of some tRNAs. Thus, tRNA modifications represent an important mechanism of translational control.

The Elongator complex is a big macromolecular complex, composed of six pairs of proteins named Elp1-6. Among other suspected activities, it has the capability to conduct 5‑carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) modifications on U34 in the anticodon loop of some tRNA species. Elp123 carries the enzymatic core of whole complex and the Elp3 subunit is responsible for the modification activity. The postulated role of Elp1 as a scaffold, the presence of numerous domains responsible for protein-protein interaction and the detection of various post translational modifications indicates that proteins responsible for regulation of Elongator activity, such as Sit4, Hrr25 and Kti12 may directly contact Elongator via their interaction with Elp1.

My project focuses on the functional and structural characterization of specific regulatory factors bound to Elongator. I plan to combine biochemistry, crystallography and electron microscopy to understand their essential contribution to the tRNA modification activity of Elongator.

Acknowledgements Current research was funded by NCN OPUS grant – UMO-2015/19/B/NZ1/00343.

Acknowledgements External funding from EMBO YIP/IG, NCN (OPUS10, UMO-2015/19/B/NZ1/00343) and FNP (First Team, FT/2016-1/2) References [1] S. Glatt & C.W. Müller; Structural insights into Elongator function; Curr Opin Struct Biol. Apr;23(2), (2013) [2] S. Glatt S, R. Zabel, O. Kolaj-Robin, O.F. Onuma, F. Baudin, A. Graziadei, V. Taverniti, T.Y. Lin, F. Baymann, B. Séraphin, K.D. Breunig, C.W. Müller; Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi. Nat Struct Mol Biol., Sep;23(9), (2016) [3] M.I. Dauden, J. Kosinski, O. Kolaj-Robin, A. Desfosses, A. Ori, C. Faux, N.A. Hoffmann, O.F. Onuma, K.D. Breunig, M. Beck, C. Sachse, B. Seraphin, S. Glatt*, C.W. Müller*; Architecture of the yeast Elongator complex, EMBO Rep. 2017 Feb;18(2)

6th Central European Congress of Life Sciences Eurobiotech 2017

170 Abstracts

L15&18.3

L15&18.4

Phage Display selection and characterization of scFv specifically recognizing extracellular domain of FGFR1

Unusual mechanical stability of an artificial model protein cage

Jakub Szymczyk, Julia Chudzian, Łukasz Opaliński, Martyna Szczepara, Małgorzata Zakrzewska, Jacek Otlewski University of Wrocław, Faculty of Biotechnology, Department of Protein Engineering, Joliot Curie 14a, Wrocław, 50-383, Poland Fibroblast growth factor receptor 1 belongs to the family of the tyrosine kinase receptors that are responsible for many crucial biological processes such as promotion of cell division and proliferation. It has been shown that FGFR overexpression or its overactivity lead to the disruption of cell signaling followed by uncontrolled cell division and mechanism of apoptosis avoidance. It was reported in many types of tumors, particularly in malignant varieties.

Therefore, the FGFR1 is a promising target for novel anti-cancer therapies. One of the commonly used anticancer therapeutic strategy includes the use of monoclonal antibodies as a drug delivering molecules. The molecules, specifically recognizing tumor associated antigens can be selected using Phage Display technique that uses phages to display and present these molecules.

In this study, two commercial phage libraries (Tomlinson I and J) presenting scFv molecules have been used.

After the selection and protein purification, scFv molecules were analyzed by surface plasmon resonance (SPR) to confirm their interaction with ligands and to determine the kinetic parameters of the interactions with FGFR1. To determine the fragment of FGFR1 extracellular domain interacting with selected scFv and to determine specificity of the interaction for FGFR1 ELISA and pull-down assay were performed. As a result, group of proteins specifically recognizing FGFR1 with three different binding sites were identified.

Acknowledgementss The work was supported by the National Science Centre, Poland (grant 2011/02/A/ NZ1/00066).

Conference costs were supported by Wroclaw Centre of Biotechnology programme “The Leading National Research Centre (KNOW) for years 2014–2018”.

Soumyananda Chakraborti, Michael Sarna, Jonathan G Heddle Malopolska Centre of Biotechnology, Jagiellonian University, 7A Gronostajowa Street, Krakow, Poland Protein cage stiffness/ mechanical stability is one of the important factors that determines their cellular internalization.[1] For example it has been found that immature HIV particles are stiffer (14-fold) than mature particles and this change in stiffness (particle softening) triggers effective internalization of mature particles.[2] Many other viruses evade host defenses simply by changing their mechanical stiffness through point mutations. From a nano-biotechnology point of view protein cages with tunable mechanical stability may be very useful for different drug delivery applications. So far, data on mechanical stability of protein cages mostly comes from virus like particles such as bacteriophage (λ, φ29) MVM, CCMV.[3, 4] Normally to determine protein cage mechanics, protein cage are subjected to physical deformation by applying an external force from outside via fine cantilever tip attached to an AFM (atomic force microscope). From the force needed to deform particles, one can calculate the spring constant k of the particle. The spring constant provides a direct estimate of the mechanical rigidity/softness of a particle. It has been found that different protein cages have different softness/rigidity and this property is also can be sensitive to whether virus is filled with nucleic acids. It has been found for CCMV Virus like particle (VLP) that a natural mutation generates a more rigid VLP with increased thermal stability.[5] Indeed there is a strong correlation between thermal stability and mechanical rigidity of protein cage, as increase in capsid stiffness may lead to “anchoring” effect preventing thermally induced deformation.

Studies to date have been almost exclusively on VLPs. Artificial protein cages are a product of synthetic structural biology and have promise as designed nano-containers. In this context an artificial protein cage made from TRAP6 is very relevant: The cage can be generated using gold nanoparticles and has been found to be highly thermostable so we can expect it should have high Young’s modulus, however it has never experimentally validated. In this work we have used AFM to determine the mechanical stiffness of a TRAP-cage (single particle) and it shows very high mechanical stability with an average value of ~0.15GPa. References [1] Ghisaidoobe AB, Chung SJ. Nanomedicine. 2015; 10(24):3579–95.

[2] Jutz G et al. Chem Rev. 2015; 115(4):1653–701.

[3] Webb, B et al. Arch. Biochem. Biophys. 1994, 309, 178.

[4] Macara, I. G et al. Biochem. J. 1972, 126, 151.

[5] Yang, Z et al. Chem. Commun. 2007, 33, 3453.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 171

L15&18.5

Keynite lecture

Biomolecular interactions analytics using microscale thermophoresis

L15&18.6

Jakub Nowak Application Specialist & Sales; NanoTemper Technologies sp. z o.o., Krakow, Poland, E-mail: jakub.nowak@nanotemper.de Keywords: biomolecular interactions, complex buffer systems, high-affinity interactions The analysis of bio-molecular interactions, such as protein-protein, protein-nucleic acid or protein-small molecule, not only helps to develop therapeutics or diagnostics techniques, but it also provides important insights into cellular processes. Here we present MicroScale Thermophoresis (MST) for the investigation of affinities of biomolecular interactions. MST analyzes the directed movement of molecules in optically generated microscopic temperature gradients. This thermophoretic movement is determined by the entropy of the hydration shell around the molecules. Almost all interactions and any biochemical process relating to a change in size, charge and conformation of molecules alters this hydration shell and is thus detectable by MST. Measurements can be performed with purified molecules as well as in close-to-native conditions (lysate, serum). The readout of the method is fluorescence, derived either from fluorescently labeled interaction partners (via chemical dyes or fluorescent fusion proteins) or from intrinsic protein fluorescence.

The presentation will cover biophysical backgrounds of the technology, highlighting benefits of the MicroScale Thermophoresis platform, followed by specific examples of its application in characterizing biomolecular interaction in various scenarios.

The fluorescence of tryptophans in a protein is strongly dependent on its close surroundings. By following changes in fluorescence, chemical and thermal stability can be assessed in a truly label-free fashion. The dual-UV technology by NanoTemper allows for rapid fluorescence detection, providing an unmatched scanning speed and data point density. This yields ultra-high resolution unfolding curves which allow for detection of even minute unfolding signals.

Furthermore, since no secondary reporter fluorophores are required, protein solutions can be analyzed independent of buffer compositions, and over a concentration range of 150 mg/ml down to 5 µg/ml. In addition, information on protein aggregation can be recorded in parallel, providing insight into colloidal stability of the sample. Therefore, nanoDSF is the method of choice for easy, rapid and accurate analysis of protein folding and stability, with applications in membrane protein research, protein engineering, formulation development and quality control.

The presentation will cover biophysical concepts of the technique showing benefits of the nanoDSF technology platform, and will be followed by specific examples of nanoDSF applications towards various experimental systems.

RiPPs are ripe for harvest: structural biology of antibiotic biosynthesis Dmitry Ghilarov Malopolska Centre for Biotechnology, Jagiellonian University, 30-387 Cracow, Poland, Contact: dmitry.gilyarov@uj.edu.pl The majority of leads for the drug development pipelines still come from natural sources such as plants, bacteria or fungi. Post-translational modification of ribosomally produced precursor peptides in bacteria generates an unprecedented diversity of yet-to-characterise compounds branded “ribosomally synthesised post-translationally modified peptides” – “RiPPs”. Genome mining techniques allowed high throughput prediction and discovery of new RiPPs based on similarity of the modifying enzymes.

Until recently, structural basis of the peptide discrimination and modification by different RiPP synthetases remained a mystery. Here I will present a model for the biosynthesis of one example RiPP antibiotic, microcin B17 (MccB17) from Escherichia coli, based on crystal structures of a full synthetic complex and a protease essential for microcin production. I will briefly discuss the current state and perspectives of studying MccB17-related compounds for the development of new antibacterials with a species-specific mode of action.

6th Central European Congress of Life Sciences Eurobiotech 2017

172 Abstracts

L15&18.7

L15&18.8

Impact of gold clusters on cell viability and motility

Targeted inhibition of STATs as a potential treatment strategy in cardiovascular diseases

Kinga Borzęcka-Solarz & Jonathan Heddle Bionanoscience and Biochemistry Laboratory, Malopolska Centre of Biotechnology UJ, 7A, Gronostajowa St., 30-387 Krakow, Poland, borzecka.kinga@gmail.com Gold nanoparticles (Au-NPs) are used in several fields including biomedical applications, nanomedicine, cancer treatment and regenerative medicine and have recently been shown to be required for formation of a novel protein “cage”, which itself has potential medical application.[1,2] However, there is still little conclusive information on their interactions with cells and potential cytotoxic profile, which depends on variety of factors, including size, shape and chemical compositions.[3,4] In our studies we selected human umbilical cord mesenchymal stem cells (hUC-MSC) and cancer cells (HeLa and PC-3) to examine the toxicity of the gold particles used in protein cage formation (specifically phosphine-stabilized ultrasmall (1–3 nm) gold nanoparticles).

The effects of gold components on cell viability, morphology and migration were assessed with a variety of methods and the results indicated that the Au-NPs rather than the phosphine ligand triggered cytotoxicity in cancer and stem cells. This toxic effects occurred in all tested cells as strong reduction of cell viability and increase of population of apoptotic cells within 24 h after treatment.

Furthermore, exposure to Au-NPs was found to induce changes in cell morphology and cytoskeleton filament disruption, which negatively affects the growth, migration and cell survival mechanisms.

Martyna Plens-Galaska1, Malgorzata Szelag1, Joanna Wesoly2, Hans A.R. Bluyssen1 Department of Human Molecular Genetics; 2Laboratory of High Throughput Technologies, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University

1

Keywords: STAT, atherosclerosis, inflammation Key factors contributing to early stages of atherosclerosis include the pro-inflammatory cytokines Interferon (IFN)α, IFNγ and Toll-like receptor 4 (TLR4) stimuli. Together, they trigger activation of Signal Transducers and Activators of Transcription (STAT) protein family.

Searches for STAT3-targeting compounds exploring the pTyr-SH2 dimerization area of STAT3 are numerous, and yielded many synthetic small molecules, including STATTIC and STX-0119.

We developed a pipeline approach combining comparative in silico docking of STAT-SH2 models with in vitro STAT inhibition validation, as a novel STAT-inhibitory selection strategy. This approach allowed us to identify a new type of non-specific STAT inhibitor, C01L_F03 targeting the SH2 domain of STAT1, 2 and 3 with equal affinity. Moreover we observed a similar STAT cross-binding mechanism for STATTIC and STX-0119, leading to genome-wide inhibition of pro-atherogenic gene expression. Consequently, a multi-STAT inhibitory strategy was applied to inhibit VSMC migration, leukocyte adhesion to ECs and impairment of aortic ring contractility

Acknowledgements This work was supported by National Science Centre (NCN, Poland) grant No. 2016/20/W/NZ1/00095 (Symfonia-4).

References [1] Malay, A. D. et al. Gold Nanoparticle-Induced Formation of Artificial Protein Capsids. Nano Lett. 12, 2056–2059, (2012).

[2] Imamura, M. et al. Probing structural dynamics of an artificial protein cage using high-speed atomic force microscopy. Nano Lett. 15, 1331–1335, (2015).

[3] Pan, Y. et al. Size-dependent cytotoxicity of gold nanoparticles. Small 3, 1941–1949, (2007).

[4] Pan. Y, et al. Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small 5, 2067–2076, (2009).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 173

L15&18.9

Posters

Small-molecule inhibitors of heme oxygenase-1 as possible anti-cancer drugs

P15&18.1

Paulina Podkalicka1, Olga Mucha1, Kalina Andrysiak1, Anna Biela1, Szczepan Kruczek1, Maciej Mikulski2, Kamil Sitarz2, Michal Galezowski2, Arkadiusz Bialas2, Tomasz Rzymski2, Krzysztof Brzozka2, Alicja Jozkowicz1, Jozef Dulak1, Agnieszka Loboda1 1 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Krakow, Poland; 2Selvita S.A., Krakow, Poland

Keywords: HO-1, tumorigenesis, anti-cancer therapy Heme oxygenase-1 (HO-1), a cytoprotective, anti-apoptotic and anti-inflammatory enzyme, is highly induced in a variety of cancer types and its increased expression is correlated with more advanced tumor development stage as well as poorer survival of patients.

As the possibilities of specific inhibition of HO-1 are strongly limited, we initiated a drug discovery program aimed at identification and characterization of novel, potent and selective small-molecule inhibitors of HO-1. Virtual High Throughput Screening (vHTS) followed by structure-based rational design led to the identification of molecules which actively inhibit the catalytic function of HO-1 at submicromolar concentrations. The best compounds were tested in vitro, on various cancer cell lines and their effects on cell viability, clonogenic potential and possibility to sensitize cells to commonly used chemotherapy was analyzed. Initial in vivo experiments aimed at checking pharmacokinetic properties suggest their usefulness in further oncological studies.

Acknowledgements Supported by the National Centre for Research and Development (NCBR) (grant no PBS2/B7/15/2014).

Effects of Rotenone in Neuroblastoma SKN-AS Cells and its Neutralization Nayat N. Ciroglu1,2, Pınar Obakan Yerlikaya2, Aylin Sendemir Urkmez3, Ajda Coker Gurkan2, Elif D. Arisan2 and Mehves E. Genc1 1 Yeditepe University,Department of Pharmacology, Istanbul Turkey; 2Istanbul Kultur University,Department of Molecular Biology and Genetics, Istanbul Turkey; 3Ege University,Department of Biotechnology, Izmir Turkey

Objective: Rotenone is a widely used pesticide and neurotoxic agent, may also affect humans. NAC is an antioxidant. It is commonly used to identify and inhibit ROS. In this study we aimed to demonstrate the potential effect of NAC addition in rotenone treated neuroblastoma (NB) SK-N-AS cells.

Methods: Rotenone-induced neurotoxicity on NB cells was evaluated by cell viability assay, growth assay, DAPI and DiOC6 staining. Furthermore, reactive oxygen species generation in rotenone treated SK-N-AS NB cells was investigated with and without NAC addition.

Results: In SK-N-AS cells, MTT assay showed a dose-dependent neurotoxicity due to rotenone application. Rotenone treatment inhibited cell proliferation in a time-dependent manner in SK-N-AS cells. DAPI and DiOC6 staining revealed highly increased cell damage in SK-N-AS cells after 24 h of rotenone treatment. The addition of anti-oxidative agent NAC in MTT assay prevented rotenone-induced apoptosis in SK-N-AS NB cells.

This finding was confirmed by cell cycle assay and ROS generation measurement with DCFH-DA staining.

Conclusion: Rotenone decreased cell viability and increased ROS generation in SK-N-AS cells as expected. The rotenone induced highly toxic effects by ROS generation in SK-N-AS cells were completely neutralized after addition of NAC. Furthermore, in SK-N-AS NB cells a highly reduced rate of rotenone-induced apoptosis could be demonstrated after NAC addition with DCFH-DA staining.

6th Central European Congress of Life Sciences Eurobiotech 2017

174 Abstracts

P15&18.2

P15&18.3

GD2 ganglioside-binding antibody and specific aurora A kinase inhibitor induce autophagy in IMR-32 neuroblastoma cells

Application of Adenovirus dodecahedron delivery vector as a new approach in overcoming drug resistance

Małgorzata Durbas1, Paweł Pabisz2, Katarzyna Wawak 2, Elżbieta Boratyn1, Iwona Nowak1, Irena Horwacik1, Hanna Rokita1

Marta Jedynak1, Patryk Dolega P1, Małgorzata Podsiadla-Bialoskorska1, Jadwiga Chroboczek1,2, Ewa Szolajska1

1 Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland; 2Departament of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland

1 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Department of Protein Biosynthesis, Poland; 2Université Grenoble Alpes, CNRS, Grenoble INP, TIMC-IMAG, EFS, France

GD2 ganglioside (GD2) and aurora A kinase are important molecular targets in neuroblastoma [1,2]. However, it has not yet been elucidated how targeting GD2 and aurora A kinase with 14G2a monoclonal antibodies (14G2a mAbs) and small-molecule inhibitor (MK-5108), respectively, may affect autophagy process in neuroblastoma cells.

In this study, we investigated the potential of 14G2a mAbs and MK-5108 inhibitor in the regulation of autophagy in IMR-32 neuroblastoma cells. Both 14G2a mAbs and MK-5108 treatment induced the expression of crucial autophagy marker, microtubule-associated protein (LC3A/B), as demonstrated by up-regulation of autophagosome-associated, converted LC3A/B-II form at 48 and 72 h upon treatment. We performed autophagic flux assay in IMR-32 cells measured by the CYTO-ID dye. 14G2a mAbs and chloroquine combined treatment indicates that autophagic flux occurs in IMR-32 cells as the fluorescence intensity of CYTO-ID dye is increased when treated with both agents as compared to control or single agent-treated IMR-32 cells. The analogical effects were observed for MK-5108 and chloroquine combined treatment. These results were confirmed by measuring CYTO-ID signal accumulation in autophagic compartments using fluorescence microscope and Western blot analysis of the expression levels of LC3A/B-II converted form.

Results presented here provide first-hand evidence that 14G2a mAbs and MK-5108 inhibitor induce autophagy process in IMR-32 cells.

Despite the progress in the field of drug delivery there is still a need in clinical applications for development of efficient, non-toxic carriers targeting cancer cells. The enormous challenge in modern chemotherapy is drug resistance of malignant tumors. A novel approach to combat cancer drug resistance is to take advantage of the ability of nanocarriers to deliver chemotherapeutic agents through endocytosis, thus sidestepping drug resistance mechanisms.

Adenoviral dodecahedron (Dd) is a non-infectious virus-like particle, endowed with remarkable cell penetration capabilities and able to deliver intracellularly numerous copies of active biomolecules (Zochowska M et al (2015) Nanomedicine 11: 67–76). Here we show that Dd endocytosis is indirectly dependant on cholesterol, while the lipid has no influence on the efficiency of vector cell attachment. Moreover we present the results of Dd in vitro studies showing efficient penetration of drug-resistant cancer cells. We demonstrate that conjugation of anticancer antibiotic, doxorubicin, with Dd results in overcoming drug resistance and significantly improves drug efficacy in drug-resistant human uterine sarcoma cells. The results concerning efficient penetration and delivery of anticancer agent to drug-resistant cells, provide a basis for the future application of Dd as a putative drug carrier in targeted cancer therapy.

Acknowledgements The work was financially supported by the National Science Centre (DEC2013/09/B/NZ3/02327).

References [1] Wu ZL et al (1986) Cancer Res 46(1): 440–3.

[2] Gautschi O et al (2008) Clin. Cancer Res 14: 1639–1648.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 175

P15&18.4

P15&18.5

Monoamine oxidase from geomyces sp.

P3 as a tool in synthesis of chiral amine building blocks of pharmaceutical drugs

Study on the effect of dental material Optibond on sulfurtransferases expression in human fibroblast cell line Hs27

Iga Jodłowska, Klaudia Szmejda, Tomasz Florczak

Anna Bentke, Kinga Kaszuba, Agnieszka Krawczyk, Maria Wróbel

The Institute of Technical Biochemistry, Technical University of Lodz, Poland

Jagiellonian University Medical College, Kraków, Poland

Keywords: flavine monoamine oxidase, psychrophilic enzyme.

In natural environment flavine monoamine oxidases catalyze reaction of oxidative deamination of primary aliphatic and aromatic, secondary and tertiary amines to imines which further reacts to ketons or aldehydes and ammonia. The reaction of oxidative deamination is highly specific, enzyme is able to successively transform/oxidize only one amine enantiomer. According to its highly specificity, and role in amine metabolism it has a great potential in green chemistry. Monoamine oxidase from psychrophilic fungus Geomyces sp. P3 is able to catalyze oxidative deamination of cyclic amines and their derivatives like pyrrolidines. What makes the enzyme a great candidate for use in stereospecific biotransformations.

On the poster I would like to present the screening of potential producer of monoamine oxidase from extremophilic fungi. Optimization of the biosynthesis process of monoamine oxidase from Geomyces sp. P3. What is more the potential of monoamine oxidase from Geomyces sp. P3 in biotransformations of cyclic amines.

Keywords: Optibond, Hs27 fibroblast cell line, sulfurtransferases The literature data indicate that fillings used commonly in dentistry may contain and release cytotoxic substances and exhibit damaging effect on living cells of the surrounding tissue – the pulp. In vitro studies showed that methacrylate resins inhibit the synthesis of DNA and proteins in fibroblasts. At higher concentrations they also lead to cell death. The aim of the study was to investigate the concentrations and incubation times of commonly used Optibond dental filling, in which they would induce a cytotoxic effect on normal Hs27 cell line and also to show its effect on sulfurtransferase enzymes expression under estimated panel of concentrations and incubation times.

Experiments were performed on an in vitro cellular model.

Changes in cellular integrity were measured by the LDH test. The expression levels of 3-mercaptopyruvate sulfurtransferase, Cystathionine γ-lyase, and other were determined by RT-PCR and Western Blot protocol. Changes in cell viability were visualized by crystalline violet staining.

The cytotoxic effect of Optibond filling was demonstrated for concentrations higher than 0.03%. Reduced number of cells and changes in the expression of the analyzed mRNA and proteins were demonstrated for the tested range of Optibond concentrations.

6th Central European Congress of Life Sciences Eurobiotech 2017

176 Abstracts

P15&18.6

P15&18.7

The expression of antioxidative enzymes and lipids peroxidation in the heart of frogs exposed to heavy metal ions

Quinaldic acid alters cell cycle progression of colorectal adenocarcinoma cells in vitro

Marta Kaczor-Kamińska , Piotr Sura , Kinga Kaszuba1, Maria Wróbel1 1

2

1 Chair of Medical Biochemistry, Jagiellonian University, Collegium Medicum, Krakow; 2Chair of Medical Biology, Jagiellonian University, Collegium Medicum, Krakow

Heavy metal ions are environmentally persistent toxins. There are three main reasons for their high toxicity in living organisms: high affinity for thiol-, amino- and carboxyl groups of amino acid residues in proteins, the effect on the antioxidant potential of cells, the competition with divalent cations of metals (copper, zinc) for active sites of proteins.

Main goal of the undertaken experiments was to confirm the oxidative stress in tissue exposed to heavy metal ions, through the determination of malone dialdehyde concentration in tissue homogenates and the investigation of gene expression of enzymes involved in antioxidation e.g.: glutathione peroxidase, catalase and superoxide dismutase. In the heart of Xenopus tropicalis the expression of sulfurtransferases (γ-cystathionase, 3-mercaptopyruvate sulfurtransferase and rhodanese) genes was also analyzed.

Results obtained showed that in the heart of animals exposed to heavy metal ions L-cysteine is transformed into sulfane sulfur containing compounds as well as glutathione. Changes found in rhodanese expression, and in the level of sulfane sulfur suggest a role in antioxidative processes.

Ewa Langner1, 2, Piotr Pożarowski3, Grażyna Rajtar1 1 Department of Pharmacology, Medical University of Lublin, ul. Chodźki 4a, 20-093 Lublin, Poland; 2Department of Medical Biology, Institute of Agricultural Medicine, Jaczewskiego 2, 20-0 90 Lublin, Poland; 3Department of Clinical Immunology, Medical University, Chodźki 4a, 20-093 Lublin, Poland

Keywords: quinaldic acid; colon cancer; cell cycle Introduction: Quinaldic acid is an endogenous tryptophan metabolite and its biological function is poorly known up to date. It has been proposed that quinaldic acid may arise via kynurenine pathway and constitute a dehydroxylation product of kynurenic acid. Recently, it has been shown that quinaldic acid in micro- and milli-molar concentrations shows antiproliferative activity in human colon cancer cells in vitro [1]. The aim of present study was to evaluate the effect of quinaldic acid on cell cycle progression of human colorectal adenocarcinoma cells.

Methods: Two colon cancer cell lines (HT-29, LS180) were used in the study. Flow cytometry analyses were applied in order to evaluate cell cycle progression upon quinaldic acid incubation. Western blot analyses were used to show the expression of several proteins within tested cancer cells.

Results: Quinaldic acid suppressed cell cycle progression of both HT-29 and LS180 cancer cells. Moreover, it induced remarkable changes in the expression of cell cycle regulatory proteins.

Conclusion: Quinaldic acid regulates the progression of cell cycle in human colorectal adenocarcinoma cells in vitro.

Acknowledgements This study was supported by the Medical University in Lublin (MNmb 621).

References Langner E et al (2015) European Journal of Pharmacology 757: 21–27

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 177

P15&18.8

P15&18.9

Neuroprotective properties of young barley water extract. In vitro study in different models of neurodegeneration

Preparation of genetic constructs for modification of animal cells using CRISPR/Cas9 technology

Marta Kinga Lemieszek , Wojciech Rzeski 1

1,2

1 Department of Medical Biology, Institute of Rural Health, Jaczewskiego 2, 20-090 Lublin, Poland; 2Department of Virology and Immunology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland

Observed recently immense popularity of young barley is the consequence of its nutrient-dense profile and multidimensional health advantages. Due to the abundance of nutrients with beneficial effect on the nervous system (e.g. folic acid, choline, Mg) the activity of young barley water extract (YB) was investigated in different models of neurodegeneration: excitotoxicity induced by glutamate; neurotoxicity induced by cisplatin, cyclophosphamide, 5-fluorouracil; trophic stress (serum deprivation); and oxidative stress induced by tBuOOH. Studies were conducted in neuronal cultures derived from human neuroblastoma SH-SY5Y cell line. Extract influence on cells viability was examined using Neurite Outgrowth Staining, MTT and LDH tests. Antioxidant capacity of YB was determined by commercial antioxidant assays. Performed studies revealed beneficial effect of YB on neurons viability in both normal and different stress conditions. The extract not only eliminated the negative effects of glutamate excess and neurotoxicity induced by cytostatics but also acted as a trophic factor. Furthermore, YB ability of reactive oxygen species neutralization was proven. Discovered neuroprotective properties of YB support the hypothesis that diet supplemented with young barley might be useful for neurodegenerative diseases prevention, however these observations have to be confirmed in animal and clinical studies.

Magdalena Hryhorowicz1, Joanna Zeyland1, Natalia Mazurkiewicz1, Agnieszka Nowak1, Ryszard Słomski1,2, Daniel Lipiński1 1 Poznan University of Life Sciences, Department of Biochemistry and Biotechnology, Poland; 2Institute of Human Genetics, Polish Academy of Sciences, Poland

Keywords: CRISPR/Cas9 system, genetic modification, guide RNA, Cas9 nuclease The application of the CRISPR/Cas9 (clustered regularly-interspaced short palindromic repeats, CRISPR; CRISPR-associated proteins, Cas) system as a genome editing method has been one of the greatest scientific advances in recent years. Modifications in a specific site in the genome are possible thanks to cellular repair processes of double strand breaks induced by the Cas9 protein. The most important advantages of the CRISPR/Cas9 system include its high specificity, easy construct design (dependent mainly on the 20 base pairs comprising guide RNA), easy preparation of genetic constructs, high transformation efficiency as well as potential application of many modifications within one experiment, which makes this approach both cost- and time-effective. Here, we review protocols for preparation of CRISPR/Cas9 reagents to create knockout or knock-in animals.

Acknowledgements This work was funded by grant for young researchers at Poznan University of Life Sciences (Grant No. 507.173.89).

Acknowledgements This work was funded by the Institute of Rural Health in Lublin (project number MN 17001)

6th Central European Congress of Life Sciences Eurobiotech 2017

178 Abstracts

P15&18.10

P15&18.11

Is liquid biopsy a good molecular monitoring tool?

Detection of L858R and T790M EGFR mutations in circulating tumor DNA derived from NSCLC patient treated with gefitinib (case study)

High-resolution protein stability analytics using nanodsf

Ewelina Nalejska1,2, Łukasz Żołna1, Aleksandra Chrząstek1, Bogdan Żurawski 3, Janusz Kowalewski1, Marzena Anna Lewandowska1,2

Application Specialist & Sales; NanoTemper Technologies sp. z o.o., Krakow, Poland, E-mail: jakub.nowak@nanotemper.de

Department of Thoracic Surgery and Tumors, Nicolaus Copernicus University, Collegium Medicum, 85-796 Bydgoszcz, Poland; 2Department of Molecular Oncology and Genetics, The Lukaszczyk Oncology Center, Bydgoszcz, Poland; 3 Outpatient Chemotherapy, The Lukaszczyk Oncology Center, Bydgoszcz, Poland 1

The most innovative and noninvasive diagnostic method is detection of circulating DNA (ctDNA) which allows to capture fragments of tumor DNA in blood and monitor cancer disease.

Our study was conducted on 20 liquid biopsy samples from 7 patients diagnosed with NSCLC receiving erlotinib, gefitinib or afatinib; quantification of absolute copy numbers of ctEGFR extracted from plasma was performed

by EntroGen’s ctEGFR Mutation Detection CE-IVD kit.

In 3/7 cases T790M mutation was found after targeted treatment implementation. In particular one patient had activating L858R EGFR mutation detected in FFPE and plasma; patient was followed by gefitinib treatment and had 35 months progression free survival. Additional inhibiting T790M mutation was found two months before the appearance of metastasis in scapula. Our clinical data showed that presence of T790M suggests approaching lack of response to first generation TK inhibitors.

Jakub Nowak

Keywords: protein stability, aggregation, distorted protein, unfolding vs refolding The fluorescence of tryptophans in a protein is strongly dependent on its close surroundings. By following changes in fluorescence, chemical and thermal stability can be assessed in a truly label-free fashion. The dual-UV technology by NanoTemper allows for rapid fluorescence detection, providing an unmatched scanning speed and data point density. This yields ultra-high resolution unfolding curves which allow for detection of even minute unfolding signals.

Furthermore, since no secondary reporter fluorophores are required, protein solutions can be analyzed independent of buffer compositions, and over a concentration range of 150 mg/ml down to 5 µg/ml. In addition, information on protein aggregation can be recorded in parallel, providing insight into colloidal stability of the sample. Therefore, nanoDSF is the method of choice for easy, rapid and accurate analysis of protein folding and stability, with applications in membrane protein research, protein engineering, formulation development and quality control.

The presentation will cover biophysical concepts of the technique showing benefits of the nanoDSF technology platform, and will be followed by specific examples of nanoDSF applications towards various experimental systems.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 179

P15&18.12

P15&18.13

Phage Display selection and characterization of scFv specifically recognizing extracellular domain of FGFR1

Cross-talk between platelets and lymphocytes in autoimmune thyroid disease

Jakub Szymczyk, Julia Chudzian, Łukasz Opaliński, Martyna Szczepara, Małgorzata Zakrzewska, Jacek Otlewski University of Wrocław, Faculty of Biotechnology, Department of Protein Engineering, Joliot Curie 14a, Wrocław, 50-383, Poland Fibroblast growth factor receptor 1 belongs to the family of the tyrosine kinase receptors that are responsible for many crucial biological processes such as promotion of cell division and proliferation. It has been shown that FGFR overexpression or its overactivity lead to the disruption of cell signaling followed by uncontrolled cell division and mechanism of apoptosis avoidance. It was reported in many types of tumors, particularly in malignant varieties.

Therefore, the FGFR1 is a promising target for novel anti-cancer therapies. One of the commonly used anticancer therapeutic strategy includes the use of monoclonal antibodies as a drug delivering molecules. The molecules, specifically recognizing tumor associated antigens can be selected using Phage Display technique that uses phages to display and present these molecules.

In this study, two commercial phage libraries (Tomlinson I and J) presenting scFv molecules have been used.

After the selection and protein purification, scFv molecules were analyzed by surface plasmon resonance (SPR) to confirm their interaction with ligands and to determine the kinetic parameters of the interactions with FGFR1. To determine the fragment of FGFR1 extracellular domain interacting with selected scFv and to determine specificity of the interaction for FGFR1 ELISA and pull-down assay were performed. As a result, group of proteins specifically recognizing FGFR1 with three different binding sites were identified.

Acknowledgementss The work was supported by the National Science Centre, Poland (grant 2011/02/A/ NZ1/00066).

Conference costs were supported by Wroclaw Centre of Biotechnology programme “The Leading National Research Centre (KNOW) for years 2014–2018”.

Małgorzata Tomczyńska, Joanna Saluk-Bijak Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Poland, Pomorska 141/143, 90-236, Lodz The blood platelets are multifunctional cells which are involved in the initiation of endothelial dysfunction, atheroma, modulation of inflammatory or immune responses.

Because of their multifaceted proinflammatory activity, platelets are also involved in the pathogenesis of autoimmune thyroid diseases (AITDs). The platelets and lymphocytes are mutually regulated the functions or activity.

Hence platelets increase adhesion, aggregation, migration of lymphocyte T, B, NK cells. The platelets impact on various functional aspects of lymphocyte subpopulations. The platelets can promote isotype shifting, enhance antibody production of B cells. The lymphocytes can regulate platelet activation or participation of platelets in immune processes. These two cell types collaborate in CD40-CD40L ligand-mediated intercellular signaling, integrins, cytokine secretion, hemokine receptors.

The aim of this current study was to clarify the aggregation status of platelet and lymphocyte subpopulations, determine the biological activity of these cells in circulating blood in patients with AITDs. Our results show an increased platelet activation and cross-link of platelets and lymphocytes in patients with AITDs. The initial analysis of their interaction involve light, transmission electron microscopy, flow cytometric analysis, which describe the degree of cell activation.

Flow cytometric analysis of peripheral blood demonstrated changes in counts of B, T cell population compared with healthy controls.

Acknowledgements The work includes results of research project No. DEC-2014/13/N/NZ5/01389 funded by the National Science Centre.

6th Central European Congress of Life Sciences Eurobiotech 2017

180 Abstracts

P15&18.14 Antiandrogens induce atresia and disturb maturation of porcine preantral ovarian follicles cultured in vitro Gabriela Gorczyca1*, Kamil Wartalski1, Zbignew Tabarowski2, MaĹ&#x201A;gorzata Duda1 1 Institute of Zoology and Biomedical Research, Department of Endocrinology, Jagiellonian University in Krakow, Poland; 2 Institute of Zoology and Biomedical Research, Department of Experimental Hematology, Jagiellonian University in Krakow, Poland

This study was conducted to determine whether experimentally induced androgen deficiency during in vitro culture of porcine ovarian follicles affects follicular antrum formation. Cortical slices obtained from porcine ovaries were cultured for 8 days with the addition of an androgen â&#x20AC;&#x201C; testosterone (T), antiandrogens: 2-hydroxyflutamide (2-Hf) or vinclozolin (Vnz), separately or in combinations with T. Cultures were terminated on days: 2, 4, 6, and 8.

FSHR, GDF-9 and BECN1, LC3 were assessed at mRNA and protein level. Moreover the studied proteins were immunolocalized and antrum formation progress was examined every 2 days. Results of the present study confirmed that androgens are involved in porcine early follicular development by: (1) indicating the effect of supplementation with exogenous androgens on the time-frame of antrum formation, (2) detecting changes in the expression of all investigated proteins in cultured ovarian follicles between control and experimental conditions, (3) showing for the first time, that an environmental antiandrogen Vnz adversely affects follicular survivability. The present study confirms deleterious effects of androgen deficiency on follicles at antrum formation stage, what may negatively influence reproductive function in mammals. In the light of accumulating evidence indicating the rising presence of EDCs in the environment, it seems important to establish how these compounds influence the initiation of folliculogenesis within the ovary.

Acknowledgements Supported by DEC-2013/09/B/NZ9/00226 from National Science Center Poland.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 181

Dual use of biotechnology Keynote lecture

Lectures

L16.1

L16.2

Dual use of scientific discoveries and activity of United Nations Biosecurity Working Group

New plant breeding techniques from the point of view of their marketability and patentability in the European Union

Ryszard Słomski1,2, Marlena Szalata1 Poznań University of Life Sciences, Department of Biochemistry and Biotechnology, Poland; 2Institute of Human Genetics, Polish Academy of Sciences, Poland 1

Mission of the IAP Biosecurity Working Group (established in 2004), is helping to mitigate the risk that developments in science and technology could be misused for biological weapons or terrorism, without impeding scientific progress. The Working Group chaired by the Polish Academy of Sciences included the national academies of Australia, China, Cuba, Egypt, India, Nigeria, Pakistan, Russia, the United Kingdom, and the United States.

In preparation for the 8th Review Conference of Biological Weapons Convention last year, the IAP Biosecurity Working Group, in collaboration with the Royal Society, the US National Academy of Sciences, and the Polish Academy of Sciences supported efforts to consider the implications of recent advances in science and technology.

The IAP undertake relevant activities such as education, conferences, joint statements, and reports on topical scientific issues such as synthetic biology, antimicrobial resistance, and gene editing. It is perhaps best known in Geneva for the workshops organized to review trends in science and technology (S&T) relevant to the operation of the Convention as an independent input to the 6th, 7th, and 8th review conferences and its contributions to the S&T discussions during the Meetings of Experts.

The IAP Biosecurity Working Group also analyzed possible risks of human gene-editing technologies, their potential use in biomedical research and medicine, and the clinical, ethical, legal, and social implications of their use.

Tomasz Zimny Institute of Law Studies, Polish Academy of Sciences, Poland I analyse various aspects of new plant breeding techniques (NPBT) from the point of view of marketability and patentability of their products in Europe. Genetic engineering is a powerful tool but the legislation regarding marketing of GMOs in EU is strict. While they are generally patentable and attractive for breeders, marketing of new GM products requires significant investments and is time consuming. Besides, GM products carry a negative stigma, which makes them even less economically feasible in Europe. The definition of a GMO and genetic modification (which determines if a certain product requires authorisation) in EU law is vague and patchy.

I present and overview of the EU GMO legislation with a focus on provisions, which have potential for classifying certain NPBTs as not leading to GMO creation, hence not requiring authorisation and labelling of products. Selected NBPTs are analysed for this purpose, e.g. oligonucleotide-directed mutagenesis, cisgenesis and intragenesis or grafting on GM rootstock. Their patentability is also tested, in the context of exclusions of varieties and essentially biological processes.

While some of analysed techniques (in particular those involving mutagenesis or not involving interventions in the genome) may slip beneath legislation’s radar, a broader application of NPBTs in the EU requires a paradigm shift within the law, where the focus would be put on features of the product itself, not on the way they were created.

6th Central European Congress of Life Sciences Eurobiotech 2017

182 Abstracts

L16.3

L16.4

Effects of microwave radiation on the bioactive properties in selected vegetable species

Preparation of genetic constructs for modification of animal cells using CRISPR/Cas9 technology

Remigiusz Olędzki1, Krzysztof Lutosławski2

Magdalena Hryhorowicz1, Joanna Zeyland1, Natalia Mazurkiewicz1, Agnieszka Nowak1, Ryszard Słomski1,2, Daniel Lipiński1

1 Institute of Chemistry and Food Technology, Department of Biotechnology and Food Analysis, Wrocław University of Economics, Poland; 2Institute of Chemistry and Food Technology, Department of Bioprocess Engineering, Wrocław University of Economics, Poland

Microwave processes are mainly used to induce thermal changes in the food, which depend on the processing time, the microwave power and the amount of processed food. Thermal changes are associated with modifications of bioactive properties in food.

The aim of the study was to examine the effect of microwave radiation on the total antioxidant capacity (TAC) and contents of polyphenol compounds in celery, carrot, broccoli, cauliflower, red and yellow peppers.

The TAC was analysed both in fresh and microwave treated vegetables. The heating was conducted in a microwave oven for 15 min. with a rated power of 800 W and a frequency of 2450 MHz. In methanol and ethanol homogenates were evaluated the antioxidant potential by DPPH, ABTS and FRAP and polyphenol content by F-C method.

Microwave radiation caused a significant (p≤0.05) changes in bioactive properties. In carrots and cauliflower, the TAC was reduced to a lower extent than in red peppers, yellow peppers, broccoli and celery. The highest (15–20%) reduction in TAC was observed in the red peppers, celery or broccoli, containing more than 90% water. This finding indicated that the vegetables with a lower water content characterized by lower deprivation of TAC than the vegetables with a higher water content. This phenomenon is probably interrelated with a high heat capacity of vegetables with a high water content.

During the microwave treatment, carrots and cauliflower retain the highest level of TAC as well as the highest polyphenol content.

1 Poznan University of Life Sciences, Department of Biochemistry and Biotechnology, Poland; 2Institute of Human Genetics, Polish Academy of Sciences, Poland

Keywords: CRISPR/Cas9 system, genetic modification, guide RNA, Cas9 nuclease The application of the CRISPR/Cas9 (clustered regularly-interspaced short palindromic repeats, CRISPR; CRISPR-associated proteins, Cas) system as a genome editing method has been one of the greatest scientific advances in recent years. Modifications in a specific site in the genome are possible thanks to cellular repair processes of double strand breaks induced by the Cas9 protein. The most important advantages of the CRISPR/Cas9 system include its high specificity, easy construct design (dependent mainly on the 20 base pairs comprising guide RNA), easy preparation of genetic constructs, high transformation efficiency as well as potential application of many modifications within one experiment, which makes this approach both cost- and time-effective. Here, we review protocols for preparation of CRISPR/Cas9 reagents to create knockout or knock-in animals.

Acknowledgements This work was funded by grant for young researchers at Poznan University of Life Sciences (Grant No. 507.173.89).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 183

Posters

P16.2

P16.1

Influence of the application of variable raw material composition on the process of HG alcoholic fermentation conducted under bioreactor conditions

The protective effect of selenium on human cells treated with zearalenone Anna Barbasz1, Barbara Kreczmer1, Barbara Dyba1, Maria Filek 2 1 Institute of Biology, Pedagogical University, Podchorążych 2, 30-084 Cracow, Poland; 2The Franciszek Górski Institute of Plant Physiology, Polish Academy of Science, Niezapominajek 21, 30-239 Cracow, Poland

Keywords: selenium, zearalenon, cytotoxicity Zearalenone (ZEA), produced by some species of Fusarium, is one of the most potent, non-steroidal substances from the xenoestrogen group. Selenate has been investigated as a protective substance, which has multiple actions ranging from the influence on gene expression, by reactions with reactive oxygen species to regulation of the immune system. The aim of the study was to investigate the protective effect of selenate on the cytotoxicity of ZEA on human cell of U-937 line. The viability assay, membrane integrity, the induction of inflammation and antioxidant activity analysis were used to evaluate the cellular response to the ZEA and Se treatment. The effect of ZEA or Se treatment were dose-dependent. It was found that ZEA acted cytotoxically above 30 μM while selenate addition at concentrations lower than 5μM partly reduced this effect.

Acknowledgements This work was supported by NCN No. 2014/15/B/NZ9/02192

Grzegorz Kłosowski, Dawid Mikulski Department of Biotechnology, Kazimierz Wielki University, ul. Poniatowskiego 12, 85-667 Bydgoszcz, Poland Keywords: high gravity fermentation process, different raw materials, volatile by-products of alcoholic fermentation None of the raw materials used in the fermentation technology provide optimum composition of the fermentation medium which would be rich with an assimilable source of nitrogen, mineral compounds and biologically active compounds. The aim of the study was to evaluate the influence of the variable raw material composition on fermentation activity of S. cerevisiae under HG (High Gravity) conditions and the composition of volatile by-products of alcoholic fermentation. The media were prepared according to the principles of pressureless liberation of starch (PLS) technology with varying percentages of different plant materials (maize, rye, wheat and molasses).

The research was conducted under bioreactor conditions.

The composition of the fermentation media was monitored using High Performance Liquid Chromatography (HPLC).

The composition of volatile by-products of fermentation using capillary gas chromatography (GC) was also analyzed.

A medium containing equal amounts of maize, rye and wheat grain, and molasses gave the highest final ethanol concentration of about 130 g/L. It was also found that irrespective of the type and content of starchy materials in the fermentation media, the addition of molasses at different concentrations (25, 33 and 50%) increased the final ethanol concentration to above 112 g/L. The use of variable raw material composition during mash preparation resulted in significant changes in the composition of the analyzed fermentation by-products, including carbonyl compounds, higher alcohols and esters.

6th Central European Congress of Life Sciences Eurobiotech 2017

184 Abstracts

P16.3

P16.4

Orthogonal, light-inducible protein expression platform in yeast Sacchararomyces cerevisiae

Effects of various parameters of acidic pretreatment on the enzymatic hydrolysis efficiency of distillery stillage of different origin

Katrin Messerschmidt, Fabian Machens, Lena Hochrein, Gita Naseri University of Potsdam, Synthetic Biosystems - Cell2Fab Research Unit, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany, Electronic address: messer@uni-potsdam.de The project Cell2Fab aims at the generation of yeast artificial chromosomes for next generation biotechnological procedures. Fundamental goal is the allocation of modular-build circular YACs that can be easily adapted to different biotechnological applications by facilitating highly regulated production of small to large cohorts of proteins and peptides in the yeast Saccharomyces cerevisiae. The long-term goal is the use of YACs for the generation of “multi-enzyme-machines” for the production of pharmacologic relevant peptides, entirely new functional units for next generation biotechnological procedures or the production of proteins for in-vitro-translation. First results for non-conventional cloning technologies and light-induced protein expression for easy and fast construct assembly and protein expression will be presented. We are very interested in meeting other people in synthetic biology and biotechnology, academia as well as industry, to find cooperation partners that are in need of cloning and expression strategies to enable their research and production.

Dawid Mikulski, Grzegorz Kłosowski Department of Biotechnology, Kazimierz Wielki University, ul. Poniatowskiego 12, 85-667 Bydgoszcz, Poland Keywords: distillery stillage, acidic pretreatment, cellulose, bioethanol An essential step in the production of bioethanol from cellulose, due to the complexity of lignocellulosic biomass, is a pretreatment of the raw material. Efficiency of the pretreatment depends primarily on the origin of plant biomass and process parameters. The aim of the study was to evaluate the influence of variable process parameters, such as temperature, exposure time and the concentration of sulfuric acid on the amount of sugars released during the pretreatment of distillery stillage and enzymatic hydrolysis of cellulose. Rye, wheat and maize distillery stillages with a known content of cellulose, hemicellulose and lignin were used as raw material. The following variable parameters were applied: temperature of 121 and 131°C, exposure time of 30 and 60 minutes and the H2SO4 concentration of 0.1 and 0.2 M. Efficiency of the process was evaluated by measuring the concentrations of sugars released during pretreatment and enzymatic hydrolysis with the use of High Performance Liquid Chromatography (HPLC) method. The highest average glucose concentrations during the pretreatment and enzymatic hydrolysis (1.3 g/L and 3.5 g/L, respectively) were obtained for rye and wheat distillery stillage by applying 0.2 M H2SO4, for 60 minutes at both 121 and 131°C. The highest efficiency of pretreatment was observed for maize distillery stillage with the highest cellulose concentration (32.2% DM) processed with 0.2 M H2SO4, for 60 minutes at 131°C.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 185

P16.5

P16.6

Optimization of the enzymatic hydrolysis process of distillery stillage of various origin subjected to the procedure of acidic pretreatment

Association of kappa –casein gene polymorphism with milk yield, fat and protein in Holstein cattle

Dawid Mikulski, Grzegorz Kłosowski

Martina Miluchová, Michal Gábor, Juraj Candrák, Anna Trakovická, Kristína Candráková

Department of Biotechnology, Kazimierz Wielki University, ul. Poniatowskiego 12, 85-667 Bydgoszcz, Poland

Department of Genetics and Breeding Biology, Slovak University of Agriculture in Nitra, Tr. A Hlinku, 2, 949 76 – Nitra, Slovakia

Keywords: distillery stillage, cellulose hydrolysis, optimization After selection of pretreatment parameters, the next step in the development of an efficient process of lignocellulosic biomass degradation is the optimization of enzymatic hydrolysis that would guarantee a high efficiency at this stage of the process. The aim of the research was to select the enzymatic hydrolysis parameters of the distillery stillage of various origin, such as hydrolysis time, enzyme and biomass concentration. Rye, wheat and maize distillery stillage with a known cellulose content (16.8, 18.6 and 32.2% of dry mass, respectively) were used. The following enzyme hydrolysis parameters were modified: biomass concentration (1, 2 and 3 g of dry mass), concentration of Cellic® CTec2 preparation by Novozymes (6, 12 and 24% w/w, 1 g of preparation per 1 g of cellulose), and hydrolysis time (24, 48 and 72 h). The efficiency of the hydrolysis process was calculated from the glucose concentration with the use of High Performance Liquid Chromatograpy (HPLC) method. It was observed that the increasing biomass concentration adversely affected the efficiency of pretreatment. A statistically significant influence of enzyme dose at the level of 24% w/w on the increase of hydrolysis efficiency after 72 h of the process for 2 and 3 g of dry mass was also reported. At the same time, a slight increase in the efficiency of the enzymatic hydrolysis (about 3%) was observed between 24th and 72nd h of the process. Optimization of enzymatic hydrolysis of cellulose allowed for the following maximum efficiency: 84% for rye distillery stillage, and ca. 70% for wheat and maize distillery stillage.

Keywords: Holstein cattle; milk production; CSN3; kappa-casein; genetic structure The goal of the paper was to evaluate the effect of genetic polymorphism of kappa-casein on the milk production in Holstein cattle. A total 210 cows of Holstein cattle were use in this study. The genotype structure of cattle population was established by PCR-RFLP analyses. In the population of cattle were detected genotypes AA (69.52%), AB (27.62%) and BB (2.86%). The frequency of allele A was 83.33% and allele B was 16.67%. Effectiveness of allele incidence and genetic diversity was evaluated with following parameters: theoretical heterozygosity, experimental heterozygosity, polymorphism information content, expected homozygosity, effective number of alleles, level of possible variability realization. The analysed population of Holstein cattle exhibit high value of homozygosity and low values of polymorphism information content, effective number of alleles and level of possible variability realization. The effect of CSN3 gene polymorphism on average breeding values for milk production traits as the yield of milk, fat and protein in kilograms as well as contents of fat and protein in percentages were analysed by SAS Enterprise Guide 5.1. Statistical analysis was confirmed that the AA genotype significantly reduces the average value of the protein content of milk on average by 0.08% compared with genotype BB. For other breeding values the impact of individual genotypes CSN3 gene on their variability was not observed.

Acknowledgements This study was supported by APVV-14-0054 and APVV-0636-11.

6th Central European Congress of Life Sciences Eurobiotech 2017

186 Abstracts

P16.7

P16.8

Effective population size and genetic drift in warmblood horses based on microsatellite DNA markers

Integrated system of sewage treatment, biogas production and methane enrichment of biogas unit

Nina Moravčíková, Radovan Kasarda, Ondrej Kadlečík Department of Animal Genetics and Breeding Biology, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia Keywords: diversity, horses, local population, microsatellite variation The aim of this study was to analyse the state of genetic diversity and potential loss of variability due to bottleneck or other genetic forces trough estimation of effective population size in three horse breeds: Lipizzan (103), Furioso (68), and Nonius (18). The genotyping data of 13 microsatellite loci have been collected from a total of 189 individuals representing the genepool of those breeds in Slovakia. Across all microsatellites the decrease of effective allele number (3.99±0.19) compared to mean number of alleles (6.26±0.35) was found. However, average value of gene diversity (0.73±0.01) indicated sufficient proportion of genetic variability within populations which confirmed also the Shannon’s index (1.47±0.05). The number of private alleles characterizing genetic uniqueness of each populations ranged from 1.31 (Lipizzan) to 0.15 (Nonius). The evidence of recent inbreeding was not found (F IS=-0.22).

The low estimates of effective population size (in average 48.27) suggested mainly small number of funders in parental populations or extensive losses of variation through random genetic drift. The analysis of bottleneck did not shown a significant putative bottleneck events. Our study is crucial for preserving the genetic diversity within analysed breeds and for preventing undesired loss of rare alleles that can be unique only for these local populations.

This study was supported by the Slovak Research and Development Agency (APVV-14-0054) and VEGA (1/0742/17).

Andrzej G. Chmielewski1, Jacek Palige1, Otton Roubinek1, Sylwia Witman-Zając1 , Janusz Usidus2, Adam Kryłowicz2 Institute of Nuclear Chemistry and Technology, Warsaw, Dorodna 16, Poland; 2 Association of Polish Electrical Engineers, Zamość, Rynek Wielki 6, Poland

1

This paper presents the project concerning research and development of the integrated plant with a capacity of approximately 100 kWe (150 kWt) for biogas production using substrates in the form of sludge from sewage treatment plants and waste and agricultural and food products.

Biogas, used as fuel in co-generators or after enrichment in methane for CNG obtaining or as addition to the local natural gas grid. In the adopted solution, biogas plant with purification system and compression of biogas is distributed energy source with a capacity of about 100 kWe. Selection of optimum working conditions and building of biogas plant based on the polish patents were made by the scientific-technical consortium consisting of the INCT and EKO-POL firm from Szewnia Dolna. The whole project is carried out within a framework of PBS III program of The National Centre of Research and Development.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 187

Stem cells in diseases and therapy Keynote lecture

Lectures

L17.1

L17.2

Molecular regulation of muscle stem cell function

Cobalt protoporphyrin increases G-CSF and mobilizes granulocytes and hematopoietic stem cells to the peripheral blood in mice

Michael A. Rudnicki Ottawa Hospital Research Institute, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8L6. mrudnicki@ohri.ca.

We discovered that a subset of satellite cells in skeletal muscle are self-renewing stem cells that give rise to myogenic progenitors through asymmetric apical-basal cell divisions. Our identification of satellite stem cells has facilitated important insights into satellite cell biology. For example, we discovered Wnt7a/Fzd7 signaling as important intrinsic control mechanism that plays a central role in regulating the pool size of the satellite stem cell compartment by stimulating symmetric stem cell expansion. Direct injection of recombinant Wnt7a protein into muscle significantly augments regeneration. Wnt7a treated muscles were larger, contained higher numbers of satellite cells, larger caliber myofibers, and were able to generate more force upon stimulation. Thus, the regulation of asymmetric stem cell division is a key control point that impacts the efficacy of the entire regenerative program. Stem cell polarity is established by the PAR complex, comprised of PAR3/PAR6/aPKC, to regulate self-renewal and expansion. We have discovered that full-length dystrophin is expressed in satellite stem cells in skeletal muscle. Dystrophin has long been known to be expressed in differentiated myofibers where it is required for sarcolemmal integrity, and loss-of-function mutations in its gene result in Duchenne Muscular Dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. We have made the seminal discovery that dystrophin regulates the establishment of PAR-mediated polarity in satellite cells. In the absence of dystrophin, the polarity effector Par1b is dysregulated, leading to the failure of Par3 to become localized to the cortex associated with the basal lamina. Importantly, this results in an abnormal increase in centrosome number, a 5-fold reduction in the proportion of satellite stem cells undergoing asymmetric divisions, and a marked decrease in the generation of myogenin-expressing progenitors. Accordingly, our data suggests that the failure of regenerative myogenesis to keep pace with disease progression in DMD is not due to muscle stem cell exhaustion, but rather is due to a cell-autonomous deficiency in asymmetric division.

Agata Szade, Krzysztof Szade, Witold N. Nowak, Karolina Bukowska-Strakova, Maciej Cieśla, Neli Kachamakova-Trojanowska, Józef Dulak, Alicja Józkowicz Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Poland Granulocyte colony-stimulating factor (G-CSF) is the mobilizing agent widely used in clinical practice to induce mobilization of cells from the bone marrow to the blood. However, G-CSF therapy is not always effective.

Here we show that administration of cobalt protoporphyrin IX (CoPP) increases the plasma concentrations of G-CSF, IL-6, and MCP-1 in mice, triggering the efficient mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC) to the peripheral blood. We directly compare the mobilization capacity of CoPP with that of recombinant G-CSF. Flow cytometry analysis of peripheral blood shows that CoPP-mobilized granulocytes have phenotype characteristic for more mature cells (with higher granularity and higher expression of Ly6G than cells mobilized with G-CSF). Furthermore, CoPP mobilizes the higher number of HSPC than G-CSF. In contrast to G-CSF, CoPP does not increase the number of circulating T-cells.

We also demonstrate that transplantation of CoPP-mobilized peripheral blood mononuclear cells (PBMC) results in a higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G-CSF. Although CoPP is commonly used to activate Nrf2/HO-1 axis, the observed effects are Nrf2/HO-1 independent, as CoPP induces efficient mobilization in both HO-1- and Nrf2-deficient mice. Concluding, CoPP increases endogenous expression of mobilization-related cytokines and has superior mobilizing efficiency compared to recombinant G-CSF. This observation could lead to the development of new therapy options for treatment of neutropenia and more effective harvesting of HSPC for transplantation.

6th Central European Congress of Life Sciences Eurobiotech 2017

188 Abstracts

L17.3

Posters

Comparison of the transcriptome of human mesenchymal cells isolated from right ventricle and epicardial fat

P17.1

Jacek Stępniewski1, Urszula Florczyk1, Karolina Bukowska-Strakova1, Krzysztof Szade1, Agnieszka Langrzyk 2, Tomasz Cichoń3, Stanisław Szala3, Agnieszka Paziewska4, Izabela Rumieńczyk4, Maria Kulecka4, Michał Mikuła4, Jerzy Ostrowski4, Marian Zembala5, Alicja Józkowicz1, Michal Zembala5 and Józef Dulak1,2 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Kardio-Med Silesia, Zabrze, Poland; 3Institute of Oncology, Gliwice, Poland; 4Institute of Oncology, Warsaw, Poland; 5Silesian Center for Heart Diseases, Zabrze, Poland 1

Mesenchymal stromal cells (MSC) isolated from different tissues are claimed to demonstrate similar therapeutic potential. However, thorough comparison of such cells are lacking. Thus, the aim of this study was to compare transcriptome of MSC with the same phenotype isolated from two different tissues, before and after culture. For that purpose cells were isolated from biopsies of right ventricle and epicardial fat collected from patients who underwent heart transplantation. Immunophenotyping revealed three distinct populations in both tissues: i) CD31-CD45CD90+CD34+CD146-, ii) CD31-CD45-CD90+CD34CD146+ and iii) CD31-CD45-CD90-CD34-CD146+, from which only the first could be grown after sorting. Thus, material for RNA-seq was collected from these cells before culture (250 cells) and on the passage 6 (5000 cells).

Transcriptomic analysis revealed that cells of the same phenotype upon isolation preferentially clustered according to the tissue of origin, not the patient they were isolated from. Additionally, cells from epicardial fat demonstrated higher heterogeneity. After 6 passages, heart- and fat-derived cells did not acquire similar transcriptome. Cell culture itself, however, significantly changed gene expression within both tissues. This is an important indication for further investigations of therapeutical potential of MSC. Accordingly, searching for the optimal cell source should be considered for any cell-based therapy.

Cobalt protoporphyrin increases G-CSF and mobilizes granulocytes and hematopoietic stem cells to the peripheral blood in mice Agata Szade, Krzysztof Szade, Witold N. Nowak, Karolina Bukowska-Strakova, Maciej Cieśla, Neli Kachamakova-Trojanowska, Józef Dulak, Alicja Józkowicz Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Poland Granulocyte colony-stimulating factor (G-CSF) is the mobilizing agent widely used in clinical practice to induce mobilization of cells from the bone marrow to the blood. However, G-CSF therapy is not always effective.

Here we show that administration of cobalt protoporphyrin IX (CoPP) increases the plasma concentrations of G-CSF, IL-6, and MCP-1 in mice, triggering the efficient mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC) to the peripheral blood. We directly compare the mobilization capacity of CoPP with that of recombinant G-CSF. Flow cytometry analysis of peripheral blood shows that CoPP-mobilized granulocytes have phenotype characteristic for more mature cells (with higher granularity and higher expression of Ly6G than cells mobilized with G-CSF). Furthermore, CoPP mobilizes the higher number of HSPC than G-CSF. In contrast to G-CSF, CoPP does not increase the number of circulating T-cells.

We also demonstrate that transplantation of CoPP-mobilized peripheral blood mononuclear cells (PBMC) results in a higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G-CSF. Although CoPP is commonly used to activate Nrf2/HO-1 axis, the observed effects are Nrf2/HO-1 independent, as CoPP induces efficient mobilization in both HO-1- and Nrf2-deficient mice. Concluding, CoPP increases endogenous expression of mobilization-related cytokines and has superior mobilizing efficiency compared to recombinant G-CSF. This observation could lead to the development of new therapy options for treatment of neutropenia and more effective harvesting of HSPC for transplantation.

Acknowledgements The study was funded by The National Centre for Research and Development via STRATEGMED programme (STRATEGMED 2/269415/11/NCBR/2015).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 189

Big Data and Biotechnology Lectures

L19.2

L19.1

High throughput estimation of biologically relevant RNA secondary structures for bioenginering and drug design

Tackling Three at Once: The Bread Wheat Genome Project Georg Haberer1, The International Wheat Genome Sequencing Consortium (IWGSC)2, The WHEALBI consortium3 Plant Genome and System Biology/PGSB, Helmholtz Center Munich, Ingolstädter Landstrasse 1, 85764 Neuherber, Germany (g.haberer@helmholtz-muenchen.de); 2 https://www.wheatgenome.org/; 3 http://www.whealbi.eu/

Marianna Plucinska, Agnieszka Chełkowska, Marek Żywicki Department of Computational Biology, Adam Mickiewicz University in Poznań, Poznań, Poland

1

In plants, our biological knowledge has been enormously accelerated by whole genome projects which provide invaluable tool boxes both for academic research as well as for agricultural and applied sciences. Success stories in the model plant Arabidopsis and Oryza sativa, a major crop for human nutrition, impressively prove such synergy effects between genome sequences and knowledge gain. Bread wheat (Triticum aestivum) has strongly shaped human civilization since its domestication at the Neolithic revolution.

Today, wheat is one of the top three crops in agriculture and provides ~20% of all calories of the world’s population.

In contrast to the other two major crops – maize and rice, whole genome projects have been impeded by several notoriously difficult properties of the wheat genome, namely its huge size five times larger than the human genome, its pronounced repetitive nature and its hexaploid karyotype.

In the framework of the IWGSC consortium, a highly improved genome assembly of bread wheat (Chinese spring variety) has been generated with a scaffold N50 of 7.4 Mb. Scaffolds have been joined and concatenated to pseudomolecules comprising ~14.5 Mb (85%) of the estimated genome size of 17 Gb. The assembly is a joint effort of many groups in the consortium using a large array of technologies including next generation sequencing of flow sorted single chromosomes, classical bac-by-bac sequencing of a minimal tiling path supported by FPC and the recently established whole genome shotgun DeNovoMAGIC™ technology (NRGene Inc.). The high accuracy of the assembly is supported by HiC chromosome capture as well as by genetic and radiation hybrid maps. Using homology and large experimental RNAseq data covering a wide variety of conditions, tissues and developmental stages, >110k high confidence gene models were predicted for bread wheat.

In collaboration with the EU project WHEALBI, exome sequence data of ~500 wheat varieties were mapped to these gene models to assess genic diversity in wheat.

The panel comprises diploid, tetraploid and 435 hexaploid accessions, representing wild species, landraces and old and current cultivars of 59 countries with an emphasis on European locations. In summary, we called for >600k genomic positions SNPs and small indels, and detected an average of ~270 absence variants for the analyzed wheat accessions. The detected variation provides a rich source to investigate wheat genome evolutionary diversity at spatio-temporal, ploidy and sub-genomic levels. The study is complemented by large field trials scoring agricultural relevant traits for the lines.

In recent years RNA has emerged as potent target for bioengineering and drug design. The ability of RNA to specifically bind small chemical compounds provide unprecedenced oportunities in design of novel systems for controlled protein expression, development of antimicrobial compounds or targeting of abberant, disease-associated transcripts. Those possibilities relay on rational design of the chemical compounds able to bind specific RNA structures. Thus, the knowledge of biologically relevant RNA structural elements is a key requirement.

Recently developed protocols for high throughput RNA structure probing provide a framework for transcriptome-wide investigation of RNA secondary structures.

However, to find the relevant structural motifs, appropriate methods for analysis of gathered large-scale data has to be applied. We have developed a complete framework for analysis of such high throughput probing data. It include a novel method for estimation of RNA probing signal from raw sequencing reads, which allows for robust estimation of both, in vitro and in vivo RNA secondary structure.

Next, we have developed a novel approach for identification of condition-dependent changes in RNA structure. Finally, based on transcritome-wide analysis, we are also able to detect the local RNA structues present in unrelated transcripts, which represent novel functional RNA motifs.

Such RNA domains and “switches” are most often involved in regulation of gene expression, thus provide a valuable target for bioingeneering and drug design.

6th Central European Congress of Life Sciences Eurobiotech 2017

190 Abstracts

L19.3

L19.4

Turning Data into Information

Evaluation of chromosome microrearrangements of an intersex horse applying array CGH

Bernal. C., Niemeijer, M., and Keijzer, T. Applikon Biotechnology B.V. Heertjeslaan 2, 2629 JG, Delft, The Netherlands.

The availability of the current parallel-processing techniques in the field of cell culture generates very complete information about the whole biological process. One normally monitors the pH, temperature and dissolved oxygen of a cell culture, but one can additionally register, for example, metabolic data from HPLC, off-gas analysis or number of cells and viability from a cell counter during the cultivation process. In the end, this approach leads to one of the scientist’s current challenges: How to handle this huge amount of data? Finding of workable solutions to this problem is essential, since this huge amount of data is retrieved from various analytical tools and multiple databases. The use of PIMS (Process Information Management Systems) offers an integrated workflow solution that covers the whole biological process: DoE and recipe planning, material preparation and tracking, data logging, plotting, monitoring, sampling, data analysis and process documentation. PIMS will enable users to simplify their bioprocess work-flow and to turn their data into information.

Katarzyna Kowalska, Klaudia Pawlina-Tyszko, Wojciech Witarski, Magdalena Wojtaszek1 Monika Bugno-Poniewierska National Research Institute of Animal Production, Department of Animal Genomics and Molecular Biology, Balice, Poland The development of high-resolution cytogenetic techniques enables to increase the accuracy of diagnosis of chromosomal disorders. The aim of the study is a comprehensive analysis of chromosome abnormalities, including unbalanced microrearrangements in individuals affected by reproductive disorders using a highly precise technique – array comparative genomic hybridization (aCGH).

The examined case was an intersex horse which had overall male body conformation; however, its external genitalia consisted of the incompletely developed vulva and penis. Cytogenetic examination using fluorescence in situ hybridization technique showed three cell lines: 63,X, 64,XX and 65,XX with a fragment of the Y chromosome. In the next step, we carried out DNA labeling using SureTag DNA Labeling Kit (Agilent). The labelled DNA was then subjected to 40 h hybridization onto 2x400K custom equine CGH microarrays (Agilent). The washed microarrays were scanned with SureScan G2565CA Scanner (Agilent). The obtained raw data were analysed using Agilent Genomic Workbench (7.0).

The analysis using aCGH method revealed 416 aberrations (256 amplifications and 161 deletions) in the genome of the investigated horse. The amplification of almost whole ECAX and the deletion of ECAY were identified. The largest number of aberrations was detected on ECA1. The obtained results allowed characterizing genes and pathways altered in the genome of the examined horse.

Acknowledgements Financed: BIOSTRATEG2/297267/14/NCBR/2016.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 191

L19.5

Posters

Common breeding history of different cattle breeds based on high-throughput genotyping data

P19.1

Veronika Kukučková1, Radovan Kasarda1, Nina Moravčíková1, Ino Curik 2, Mojca Simčič3, Gábor Mészáros4 1 Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, Department of Animal Genetics and Breeding Biology, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic; 2 University of Zagreb, Department of Animal Science, Svetošimunska 25, 100 00 Zagreb, Croatia; 3 University of Ljubljana, Department of Animal Science, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; 4 University of Natural Resources and Life Sciences, Division of Livestock Sciences, Gregor-Mendel Str. 33, 1180 Vienna, Austria

The power of high-throughput molecular information in clustering cattle breeds up to very weak differentiation was demonstrated. The presented BovineSNP chip data were analysed using a genetic mixture model for unlinked markers to determine the degree of genetic differentiation among 4 European local populations. Bayesian clustering performed by the admixture analysis of 9,433 markers was used to characterise genetic diversity and population structure of Slovak as well as Austrian Pinzgau, Cika and Piedmontese cattle. Generally, Slovak Pinzgau and Cika have similar origin in Austrian Pinzgau, and therefore both are known as a separate breed shorter time than Austrian Pinzgau or Piedmontese. The actual rates of admixture associated with direction of gene flow were randomly generated from a uniform distribution. Two ways of gene flow were noticed between each pair of populations except connection with Piedmontese. This breed more or less contributed to formation of analysed populations while no of them contributed to the formation of Piedmontese.

The obtained genetic data clarified some issues related to the local Pinzgau breeds origin and structure. The study proved Slovak as well as Austrian cattle are important and viable targets for conservation for they display special traits both phenotypic and of cultural and historical nature that should earn conservation efforts. This work has been supported by the Slovak Research and Development Agency (Contract No. APVV-14-0054).

Common breeding history of different cattle breeds based on high-throughput genotyping data Veronika Kukučková1, Radovan Kasarda1, Nina Moravčíková1, Ino Curik 2, Mojca Simčič3, Gábor Mészáros4 1 Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, Department of Animal Genetics and Breeding Biology, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic; 2 University of Zagreb, Department of Animal Science, Svetošimunska 25, 100 00 Zagreb, Croatia; 3 University of Ljubljana, Department of Animal Science, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; 4 University of Natural Resources and Life Sciences, Division of Livestock Sciences, Gregor-Mendel Str. 33, 1180 Vienna, Austria

The power of high-throughput molecular information in clustering cattle breeds up to very weak differentiation was demonstrated. The presented BovineSNP chip data were analysed using a genetic mixture model for unlinked markers to determine the degree of genetic differentiation among 4 European local populations. Bayesian clustering performed by the admixture analysis of 9,433 markers was used to characterise genetic diversity and population structure of Slovak as well as Austrian Pinzgau, Cika and Piedmontese cattle. Generally, Slovak Pinzgau and Cika have similar origin in Austrian Pinzgau, and therefore both are known as a separate breed shorter time than Austrian Pinzgau or Piedmontese. The actual rates of admixture associated with direction of gene flow were randomly generated from a uniform distribution. Two ways of gene flow were noticed between each pair of populations except connection with Piedmontese. This breed more or less contributed to formation of analysed populations while no of them contributed to the formation of Piedmontese.

The obtained genetic data clarified some issues related to the local Pinzgau breeds origin and structure. The study proved Slovak as well as Austrian cattle are important and viable targets for conservation for they display special traits both phenotypic and of cultural and historical nature that should earn conservation efforts. This work has been supported by the Slovak Research and Development Agency (Contract No. APVV-14-0054).

6th Central European Congress of Life Sciences Eurobiotech 2017

192 Abstracts

P19.2

P19.3

Analysis of genetic basis of natural herbicide resistance in maize by high-throughput sequencing technology

Can ecotoxicity tests performed in artificial soils may be used for prediction of metals toxicity in natural soils?

Medhat Mahmoud1,2, Marek Żywicki2, Wojciech M.Karłowski2

Mateusz Sydow1, Łukasz Chrzanowski1, Nina Cedergreen2, Mikołaj Owsianiak3

Institute of Bioorganic Chemistry Polish Academy of Sciences, Department of Protein Biosynthesis, Poland; 2 Institute of Molecular Biology and Biotechnology Faculty of Biology A. Mickiewicz University, Department of Computational Biology, Poland

1 Institute of Chemical Technology and Engineering, Poznan University of Technology, Poland; 2Department of Plant and Environmental Sciences, University of Copenhagen, Denmark; 3Division for Quantitative Sustainability Assessment, Department of Management Engineering, Technical University of Denmark, Denmark

1

Keywords: Maize, genomic variation, next generation sequencing, genotyping by sequencing, glyphosate, natural resistance to herbicides Maize is one of the world’s leading grains and in Europe is third only to wheat and barley. It is mainly used as food, feed source and bio-fuel. Maize has a human size genome and among the crop species is characterized by one of the highest genomic variability, mainly due to transposon-richness. The genome organization is complex and composed of approximately 85% repetitive sequences. Activation and loss of the transposable elements (TE) combined with their ability to shuffle gene fragments has a great impact on the dynamics of maize genome. Genomic sequences of different maize lines can differ from 25% up to 84%. Many studies have showed that such genomic variability (e.g., SNPs, indels, CNVs) may be associated with wide range of plant phenotypic traits and involved in metabolic fluctuations. In 1998 Monsanto first introduced glyphosate-resistant corn and now in USA 93% of cultivated maize is engineered to be glyphosate resistant.

It has been shown that intensive, continuous application of glyphosate may leads in some cases to development of higher levels of resistance to the herbicide. Here, we studied the nucleotide and genomic structural variations between two maize lines naturally differing in tolerance to glyphosate using high-throughput sequencing technologies: Illumina and SMRT PacBio generating short and long reads, respectively to deal with maize genome complexity. We found 8 and 30 unique structure variations in tolerant and sensitive lines, respectively and we identified more than 800,000 unique indels in tolerant line along with more than 4 million SNPs. Putative influence of the identified polymorphic sequences and there phenotype will be discussed.

Development of terrestrial ecotoxicity indicators of metals in natural soils is jeopardized by limited availability of predictive models or data of sufficient quality for speciation calculations needed for deriving metal free ion concentrations, which correspond to toxic metal forms. To compensate for limited number of data, toxicity tests conducted in artificial soils, like OECD standard soil, could be tapped as a potentially useful source of ecotoxicity data for prediction of metals EC50 values in natural soils using Free Ion Activity Model (FIAM). However, potential differences in metal exposure between artificial and natural soils may still limit the use of FIAMs developed in artificial porous matrices. The aim of the study was to assess the performance of FIAMs and empirical regression models (ERMs) developed basing on effect data measured in artificial soils for prediction of metal ecotoxicity toward different groups of terrestrial organisms inhabiting natural soils. First, empirical data from a literature review was collected and FIAMs and ERMs basing solely on data from experiments performed in artificial soils were developed using WHAM7 speciation software. Next, both models were validated for prediction of total metal based EC50 values in natural soils. The prediction with the use of both models was within two orders of magnitude suggesting that test conducted in artificial soils may not always be a useful source of data for environmentalists and ecotoxicologists.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 193

P19.4

P19.5

Analysis of the influence the rotating magnetic field (RMF) on the horseradish peroxidase catalytic properties

Modification of catalytic properties of laccase with use the rotating magnetic field

Agata Wasak , Radosław Drozd , Rafał Rakoczy 1

1

2

West Pomeranian University of Technology, Department of Immunology, Microbiology and Physiological Chemistry, Piastów Avenue 45, 70-311 Szczecin, Poland; 2West Pomeranian University of Technology, Institute of Chemical Engineering and Environmental Protection Processes, Piastów Avenue 42, 71-065 Szczecin, Poland 1

Horseradish peroxidase (HRP) is very well known heme containing enzyme, which use H2O2 to oxidise a variety of organic and inorganic compounds. HRP is widely used in organic synthesis, molecular biology as a biocomponent of immunoassays and biosensors. Despite the fact that HRP has been studied for more than a century, we still not know much about the influence of different kind of electromagnetic fields on the activity of this commonly occurring enzyme.

The purpose of this study was analysis of an effects the rotating magnetic field (RMF) on activity and kinetic parameters horseradish peroxidase under different frequency (1–50 Hz) and different exposure time (1–8h).

The obtained results showed that exposure of HRP at the RMF at frequency 40 and 10 Hz, reduced the activity of enzyme about 10% and 20% respectively. We also investigated the optimum pH of HRP in different frequencies.

It has been observed that the enzyme activity under exposure at RMF (50–1 Hz) in from 4.0 to 7.0 pH was constant, while for the control was highest in 4.0 pH, after exceeding this value drastically decrease to 25% of initial value at pH 7.0.

The results showed that RMF at frequency 40 and 10 Hz influence on decrease activity of HRP, however exposure of this enzyme to this factor increased its pH stability over a wider range.

Acknowledgements We are grateful for the financial support of the National Science Centre Poland within the PRELUDIUM Programme (Grant N. 2016/21/N/ST8/02343).

Agata Wasak1, Radosław Drozd1, Rafał Rakoczy2 1 West Pomeranian University of Technology, Department of Immunology, Microbiology and Physiological Chemistry, Piastów Avenue 45, 70-311 Szczecin, Poland; 2West Pomeranian University of Technology, Institute of Chemical Engineering and Environmental Protection Processes, Piastów Avenue 42, 71-065 Szczecin, Poland.

Laccases (EC 1.10.3.2) have a great biotechnological potential due to their ability to oxidize a broad range of substrates. Therefore, scientist are looking for ways to increase their activity and modification of catalytic properties for the technological process. The increase of laccase activity is obtained through optimization of culture conditions, e.g. addition of copper ions or directly by the use of mediators, which are expensive and dangerous for environment. The alternative may be the use of electromagnetic fields (EMF) which are capable of inducing and modifying the course of enzymatic reactions. At present, a research are underway on the use of EMF in optimization of reactions, by a change catalytic properties of the biocatalysts.

The aim of study was to analysed the effect of the rotating magnetic field (RMF) on the activity of laccase, as an attractive enzyme for industry and biotechnology.

We have examined RMF effect of low frequency and different exposure time on laccase. The activity of enzyme increased under RMF exposure about 21; 24; 20% for 20, 30 and 50 Hz respectively. It has also been observed that exposure at 10 Hz affects the increase of stability of the laccase in acidic pH. The obtained results indicate that exposure in RMF increase activity and stability of laccase, improving the technological value of the enzyme.

Acknowledgements We are grateful for the financial support of the National Science Centre Poland within the PRELUDIUM (Grant No. 2016/21/N/ST8/02343).

6th Central European Congress of Life Sciences Eurobiotech 2017

194 Abstracts

P19.6

P19.7

Greater Poland Voivodeship as the bioenergy producer

Network analysis of Arabian horses family ties

Ewa Woźniak, Eliza Kalbarczyk Institute of Socio-Economic Geography and Spatial Management, Faculty of Geographical and Geological Sciences of Adam Mickiewicz University The main goal of poster is to show the production and location of bioenergy sector in regional approach for Greater Poland Voivodeship in comparison to all provinces in Poland. The analyses take into account the biogas and biomass power plants. White biotechnology makes that industrial processes are more friendly to environment and less expensive. With regard to bioenergy in Poland, the important issue is the production of energy from renewable sources. This is developing quite rapidly. Renewable sources of energy produced in 2015 accounted for more than 11% of total energy production. The dominant source of energy is still coal. Poland is obliged to reach a 15% share of renewable sources in energy consumption by 2020 as a result of the Climate Package. According to the data from Energy Regulatory Office, in Poland in 2016 there were 303 biogas plants with a total power of 240 MW and 41 biomass power plants with total power of 1 281 MW. There are distinguished 4 types of biogas plants due to the production. There are biogas plants generating energy from: sewage treatment plants, agricultural biogas, landfill biogas and mixed biogas.

Forestry-wood sector is the main source of biomass for bioenergy. There are also few sources of biomass, like energy crops, waste from forest, agriculture and garden or municipal and industrial waste. According to the data from Energy Market Agency, production of biomass energy decreased by 50% in 2016. It was related to the new Act on Renewable Energy Sources.

Kacper Żukowski1, Katarzyna Pietrzyk1, Monika Stefaniuk-Szmukier2, Agata Sosińska2, Katarzyna Piórkowska1, Katarzyna Ropka-Molik1, Monika Bugno-Poniewierska1 National Research Institute of Animal Production, Cracow, Poland; 2University of Agriculture in Cracow, Poland

1

The aim of the analysis was to identified connections between Arabian horses, show the most influential sires and dams, indicate clusters of tightly connected and a few key mares and stallions.

The analysis was based on 1412 animals. The pedigree structure was available from Janów Podlaski pedigree database. The few methods like node degree, closeness, betweenness, diameter, transitivity, page rank, eigenvector centrality and clustering were used in analysis. All this statistics were shown in graphical form to better understood relationship networks and breeding structure.

Moreover, the new visualisation technologies allows to present interactive plots.

The study showed that some core mares and stallions were extremely important as breeding animals. High correlation between used methods like between page rank and eigenvector was observed. The diameter of a network which represents the longest of all the calculated shortest paths between horses was based on 23 animals. Due to the confidentiality agreement some of the results does not present.

Acknowledgements The study was supported by BIOSTRATEG2/297267/14/NCBR/2016.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 195

P19.8

P19.9

The utilization of multi-dimensional scaling for colour coat database misclassification in Holstein cattle

POLGENOM – Polish genomic reference database – as a result of whole genome sequencing of successfully aging long-lived Polish Caucasians

Kacper Żukowski, Justyna Dulska, Artur Gurgul, Katarzyna Piórkowska, Katarzyna Ropka-Molik National Research Institute of Animal Production, Cracow, Poland The goal of the present study was to recognize genomic SNP pattern of colour coat of red and white or black and white Holstein-Friesian cattle. Moreover, indicate misclassified animals and to correct their database entries.

In total 455 animals, 361 bulls and 94 cows, were genotyped using Illumina BovineHD which contains over 777K SNPs. Two additional subsets of SNPs were extracted from the main dataset to better simulate the real utilization of SNP chips in genomic selection practice. The additional subsets consider over 50K SNP panel like in Illumina Bovine50SNP chip and over 7K SNP panel like in Illumina BovineLD chip. The multi-dimensional scaling was applied to quantify the colour coat substructure.

The study showed that the genomic structure of colour coat has got strong evidence reflected by the SNP genotypes. There was no significant differences between proposed panels witch indicates that even low density panels – daily used to routine cattle evaluation could be used for MDS scaling. The misclassified animals were recognized and database entries were corrected. It is extremely important to routine process when genetic material of death animals is available without description of phenotype.

E. Matczynska1, P. Lyszkiewicz2, M. Krawczyk1, D. Kowalczyk 2, R. Szymańczak1, M. Mossakowska3, M. Puzianowska-Kuznicka4, M.Zagulski1 and A.Boguszewska-Chachulska1 Genomed SA, Warsaw, Poland; 224 Godziny Sp. z o.o., Warsaw, Poland; 3International Institute of Molecular and Cell Biology, Warsaw, Poland; 4Mossakowski Medical Research Centre, PAS, Warsaw, Poland 1

The aim of the project was to characterise genome sequence variation in the Polish population and to combine it with collected clinical data for prospective usage in diagnostics and personalised medicine.

Whole genomes of 130 successfully aging Polish 90+ years old people were sequenced with a mean 30x coverage using the Illumina technology. An in-house developed bioinformatic pipeline was applied to identify and annotate sequence variants.

The genomic data were combined with a large set of collected clinical and biochemical data. Mitochondrial variants were added to the database. An analysis of structural and copy number variations was performed.

Approximately 16.7 mln of variants in the nuclear genome were found (22 millions before filtering), including 15.3 million SNVs and 1.4 million small insertions and deletions (indels). 81% of variants have been already present in the dbSNP database, 73% - in the 1000 Genomes Project (Phase 3). Almost 2.76 mln novel SNVs and 0.34 mln novel indels were detected.

Rare variants categorized by the Human Gene Mutation Database as disease-causing were analysed, yielding 818 variants with MAF<0.5%, which corresponds to 6 distinct rare DM variants per each long-lived person.

The data obtained in frame of this project shed a new light on the effect of previously characterised disease-causing mutations on the individual health and life span.

Acknowledgements Project supported by the National Centre for Research and Development INNOTECH programme.

6th Central European Congress of Life Sciences Eurobiotech 2017

196 Abstracts

Tools in the treatment of human disorders Lectures

L20.2

L20.1

Age-dependent posttranslational protein modification – driving force and target in neurodegeneration

Striving for excellence in Health care Fiona Wood Director of the Burns Service of Western Australia, Fiona Stanley and Princess Margaret Hospitals; Director of the Burn Injury research Unit University of Western Australia We are living in a time where science and technology are advancing at an ever increasing rate. Harnessing the power of that science and technology into clinical practice is an ever increasing challenge.

Every intervention from the time of injury will influence the scar worn for life. Therefore there are therapeutic opportunities for development of innovative therapies at every step along the clinical pathway.

Just as complex tissues have self-organised into the adult body, there are parallels with tissue repair requiring understanding in order to effectively implement tissue engineering and traditional skin grafting strategies.

From basic science and clinical research we understand that burn injury is related to an aggressive and prolonged inflammatory response that results in the development of a scar with an abnormal extracellular matrix (ECM).

From population health data linkage studies we know that burn injury has a negative impact on lifelong survival.

The ongoing abnormal ECM (in shape and chemistry) is permitted by the immune system for which we now ask: • Does the permissive immune system lead to an increased lifetime risk related to an alteration in the immune response e.g. cancer? Conversely, will a recovered immune system mitigate against this risk?

• Can the CNS 3D information housed in the homunculus be used to drive regenerative nerve repair within the skin?

• Will shape recognition at the cellular and tissue level facilitate a regenerative healing pattern?

• Will regeneration of normal ECM structure be associated with normal phenotypic expression of skin cells?

• Will tissue regeneration be associated with or even dependent upon systemic recovery of the immune system?

However, the challenges we face relate not only to the individual and the health system, but to the whole community. Understanding the drivers to health and wellness and the decisions made individually and collectively underpin the opportunities for improvement in health

Hans-Ulrich Demuth Fraunhofer Institute for Cell Therapy and Immunology (IZI) Leipzig, Department of Drug Design and Target Validation (IZI-MWT), Weinberger 22, Biocenter, Halle (Saale) Germany The Alzheimer syndrome is the most frequent age-related neurodegenerative disorder which affects today more than 35million patients world-wide. In Germany alone this number will exceed 3million people in 2050.

Up to now, there is no medication available which would hold or even reverse the progressive disease. In recent decades attempts to find disease-modifying drugs have failed.

Many of them have been directed against the deposited Abeta peptides (Aβ), which are product of the turnover of a membrane protein (APP) of neurons. Some drug approaches where developed to slow down this protease-driven turnover using small molecule inhibitors, some to immunize against the Aβ peptides.

However, all of them did not specifically target the very different posttranslational modifications of Aβ or the Amyloid Precursor Protein (APP). Recent research uncovered among these modifications very aggregation prone forms which in a prion-like manner induce the formation of soluble and metastable oligomeric aggregates, which are acknowledged today as the most neurotoxic agents in the human brain.

Among them, pyroglutamate-3 amyloid-β (pGlu-3 Aβ) is an N-terminally truncated and pyroglutamate-modified Aβ species which has been shown to be a major component of Aβ deposited in the plaques and blood vessels of Alzheimer’s disease and Down syndrome brains (Saido et al., 1995; Lemere et al., 1996).

Formation of pGlu-3 Aβ is a multi-step process whereby the first two N-terminal amino acids, aspartate and alanine, are cleaved off, exposing glutamate at position 3 on the N terminus of Abeta. Subsequently, glutamate is post-translationally modified to N-terminal pyroglutamate by cyclization of the exposed glutamate residue by glutaminyl cyclase and resulting altered biochemical properties including increased hydrophobicity, high aggregation propensity and neurotoxicity (Russo et al., 2002; Schilling et al., 2006).

Prevention and therapeutic trials previously conducted in different laboratories showed that prevention of pGlu-3 Aβ formation by inhibiting the enzyme Glutaminyl Cyclase and pGlu-3 Aβ mAb immunizations resulted in a reduction in general Aβ and pGlu-3 Aβ pathology (Nussbaum et al., 2012; Frost et al., 2012). This suggests that presence of pGlu-3 Aβ contributes to increased plaque burden. Both approaches have recently reached clinical development stage.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 197

L20.3

L20.4

From the basic research to potential prophylaxis and/or treatment of periodontal disease – targeting bacterial glutaminian cyclase

In vitro cultured epidermal cells for skin regeneration – clinical applications

Jan Potempa Faculty of Biochemistry, Biophysics & Biotechnology, Jagiellonian University, Krakow Poland and Department of Oral Immunology and Infectious Diseases, University of Louisville School o Dentistry, Louisville, KY, USA Porphyromonas gingivalis, a major periodontopathogen, secretes a variety of proteins, including all major virulence factors such as gingipains (RgpA, RgpB and Kgp), with pyroglutamate (pGlu) at N-termini. This modification was linked to a gene (PG1885) encoding a protein homologous to human glutaminyl cyclase (QC). PgQC is essential for P. gingivalis vitality and predicted to be a lipoprotein.

W have experimentally demonstrated that PgQC is anchored the inner bacterial membrane facing the periplasm.

The crystal structure of recombinant PgQC confirmed that PgQC is indeed closely related to the human enzyme. To verify PgQC as a potential target for drug development we have tested inhibitory compounds developed by Fraunhofer one of which was shown to be an effective inhibitor for QC activity in vivo. P. gingivalis treatment with the compound exerted a profound effect on gingipains maturation and secretion. More importantly, however, the compound significantly inhibited P. gingivalis growth. Collectively our findings indicate that PgQC plays an important role in maturation of gingipains and constitutes an attractive target for a novel approach to treat and/or prevent periodontitis.

Acknowledgements Supported by FP7-HEALTH-F3-2012-306029 “TRIGGER” and 2975/7.

PR/13/2014/2 from European Commission and Polish Ministry of Science and Higher Education

Justyna Drukała Cell Bank, Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Malopolska Centre of Biotechnology, Jagiellonian University Improvement of isolation methods and human cell cultures and the use of material bioengineering to create scaffolds for cells and factors regulating a suitable in vitro tissue reconstruction have allowed the development of tissue engineering. It is a promising alternative for conventional methods of tissue and organ transplantation.

Skin is the largest organ in the human body which determines its integrity; its large injuries disturb the whole homeostasis of the organism. Wound closure is determined by the epidermis forming a specialized protective barrier.

The characteristic feature of epidermis is a fast regeneration conditioned by a large number of stem cells. Isolation of epidermal stem and progenitor cells residing in the germinal layer enables the reconstruction of epidermis in vitro and its application in large wound healing. The possibility of having large-scale cultures of proliferating cells from small tissue biopsy and permanent covering of the wound, without a risk of graft rejection, is an important advantage of skin reconstruction with use of autologous cells. The most obvious clinical application of cultured autografts is the treatment of life-threatening burns. Cultured keratinocytes derived from the biopsy of a small sample of healthy skin have been used to provide permanent wound coverage for medium-to-full deep, extensive burns. Cultured epithelial autografts (CEA) are transplanted onto the wound beds, as a cell suspension in fibrin glue – what is practiced here for many years. The ability of CEA to ensure confluent wound healing with minimal hypertrophy, makes it an attractive alternative to conventional skin grafts in the treatment of acute wounds. The use of CEA is also possible onto a simple granulation tissue or other vascularised wound bed, in case of deeper wounds. However, because of the lack of dermal wound bed elements, it often effects in unstable and deforming, true scars formation. Therefore, in such cases, so important might be use of the INTEGRA Dermal Regeneration Template®, which is an advanced biocompatible wound care product, “the device” that facilitates regeneration of the dermal layer by patient’s organism. The collagen-glycosaminoglycan biodegradable matrix provides a scaffold for cellular invasion and capillary growth. After collagen layer remodeling, silicon layer is replaced with ultra-thin epidermal autografts, and in selected cases – with in vitro cultured autologous epidermal progenitor cell suspension. In final effect we obtain a kind of a new multilayered skin, without the true scar formation, minimizing the risk of severe esthetic and functional sequelae. Such an model of local treatment of extensive wounds (most often reconstructive, post-burn) is clinically practiced in Pediatric Burns Centre since four years, with medico-biological cooperation, and is under multi-directional investigation.

6th Central European Congress of Life Sciences Eurobiotech 2017

198 Abstracts

L20.5

Posters

Reversing cancer drug resistance using novel flavonoid dimers

P20.1

Iris Wong, Jason Kan, Clare Yan, Xuesen Hu, Tsz Cheung Chong, Kin-Fai Chan, Tak-Hang Chan and Larry Ming Cheung Chow Department of Applied Biology and Chemical Technology The Hong Kong Polytechnic University, Hong Kong SAR, China Chemotherapy resistance is a major problem in cancer treatment. Overexpression of three ATP-binding cassette (ABC) transporters (P-gp, MRP1 and BCRP) are associated with chemotherapy resistance. These ABC transporters can actively efflux various anticancer substrates, thereby reducing intracellular drug concentration, causing chemotherapy resistance.

We have been developing novel synthetic flavonoid dimers that can target the dimeric structure of these ABC transporters, thereby reversing cancer drug resistance.

The diet-derived flavonoid dimers which can target P-glycoprotein (p-gp) and Breast Cancer Resistance Protein (BCRP)’s dimeric structure. Due to the unique design of dimeric in structure which can specifically bind to the pseudodimeric P-gp and BCRP, these flavonoid dimers can inhibit P-gp and BCRP, and reverse cancer drug resistance with very high potency and low toxicity.

Flavonoid dimer can also change the way of chemotherapy treatment. Some anticancer drugs cannot be absorbed in the digestion system of human body and must be administered intravenously. Flavonoid Dimer can act as an absorption enhancer and enable the drugs to be taken orally, reducing the risks associated with intravenous injection, and making the treatment less time-consuming, more convenient and comfortable.

In addition, the presence of P-gp and BCRP in the blood brain barrier prevents the accumulation of anticancer drugs in the brain, thereby limiting the choice of anticancer drugs available for treating glioblastoma. Using dual-selective flavonoid dimers that target both P-gp and BCRP, we can increase brain accumulation of anticancer drugs and demonstrate that it can be used in treating orthotopic xenograft of human glioblastoma in nude mice model.

In summary, we have designed and characterized different flavonoid dimers that can be used to inhibit ABC transporters and used them to either reverse cancer drug resistance, improve oral bioavailability of cancer drugs and increase the penetration of cancer drugs in the brain for treating glioblastoma.

Targeted inhibition of IRF1 as a potential treatment strategy in cardiovascular disease Antonczyk A.1, Szelag M.1, Wesoly J.2, Bluyssen H.A.1 Department of Human Molecular Genetics; Laboratory of High Throughput Technologies, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University Poznan

1 2

Keywords: IFNγ, IRF, inhibitor Interferon (IFN)γ triggers activation of members of the Interferon Regulatory Factor (IRF) family, including IRF1 and contributes to onset and progression of vascular dysfunction. Thus, IRF1 targeted inhibition can serve as a novel therapeutic strategy in cardiovascular disease (CVD).

To date no IRF inhibitors have been described, illustrating the need for IRF modeling, screening and validation tools to find IRF inhibitors. Therefore, we generated new 3D structure models for all IRF-DNA binding domains (DBD) and developed a pipeline approach that combines comparative in silico compound docking to IRFDBDs with in vitro inhibition validation to select specific IRF1 inhibitors.

Accordingly, after screening a ‘natural product’ compound library we were able to select the most promising candidate IRF1-DBD inhibitor I1_04, which suppressed IRF1 expression at the protein level and diminished IRF1-target gene expression.

Further characterization and optimization of I1_04 should lead to a novel class of IRF1 inhibitors with high specificity, potency and excellent bioavailability as a promising approach to combat CVD.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 199

P20.2

P20.3

Modulation amino acid pool of blood plasma adding pyridoxine phosphate in composition of amino acids

Mechanisms of cellular uptake of S-glycosylated thiosemicarbazides

Anna Buklaha1, Anastasiya Pauliukavets1, Vladimir Sheibak 2

Anna Czubatka-Bienkowska1, Joanna Sarnik1, Anna Macieja1, Zbigniew J. Witczak 2, Tomasz Poplawski1

1 Grodno State Medical University, Department of Microbiology, virology and immunology named after SI. Gelberg, Belarus; 2Grodno State Medical University, Department of Biochemistry, Belarus

The aim of the study was to analyze the effect of the adding pyridoxine phosphate in composition amino acid (tryptophan, taurine and arginine) and microelement (zinc sulfate) on free amino acids and their major metabolites in blood plasma.

Intragastric administration composition consisting of amino acids (tryptophan, taurine and arginine) and microelement (zinc sulfate) within 10 days increases the total amount of nitrogen-containing derivatives of amino acids, sulfur-containing amino acids, tryptophan, cysteic acid, taurine and decrease serine, glycine, valine, ornithine in blood plasma.

Intragastric administration composition amino acid (tryptophan, taurine and arginine) and microelement (zinc sulfate) in combination with pyridoxine phosphate within 10 days increases the total amount of free amino acids and nitrogen-containing derivatives, proteinogenic amino acids (essential and non-essential), the total amount BCAA, arginine, taurine, tryptophan, alanine, ornithine and decrease asparagine, histidine, glycine, tyrosine in blood plasma.

Adding pyridoxine phosphate in composition amino acid tritarg (arginine, taurine, tryptophan, zinc aspartate) increases non-proteinogenic functions of amino acid and largely modifies ratios of the various groups of amino acids in the blood plasma.

University of Lodz, Department of Molecular Genetics, Poland; 2Nesbitt School of Pharmacy, Wilkes University, Department of Pharmaceutical Sciences,USA.

1

We have designed and synthesized novel S-glycosylated thiosemicarbazides. They induced DNA damage on ovarian cancer cell line (A2780) in concentration dependent manner. Moreover they increased expression of heat shock and endoplasmic reticulum stress genes. However their mechanism of cellular uptake is still unknown. In this work we analyzed this mechanism.

We focused on glucose transporter proteins (GLUTs) as these proteins transport carbohydrates derivatives to the cell. We used three GLUT inhibitors: cytochalasin B, fasentin and forksolin. They inhibit glucose transport in different ways. The first one destroy actin filaments and inhibits glucose transport, the second is GLUT1 inhibitor and the last one blocks the transporter GLUT1 and activates adenylate cyclase. We have compared cytotoxicity and genotoxicity of most potent S-glycosylated thiosemicarbazides on A2780 cells incubated with or without GLUT inhibitors. The observed changes in cytotoxic and genotoxic effect suggest that S-glycosylated thiosemicarbazides are transported via GLUT. Acknowledgementss This work was supported by Grant from The Polish National Science Centre, decision nr DEC-2014/15/N/NZ7/02949.

6th Central European Congress of Life Sciences Eurobiotech 2017

200 Abstracts

P20.4

P20.5

Microvesicles stimulates endothelial cell migration in hyperglycemic conditions

The role of red fox as a zoonotic reservoir for Alaria alata flukes

Anna Drozdz1, Robert Jach2, Hubert Huras3, Ewa Stepien1

Dorota Dwużnik, Damian Kakietek, Maciej Religa, Ewa Mierzejewska, Anna Bajer

Jagiellonian University, Department of Medical Physics, Faculty of Physics, Astronomy and Applied Computer Science, Poland; 2 Jagiellonian University Medical College, Department of Gynecological Endocrinology, Faculty of Medicine, Poland; 3 Jagiellonian University Medical College, Department of Obstetrics and Perinatology, Faculty of Medicine, Poland

Institute of Zoology, Department of Parasitology, Faculty of Biology, University of Warsaw, Ilji Miecznikowa 1street, 02-096 Warsaw, Poland

1

Cell migration is one of the crucial mechanisms for wound healing. It strongly depends on the biochemical composition of environment. During diabetic mellitus cell migration is impaired, which could be linked to the microvesicles (MVs) activity.

Studies were performed on Human Umbilical Vein Endothelial Cells (HUVECs). The scratch assay was used to assess cell migration and confluence index (CI) were calculated. Culture media contained additional factors: glucose (25 mM/ml), MVs isolated from hyperglycemic (HG) (25 mM/ml) and normoglycemic (NG) cell conditioned media.

Cells were recorded immediately after the scratch and after 14 hours. As a long time assessment of cell metabolism viability tests were performed.

The results show that MVs influence cell migration in a different manner depending on the culture conditions: a) with the addition of NG MVs, NG cells cultured in NG conditions have significantly higher CI then cells cultured without the addition of MVs (25.25±5.92% to 18.08±5.12%; p=0.018, respectively) b) cells cultured in long term HG conditions tested in NG condition showed statistically significant CI increase c) opposite reaction was observed for NG cell tested in HG conditions. No differences in cell metabolism and proliferation were observed in viability tests.

Results show that impaired cell migration during diabetes is not caused by reduced proliferation and viability of the cells but might be a results of impaired cell to cell communication mediated by MVs.

Red fox (Vulpes vulpes) plays an important role in spread of many dangerous parasites, including the Alaria alata flukes, which cause alariosis. Alariosis belongs to a group of „emerging diseases”. Common symptoms of infection in humans include: muscle pains, chest pains, shortening of breath and skin rashes. Diagnosing is difficult and frequently confused with different conditions.

Main goal of our research was to determine the role of the red fox as a reservoir of A. alata in Poland and evaluation of health risk for public.

During hunting seasons 2015/2016 and 2016/2017, 58 foxes were derived from 3 regions of Poland (mazowieckie, warmińsko- mazurskie and kujawsko- pomorskie voivodeships). After freezing over the period of two weeks at the temperature -80ºC, inspections and autopsies of foxes were carried out. Intestinal tracts were removed, dissected and all worms counted and fixed.

Results and conclusions: High prevalence of over 90% was detected in foxes in Poland. High abundance of A. alata invasion up to 759 flukes/ individual was recorded.

Such high level of infection in foxes poses a serious health risk problem for humans and needs further monitoring.

Acknowledgements The study was funded by the National Science Centre (NCN) grant Sonata Bis 2014/14/E/NZ7/00153.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 201

P20.6

P20.7

De novo method of protein folding simulations

Antioxidant properties of hyaluronic acid in the human cancer cells in vitro

Małgorzata Gadzała1, Dawid Dułak 2, Barbara Kalinowska3, Irena Roterman-Konieczna2 ACC Cyfronet AGH, Nawojki 11, Cracow, Poland; 2 Department of Bioinformatics and Telemedicine – CM – Jagiellonian University – Lazarza 16, Cracow, Poland; 3 Faculty of Physics, Astronomy and Applied Computer Science – Jagiellonian University, S. Łojasiewicza 11, Cracow, Poland

1

A protein folding simulation is a natural biological process that shapes a linear chain of amino acids into a fully functional protein. In silicomethods of the protein folding are important for drug design pharmaceutical industry, especially in an aspect of personal therapy. There are many types of in silico approaches and one of them, de novo, is still being a challenge since the results are far from satisfying. However, successful de novo simulations can give the best answer to the question of how the natural process really goes.

We propose de novo approach that is composed of two stages: Early and Late [1]. The Early stage aims at creation of secondary structures starting from amino acid sequence. The Late stage takes that initially formed structure and optimize it both in an internal force field (electrostatic, torsional potential and van der Waals forces) and an external force field (influence of water surrounding based on 3D Gauss hydrophobicity model). The optimizations are repeated until the process fulfills a defined termination condition. As a result the tertiary structure is generated.

A current implementation of this method was tested on a set of proteins and for several of them it has given promising results.

Gabriela Gajek, Renata Kotek Laboratory of Cytogenetics, University of Lodz, Poland Keywords: hyaluronic acid, antioxidant, cancer Hialadherin overexpression (mainly CD44 and RHAMM) takes place on the surface of many tumor cells.

Hyaluronan – having affinity for these receptors – can therefore be used as a carrier of anti-cancer drugs in targeted therapy. It seems that this approach will increase the effectiveness and reduce the toxicity of chemotherapeutics. Many chemically modified hyaluronan molecules have been engineered to be good carriers for antitumor drugs.

The aim of the study was to evaluate the antioxidant properties of hyaluronic acid in colorectal carcinoma cancer cells (HCT116), colorectal adenocarcinoma cancer cells (HT29) and gastric adenocarcinoma cancer cells (AGS) under in vitro oxidative stress conditions. Measurement of oxidative stress was performed using H2-DCFDA, which is a chemically reduced form of fluorescein, which very quickly detecting intracellular oxidants.

The results showed that hyaluronan possesses strong antioxidant properties with respect to induced H2O2 reactive oxygen species in all tested tumor cells. However, these properties may adversely affect therapy, reducing the toxic effect of the drug in tumor cells, when we possibly use it as drug carrier.

References Karbownik M S et al (2011) Journal of Oncology 4: 380–395 Jóźwiak-Bębenista M et al (2010) Farmacja Polska 66: 882–93 Stern R et al (2008) Seminars in Cancer Biology 18: 237

References: Roterman I, Konieczny L et al (2011). Two-intermediate model to characterize the structure of fast-folding proteins. J Theor Biol 283(1), pp.60–70.

6th Central European Congress of Life Sciences Eurobiotech 2017

202 Abstracts

P20.8

P20.9

Preliminary analysis of Naja ashei venom proteome in terms of its pharmacological potential

The expression and activity of hydrogen sulphide generating enzymes in INFγ- and LPS-stimulated murine macrophage J774A.1 cells

Konrad K. Hus1, Justyna Buczkowicz1, Andrzej Łyskowski1, Vladimír Petrilla2,3, Monika Petrillová4, Jaroslav Legáth1,5, Aleksandra Bocian1 1 Rzeszow University of Technology, Faculty of Chemistry, Poland; 2University of Veterinary Medicine and Pharmacy, Department of Physiology, Slovak Republic; 3Zoological Garden Košice, Zoological Department, Slovak Republic; 4 University of Veterinary Medicine and Pharmacy, Department of General Education Subjects, Slovak Republic; 5University of Veterinary Medicine and Pharmacy, Department of Pharmacology and Toxicology, Slovak Republic;

One of the most common approaches in drug design is to search for biologically active compounds from nature.

Snake venom is not only essential for preparation of antivenin used in treatment of snake bites but has also proven its value in hypertension therapy. Lately it emerges as a promising source of drugs for other medically important issues like cancer or neurodegenerative diseases therapy.

Naja ashei belongs to the group of African spitting cobras which are widely distributed throughout sub-Saharan region. It is known that venom of N. ashei, similarly to other African spitting cobras, has mainly cytotoxic effect.

However, data about its specific composition are not yet available. Thus, the aim of this study was to determine the venom proteome of this snake using 2-D electrophoresis and MALDI ToF/ToF mass spectrometry techniques. Proteomic analysis revealed that the most abundant groups of proteins in N. ashei venom are represented by three-finger toxins and phospholipases A 2, however the presence of cysteine-rich venom proteins, 5’-nucleotidase and metalloproteinases has also been detected. Interestingly, analysed venom contained proteins which were not described before in African spitting cobras – cobra venom factor and venom nerve growth factor. To our knowledge there are no other reports concerning the composition of this venom and thus it could be a first step in research on clinical application of identified proteins.

Maria Wróbel1, Janusz Marcinkiewicz2, Halina Jurkowska1, Patrycja Bronowicka-Adamska1, Kinga Kaszuba1, Dominika Szlęzak1, Paulina Skalska2 1 Chair of Medical Biochemistry, Jagiellonian University Medical College, Kopernika 7, 31-034 Kraków, Poland; 2 Department of Immunology, Jagiellonian University Medical College, Czysta 18, 31−121 Kraków, Poland

Keywords: hydrogen sulphide, inflammation, interferon, lipopolysaccharide, sulfurtransferases Hydrogen sulphide (H2S) plays an important role in pathogenesis of inflammation – depending on the concentration it can have pro- or anti-inflammatory effects.

Its synthesis is carried out with the participation of enzymes: cystathionine beta-synthase (CBS, EC 4.2.1.22), gamma-cystathionase (CTH, EC 4.4.1.1) and 3-mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2).

The expression and activity of MPST, CTH, and rhodanese were investigated in lipopolysaccharide (LPS) and interferon-gamma (IFNγ)-stimulated J774A.1 cells.

The expression and activity of MPST were significantly decreased in J774A.1 cells in the presence of INFγ and LPS. Loss of activity of both MPST, as well as rhodanese can be associated with the oxidative stress, confirmed by large amount of pro-inflammatory cytokines, TNFα and interleukin 6. INFγ up-regulates CTH expression and activity in J774A.1 cells. This was correlated with increased hydrogen sulfide production in J774A.1 cells, what suggests that CTH is major enzyme involved in hydrogen sulphide production in this cells.

This studies contribute to the explanation of the role of hydrogen sulfide in the process of inflammation by determination of changes in the expression/activity of enzymes involved in the production of H2S in J774A.1 cells.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 203

P20.10

P20.11

The inhibition of cancer cell cycle and induction of apoptosis by 4-hydroxybenzyl isothiocyanate, a natural H2S-donor

Spectroscopic and proteomic analysis of urinary extracellular vesicles-potential biomarkers of renal failure

Halina Jurkowska1, Dominika Szlęzak1, Maria Wróbel1, Ewa Jasek-Gajda2

Agnieszka Kamińska1, Mark Platt2, Joanna Kasprzyk3, Anna Drożdż1, Katarzyna Dziedzic Kocurek1, Benedykt R. Jany4, Wojciech Piekoszewski3,5, Marek Kuźniewski6,Franciszek Krok4, Ewa Ł. Stępień1

1 Chair of Medical Biochemistry, Jagiellonian University Medical College, 7 Kopernika St., 31-034 Kraków, Poland; 2 Department of Histology, Jagiellonian University Medical College, 7 Kopernika St., 31-034 Kraków, Poland

Keywords: isothiocyanate, apoptosis, cell cycle, hydrogen sulfide, thiosulfate, sulfurtransferases.

The effect of 4-hydroxybenzyl isothiocyanate (HBITC), a natural product obtained from white mustard seeds (Sinapis alba), on the proliferation of human glioblastoma (U87MG) and neuroblastoma (SH-SY5Y) cells was studied and the possible mechanisms of its activity was suggested.

Our results show that 4-hydroxybenzyl isothiocyanate inhibits proliferation of U87MG and SH-SY5Y cells by blocking the cell cycle and induction of apoptosis. Inhibition of cell proliferation was associated with an increase of p21 protein level, inactivation of Bcl-2 and reduction of mitochondrial membrane potential. Interestingly, in both cancer cell lines the level of p53 protein was decreased in the presence of HBITC and this effect was concentration-dependent. It suggests modification of sulfhydryl groups of p53 protein via S-sulfuration and inactivation of p53 protein. Thus, observed in our study p21 induction can be mediated by p53-independent mechanisms. Anticancer effect of HBITC was also associated with increased hydrogen sulfide level, thiosulfate production, and decreased level of rhodanese- enzyme involved in degradation of hydrogen sulfide.

1 Jagiellonian University, Marian Smoluchowski Institute of Physics, Department of Medical Physics, Poland; 2 Loughborough University, Department of Chemistry, United Kingdom; 3Jagiellonian University, Faculty of Chemistry, Laboratory of High Resolution Mass Spectrometry, Regional Laboratory of Physicochemical Analysis and Structural Research, Poland; 4 Jagiellonian University, Marian Smoluchowski Institute of Physics, Department of Solid State Physics, Poland; 5Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University, Poland; 6Department of Nephrology, Jagiellonian University Medical College, Poland

Keywords: biomarker, diabetes, extracellular vesicles, nephropathy Nephropathy is a major complication of diabetes which develop in 30–40% of patients. Long-term hyperglycemia induces renal damage directly or by hemodynamic modifications. Extracellular vesicles (EVs) are nano-sized structures released by cells which amount or molecular content can be specific with disease progression. The aim of the study was to find characteristic proteomic and chemical profile of EVs and correlation between their number and the progress of renal failure in diabetes. Diabetic patients with (RF) and without renal failure (NRF) and healthy subjects were enrolled to this study. The number and size of EVs were determined by TRPS technology. Nano-liquid chromatography with mass spectrometry were used for proteomic analysis. EVs were visualized by Scanning and Transmission Electron Microscopy. The number of EVs was measured by flow cytometry system. Infrared Spectroscopy was used to detect specific chemical groups in EVs samples. RF patients had significantly lower number of EVs compared to NRF. Mass spectrometry analysis showed decreased uromodulin concentration in RF patients and number of unique proteins related to cell stress and secretion were detected in the EVs samples. Preliminary Infrared Spectroscopic measurements showed a difference in the infrared spectrum between diabetic patients and healthy control. Our results indicate that the number and a protein profile of EVs can be considered as potential early renal failure biomarkers in diabetic patients.

6th Central European Congress of Life Sciences Eurobiotech 2017

204 Abstracts

P20.12

P20.13

Human osteosarcoma U-2 OS cells with acquired resistance to RG7388, a small-molecule inhibitor of p53-MDM2 interaction

Association between polymorphisms in TIMELESS and asthma risk in the population of Polish children

Justyna Kocik, Justyna Polak, Tad A. Holak, Lukasz Skalniak Chemical Biology and Drug Discovery Group, Department of Organic Chemistry, Faculty of Chemistry of the Jagiellonian University in Krakow, Poland The aim of this study was to characterize human U-2 OS (osteosarcoma) cell populations with acquired resistance to RG7388. This compound is a small molecule inhibitor of p53-MDM2 interaction and belongs to the most promising potential anti-cancer drugs among synthetic MDM2 antagonists. The compound is currently being tested in clinical trials as monotherapy, as well as in combination therapies. However, no studies have been conducted to determine the acquisition of resistance by tumor cells to the treatment with RG7388. Experiments carried out in our laboratory proved that this phenomenon takes place and allowed to develop several populations of cells capable of growing in the presence of the compound. The MTT assay was performed to demonstrate that the derived populations exhibit improved survival in the presence of RG7388 compared to the U-2 OS parental cell line. Western blot analysis verified the potential of the compound to increase p53 protein level in tumor cells. It was also observed that p53 is not capable to activate p21 expression in the resistant cells. Results of flow cytometry analysis confirmed that the populations were resistant to RG7388-induced cell cycle arrest. We justify the need for focusing on strategies minimizing the phenomenon of drug resistance, e.g. by interactions with other bioactive substances.

Wojciech Langwinski, Beata Narozna, Paulina Sobkowiak, Anna Breborowicz, Aleksandra Szczepankiewicz Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical Sciences, Poland Keywords: circadian clock, asthma, SNP genotyping Introduction. Bronchial asthma is a chronic respiratory disease with symptoms showing circadian oscillations.

For this, disorders in the clock genes, may be responsible.

Central and peripheral circadian clock is self-regulating axis of many genes. Among them, CLOCK, BMAL1, PER3 and TIMELESS, constitute a core of circadian genes that regulate the oscillation cycles.

Material and methods. DNA samples were collected from 165 children with bronchial asthma and 138 from the control group. Genotyping was performed for 32 single nucleotide polymorphisms in PER3, CLOCK, BMAL1 and TIMELESS using TaqMan probes. Statistical and haplotype analysis was done using Statistica 12 and Haploview 4.2, respectively.

Results. All analyzed SNPs were in Hardy-Weinberg equilibrium. We found 3 SNPs (rs10876890, rs2291739 and rs11171856) in TIMELESSassociated with asthma risk. Moreover, we found 2 haplotypes, TTT and TAC, that occur frequent in asthmatic children in comparison to the control group.

Conclusion. This is the first study showing association of circadian clock gene, TIMELESS with asthma. It may suggest involvement of genetic predisposition on circadian clock regulation in asthma.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 205

P20.14

P20.15

Comparison of two HRR (homologous recombination repair) inhibitors as a potential therapeutic agents against cisplatin-sensitive and cisplatin-resistant human ovarian carcinoma cells

Antiproliferative capacity of Sea buckthorn (Hippophae rhamnoides L.) extracts in selected human cell lines

Anna Macieja, Joanna Sarnik, Anna Czubatka-Bieńkowska, Tomasz Popławski University of Lodz, Department of Molecular Genetics, Poland An induction of the double-strand breaks (DSBs) in cancer cells is one of the most significant strategies in the therapy of human ovarian cancers. DSBs are the most troublesome type of DNA damage and can lead to cell death, if not repaired. There are two main pathways involved in DSBs repair: the homology-dependent DNA repair (HDR) and non-homologous end-joining (NHEJ). Inhibition of DNA repair in cancer cells may improve the effectiveness of standard anticancer therapies.

In this study, we compared the effect of two HRR inhibitors on the activity of etoposide/cisplatin in human ovarian carcinoma cell lines. We used A2780 and A2780cis cell lines, which are cisplatin-sensitive and resistant, respectively. We tested HAMNO and RI-2 inhibitors. HAMNO is a potent and selective inhibitor of replication protein A (RPA) interactions with proteins involved in the replication stress response. RI(dl)-2 selectively inhibits Rad51-mediated D-loop formation without affecting its ssDNA-binding activity.

Tested compounds caused an increase of the cytotoxicity of cisplatin and etoposide, when combined. HAMNO and RI(dl)-2 caused also an increase in DSBs level in ovarian carcinoma cells. Mechanism of their action include accumulation in the G2/M checkpoint of cell cycle. Effects described above were also observed in the case pretreatment with both of inhibitors, but in the case of RI(dl)-2 this effects were much more pronounced. It may suggest, that RI(dl)-2 is more potent as the DSBs inhibitor, and could be used to overcome cisplatin resistance in cancer cells.

Acknowledgements This work was financially supported by the National Science Centre (Poland), according to the decision No. DEC-2016/23/N/NZ7/02023.

Beata Marciniak, Renata Kontek Laboratory of Cytogenetics, University of Lodz, Poland Keywords: cytotoxicity, sea buckthorn extracts, MTT assay Sea buckthorn is a plant which all parts are rich in many nutrients (vitamins, flavonoids, carotenoids, fatty acids, macro- and microelements) and is a treasure trove of essential health-promoting bioactive compounds[1, 2, 3].

The purpose of this study was to investigate the differences between the ability of extracts of sea buckthorn to inhibit of proliferation in normal and neoplastic cells. Lipid and phenolic fractions from fruits, leaves and branches were tested. The study was performed on normal human cells such as peripheral blood lymphocytes or HS27 cell line and on selected cancer lines, i.e. HT29, PC-3 and MCF7. The IC50 value of examined extracts were determined by MTT assay. The aim of this method is to determine metabolic energy of living cells by measuring the activity of mitochondrial dehydrogenase. Obtained data showed that the most active against all tested cell lines proved to be fruit extracts. In general, stronger inhibition of cell proliferation was observed in the presence of lipid fraction than phenolic one. Moreover, the cancer cell lines have been found to be more sensitive to the effects of the extracts tested. The lowest IC50 value – 18 μg/mL was obtained for the HT29 cell line after treatment by lipid fruit fraction. These findings clearly demonstrate that the sea buckthorn extracts possesses pharmacological potential. Therefore further research in this field should be continued.

Acknowledgements Work funded by the NSC: No: UMO-2015/19/B/NZ9/03164.

References [1] Olas B (2016) Food and Chemical Toxicology 97: 199–204 [2] Suryakumar G and Gupta A (2011) Journal of Ethnopharmacology 138: 268– 278 [3] Perk AA et al (2016) African Journal of Biotechnology 15: 118–124

6th Central European Congress of Life Sciences Eurobiotech 2017

206 Abstracts

P20.16

P20.17

Dendritic cells activation with parasite components

New betulin derivatives as candidates for anticancer agents

Marta Maruszewska-Cheruiyot1, Katarzyna Donskow-Łysoniewska2,1, Katarzyna Krawczak1, Maria Doligalska1 Department of Parasitology, Faculty of Biology University of Warsaw, Poland; 2 Independent Laboratory o f Parasitology, The General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, Poland.

1

Parasitic infection is successfully used in Inflammatory Bowel Diseases therapy; one group of autoimmunological disorders. Parasites inhibit inflammation as a result of immunosuppressive activity of generated antigens. However, helminth therapy with live worms has many disadvantages hence an alternative safer option is using dendritic cells stimulated with parasitic products. Dendritic cells as main group of antigen presenting cells play a crucial role in immunomodulation process practice by nematodes. Presence of helminths leads to DC activity modification in consequence with differentiation of T cells into Treg lymphocytes hence balance reestablished in the large intestine.

The aim of this study was to evaluate therapeutic potential transfer of 1 million dendritic cells JAWSII line stimulated with male and female live Heligmosomoides polygyrus L4 larvae to BALB/c mice with colitis. Immature DC JAWSII was cultured with H. polygyrus L4 larvae isolated from BALB/c mice intestines. Colitis in the animals was inducted by 3% dextran sodium sulfate. Symptoms of disease, surface receptors expression by flow cytometry and cytokine production by T cells isolated from spleen and mesenteric lymph nodes by ELISA method, were examined.

Piotr Paduszyński1, Katarzyna Jelonek 2, Marzena Jaworska-Kik1, Arkadiusz Orchel1, Elwira Chrobak3, Ewa Bębenek3, Stanisław Boryczka3, Ewa Chodurek1, Janusz Kasperczyk1,2 1 Department of Biopharmacy, SPLMS in Sosnowiec, SUM, Poland; 2Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland; 3Department of Organic Chemistry, SPLMS in Sosnowiec, SUM, Poland

Betulin has been shown to elicit anticancer properties by inhibiting cancer cells growth. New betulin derivatives have been synthesized, which exhibit even better cytotoxity compared to betulin. This project aims to develop micellar drug delivery systems for cancer therapy with betulin derivatives based on amphiphilic degradable polymers.

MCf-7 and SK-BR-3 cells were treated with phosphate and phosphonate derivatives of betulin. Cytotoxity effect was determined by Sulforhodamine B based and Cell Death Detection Elisa Plus assays. All of tested compounds had an influence on MCf-7 and SK-BR-3 cell growth. Results suggest that E-29-diethoxyphosphoryl-28-O-propynoylbetulin and 30-diethoxyphosphoryloxy-28-O-propynoylbetulin have strong cytotoxic activity and are promising candidates for drug delivery system.

Acknowledgements The work is the result of the research project No. 2015/18/M/ST5/00060 funded by the National Science Centre.

Transfer of stimulated JAWSII with H. polygyrus L4 JAWSII resulted in changes in immunological response of mice.

Acknowledgements Supported by Polish National Science Centre Grants No. 2013/09/B/NZ6/00653 (218668).

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 207

P20.18

P20.19

Dietari supplement on the basis of amino acids and zinc

Improvement in behavior and biochemical parameters in the streptozotocin-induced rat model of Alzheimer disease treated with genistein

Anastasiya Pauliukavets1, Vladimir Sheibak 2, Anna Buklaha1 1 Grodno State Medical University, Department of Microbiology, virology and immunology named after SI. Gelberg, Belarus; 2Grodno State Medical University, Department of Biochemistry, Belarus

Amino acids and their derivatives are relatively long-standing and quite effectively used in medical practice in the form of independent drugs or as complex medicines. The first group includes methionine, cysteine, glycine, taurine, glutamine, leucine, tryptophan; white the second includes gepavalag, inocardine, valikar, neuramin, tetracardium, asparcam. Preparations created on the basis of amino acids, are as a rule low-toxic, they promote the normalization of metabolism and are good for the prolonged use.

We have developed composition consisting of amino acids (tryptophan, taurine and arginine) and microelement (zinc sulfate). The dose-dependent immunomodulating effect and the effect on the structure of the amino acid pool in the cells of the immune system have been revealed. The protective effect of the supplement on the liver with the administration of the cytostatic cyclophosphamide, as well as the reduction of the negative effect of lead acetate on the animal body, have been revealed.

Thus, the composition consisting of functional amino acids (tryptophan, taurine and arginine) and zinc sulfate has a pronounced biological activity and protective properties under the conditions of exposure to negative environmental factors on the animal organism.

Karolina Pierzynowska1, Magdalena Podlacha2, Dorota Myślińska2, Irena Majkutewicz2, Jagoda Mantej1, Grzegorz Węgrzyn1 Department of Molecular Biology, University of Gdansk, Gdansk, Poland; 2 Department of Animal and Human Physiology, University of Gdansk, Gdansk, Poland

1

Alzheimer disease (AD) is the most common cause of dementia in humans. It is caused by accumulation of amyloidal plaques formed by insoluble forms of beta-amyloid (βA) and neurofibrillary tangles composed of hyperphosphorylated tau protein (P-tau) in the brain. Accumulation of these proteins impairs functions of neurons leading to neurodegeneration and a battery of symptoms, including memory deficiency. No effective AD therapy has been developed till now, and the treatment is focused only on alleviation of symptoms. Genistein, an isoflavone, stimulates lysosomal biogenesis and expression of genes coding for lysosomal hydrolases. It leads to the stimulation of the autophagy which is effective in protein aggregates reduction. The aim of the study was to test whether genistein effectively reduces βA and P-tau in brains of streptozotocin-induced rat model of AD and corrects behavior of these animals. Genistein or water were administered orally. In the Morris water maze, genistein-treated AD rats were characterized by the significant improvement of long-term memory compared to the control group. In the novelty test, there were no significant differences between genistein-treated AD rats and healthy animals, while untreated AD rats differed considerably. Levels of βA and P-tau in brains were significantly lower in genistein-treated AD animals relative to untreated AD rats.

In conclusion, genistein can be considered as a putative anti-AD therapeutic.

6th Central European Congress of Life Sciences Eurobiotech 2017

208 Abstracts

P20.20

P20.21

Lack of miR-378 mitigates dystrophic phenotype in mdx mice – transcriptome analysis

The analysis of cell cycle arrest induction by RG7388 using the selected cell line models

Paulina Podkalicka1, Olga Mucha1, Katarzyna Pietraszek-Gremplewicz1, Magdalena Kozakowska1, Iwona Bronisz1, Michal Mikula2, Alicja Jozkowicz1, Agnieszka Loboda1, Jozef Dulak1

Justyna Polak1, Justyna Kocik 2, Tad A Holak3, Lukasz Skalniak4

1 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Krakow, Poland; 2 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Warsaw, Poland

Keywords: Duchenne muscular dystrophy, miRNA, miR-378 Severity of Duchenne muscular dystrophy (DMD) – incurable disease which results in walking and breathing difficulties, might be affected by several factors, including microRNAs (miRNAs). Among them, miR-378 is thought to play an important role in muscle development, differentiation and regeneration; however, its role in DMD and its murine model (mdx mice) has not been thoroughly addressed.

We have demonstrated that mice lacking miR-378 are able to run longer distance than their wild-type counterparts in a downhill treadmill test. More strikingly, double knockout animals (mdx/miR-378 -/-) were stronger than mdx mice and ran similar distance to wild-type animals.

To shed more light on the molecular pathways responsible for the improved performance of those mice and to identify differentially expressed genes in gastrocnemius of wild-type, mdx, miR-378 -/-and mdx/miR-378 -/- animals we have performed the comparative transcriptome analysis. RNA-sequencing revealed many genes differentially expressed between mice genotypes. Further verification using real-time PCR method as well as histological and immunofluorescent analysis showed, among others, decreased inflammation and fibrosis in mdx/miR-378 -/-mice in comparison to mdx mice, what at least partially could be responsible for less severe injury and greater muscles functionality of dystrophic mice lacking miR378.

1,2 Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University in Kraków, Poland; 3,4Department of Organic Chemistry, Faculty of Chemistry; Malopolska Centre of Biotechnology, Jagiellonian University in Krakow, Poland

The aim of this study was to evaluate the potency of MDM2 antagonist, RG7388, in different p53wt cancer cell lines: HCT 116, U-2 OS, MCF-7 and SJSA-1. Even though RG7388 efficacy is being assessed in clinical trials, there is still little known about it in in vitro studies. Therefore, we investigated the vulnerability of the selected models to undergo cell cycle arrest when treated with the compound, and their ability to break the arrest during and after treatment.

The activity of RG7388 as MDM2 antagonist was tested by analysing the changes in p53 and p21 proteins levels.

In all cell lines RG7388 caused very strong accumulation of p53 and p21 in a dose-dependent manner. Next, it was examined if these changes are consistent with the induction of cell cycle arrest. MDM2 antagonist caused efficient cell cycle arrest in three out of four examined cell models.

However, RG7388 did not lead to irreversible growth arrest as the cells resumed proliferation upon drug removal (except for SJSA-1, which all died). HCT 116 line, even in the presence of RG7388 in the culture medium, continued proliferation. MCF-7 and U-2 OS cells showed comparable responses.

Our results show that although RG7388 is a potent inhibitor of cell cycle progress in p53wt cells, its action is cell type-limited and may be overcame after the drug removal. Cell line models, especially U-2 OS cell line, seem suitable for long-term treatment strategies regarding the development of secondary resistance to RG7388.

Acknowledgements Supported by grant No. 2012/06/A/NZ1/0004 (JD) and 2016/21/B/NZ1/00293 (AL) from the National Science Center.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 209

P20.22

P20.23

Inhibitory effect of Functional carb-pharmacophores with sulfur atom on proliferation of cancer cells

Influence of chlorpyrifos on the level of expression of vitamin D3 receptor in skin cells

Joanna Sarnik1, Anna Czubatka-Bieńkowska1, Anna Macieja1, Zbigniew J Witczak 2, Tomasz Popławski1

Krzysztof Sawicki1, Arkadiusz Czerwonka2, Marcin Kruszewski1,3,4, Lucyna Kapka-Skrzypczak1,3

University of Lodz. Department of Molecular Genetics.

90-236 Lodz. Poland; 2Nesbitt School of Pharmacy.

Wilkes University. PA 18766 Wilkes-Barre. USA.

1 Institute of Rural Health, Department of Molecular Biology and Translational Research, Lublin, Poland; 2Maria Curie-Skłodowska University, Department of Virology and Immunology, Lublin, Poland; 3University of Information Technology and Management, Department of Medical Biology and Translational Research, Rzeszow, Poland; 4 Institute of Nuclear Chemistry and Technology, Centre for Radiobiology and Biological Dosimetry, Warsaw, Poland

1

Defining the biological function of carbohydrates in the cell allowed the development the ideas connected with use of thesecompoundsas a drug for many human pathological conditions including cancer. For our investigation we choose group of compounds represented by1-4-S-thiodisacharidesand anhydrosugar with salicylic group connected via sulfur bridge. These compounds in our preliminary research were cytotoxic against tumor cells and their mechanism of action is linked with oxidative stress generation.

The main aim of this work is to examine the mechanism of 1-4-S-thiodisacharides(FCP) anticancer activity.

As an in vitro model we chose astrocytoma tumor HTB14 cell line. Astrocytoma cancer has a poor prognosis and a very small pool of effective drugs.Except cytotoxicity we determine abilities of FCPs of cell proliferation inhibition.

We investigate effect of the enzymatic antioxidant system in tumor cells assisted by low molecular antioxidants, including thiole and thioredoxin (TRX). Our result suggest that FCPs inhibit the proliferation of cancer cells and their activity is connected with generation of oxidative stress by inhibition of antioxidant system. After analysis of activity of tested compounds we noticed that FCP6 is the most promising anticancer agents and deserve further biological investigation.

Acknowledgements This work was supported by The Polish National Science Centre, decision nr Dec2014/15/N/NZ7/02948.

Keywords: chlorpyrifos, VDR, vitamin D3 The aim of the study was to investigate the effect of chlorpyrifos (CHP), an organophosphorus (OPs) insecticide on the expression of vitamin D3 receptor (VDR) in the skin cells. VDR is nuclear receptor (NR1l1) that mediates the biological action of the active form of vitamin D3 in numerous physiological and pharmacological processes, including calcium metabolism, cellular growth and differentiation, immunity and cardiovascular function.

Studies were performed on BJ (fibroblast) and HaCaT (keratinocytes) cell lines. Cells were pre-incubated with the precursor of vitamin D3(7-DHC) and the two concentrations of CHP (50, 250 µM). The synthesis of vitamin D3 in cells was initiated by UVB irradiation with intensity 5 or 20 mJ/cm2 for BJ and 15 or 20 mJ/cm2 for HaCaT. Control cells were UVB irradiated after preincubation with 7-DHC alone. To investigate the effect of CHP on the expression of VDR, samples were analyzed by Real-Time PCR and Flow cytomery.

A statistically significant and dose-dependent increase of VDR expression was observed in both fibroblast and keratinocytes cell lines at the mRNA and protein level.

Our research showed that exposure to CHP induced expression of VDR nuclear receptor in the BJ and HaCaT cell lines. It can therefore be concluded that OPs pesticide might interfere with vitamin D3 metabolism in the skin cells.

Acknowledgements This study was financed by the National Science Centre Fund, Project No. DEC2013/09/N/NZ7/03565

6th Central European Congress of Life Sciences Eurobiotech 2017

210 Abstracts

P20.24

P20.25

Deregulated apoptosis of neutrophils as a potential mechnism underlying the pathobiology of periodontitis

Comparative analysis of gene expression of human capillary (CAP) and high endothelial cells (HEC)

Maja Sochalska1, Magdalena Stańczyk1, Maria Użarowska1 & Jan Potempa1,2 Department of Microbiology. Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 2University of Louisville, School of Dentistry, Louisville, KY, USA, maja _ sochalska@yahoo.com 1

The pathogenesis of the periodontal disease is associated with over-reactive host inflammatory response to periodonal pathogen Porphyromonas gingivalis. Current literature implicates hyper-reactive neutrophils as the main immune cell type responsible for the tissue damage and disease progression. Despite the clear involvement of anti-apoptotic Bcl-2 family proteins in control of neutrophil apoptosis, the failure to properly regulate neutrophil survival and function in the context of periodontitis remains unclear [1].

However, studying neutrophil function ex vivo is difficult since these cells get easily activated during isolation and die rapidly in culture. Therefore, in order to investigate neutrophils contribution to periodontitis we use a nove conditional oestrogen-inducible HoxB8 overexpression system, which allows to immortalise murine bone marrow progenitors [2]. Withdrawal of oestrogen from cell cultures shuts down the HoxB8 expression and triggers differentiation into mature neutrophils, with all of their morphological and functional hallmarks such as multilobed nucleus, phagocytic capacity or ROS production. Of importance, HoxB8 progenitor lines can be established from any genetically modified mouse strain and may also be easily further genetically modified in vitro.

The establishment of new prognostic and therapeutic markers for periodontal disease will bring considerable benefits for public health and for the quality of life of patients. Our most recent results will be presented.

Agata Szade, Kevin Brulois, Mike Lee, Milladur Rahman, Eugene Butcher Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford and The Center for Molecular Biology and Medicine, VAPAHCS, Palo Alto, USA High endothelial cells (HEC) are a specialized type of blood endothelial cells (EC) that are responsible for the control of leukocyte homing into lymphoid tissues. The aim of this study is to compare the expression profiles of human HEC and capillary EC (CAP).

HEV and CAP cells were sorted from the fresh tonsil or appendix samples and isolated RNA was analyzed using Human Gene 1.0 ST Array GeneChip (Affymetrix) microarrays and Gene Spring software.

Preliminary analysis shows differential gene expression between HEV and CAP cells, as well as differences between EC subsets of different tissues (appendix or tonsil). We have observed a ≥2 fold increase in expression of 489 genes in HEV cells compared to CAP cells in tonsil samples and 186 genes in appendix samples. Among these genes, expression of 122 was elevated both in the tonsils and in the appendix, and these included genes encoding for cytokine receptors and cell adhesion molecules.

To conclude, microarray analysis revealed distinct transcriptional profiles of HEC and CAP cells in the human lymphoid tissues. The study identifies key cytokines, adhesion receptors, glycosylation and signaling pathways that define HEC specialization and may regulate their function in lymphocyte traffic.

Acknowledgements This work was supported by funding grants Foundation for Polish Science (Homing/2016-1/9) to MS, US NIH/NIDCR (DE 022597 and DE 023207), and National Science Center (2012/04/A/NZ1/00051, NCN, Krakow, Poland) to JP. Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education.

References [1] Sochalska, M. and J. Potempa Front Cell Infect Microbiol (2017) 7: 197 “Manipulation of Neutrophils by Porphyromonas gingivalis in the Development of Periodontitis.”

[2] Wang et al, Nat Methods, 3 (2006) 287–93.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 211

P20.26

P20.27

Association study of polymorphisms in genes involved in lipid metabolism

Endocan: an early marker of bacterial infection in decompensated cirrhosis

Anna Trakovická, Nina Moravčíková, Kristína Candráková, Martina Miluchová, Michal Gábor, Radovan Kasarda

Jolanta Zuwala-Jagiello1, Joanna Górka-Dynysiewicz1, Ewa Grzebyk1, Eugenia Murawska-Ciałowicz2, Monika Pazgan-Simon3

Department of Animal Genetics and Breeding Biology, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia

1 Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Poland; 2Department of Physiology and Biochemistry, University of Physical Education, Wroclaw, Poland; 3 Clinic of Infectious Diseases, Liver Diseases and Acquired Immune Deficiency, Wroclaw Medical University, Poland

Keywords: ghrelin, genetic polymorphism, human genome, leptin, lipids, metabolic disorders The aim of this study was to analyse the genetic polymorphisms in genes encoding leptin (LEP), leptin receptor (LEPR) and ghrelin receptor (GHSR) as markers involved in genetic control of human lipid metabolism. In total, 236 DNA samples were used for SNPs genotyping based on PCR-RFLP method. The subsequent association analysis was carried out to assess the impact of those SNPs on biochemical parameters including body mass index, total cholesterol, HDL and LDL cholesterol. The value of observed heterozygosity (0.417±0.075) and Wright’s FIS index (0.165±0.015) indicated the prevalence of homozygote genotypes within population. The median levels of polymorphic information content (0.37) reflected mainly the high effective allele number (1.995±0.004) of each SNPs. The association analysis performed by GLM procedure proved significant impact of loci on analysed parameters. Except SNPs genotypes, the age and gender significantly affected each trait of interest. However, our study indicated that the SNPs in LEP, LEPR and GHSRgenes affected the human nutritional status regardless of age or gender population structure and therefore can be considered as candidate for lipid metabolism disorders as well as obesity. This study was supported by the Slovak Research and Development Agency (Contract No. APVV-14-0054 and APVV-0636-11).

Background. Endocan is a newly recognized biomarker of sepsis. However, there have been no studies of the trends in endocan levels in cirrhotic patients with bacterial infection and their associations with markers of infection and inflammation.

Methods. This study sought to assess the diagnostic value of serum levels of endocan, procalcitonin (PCT), C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) in 126 patients with cirrhosis: 51 with decompensated infected cirrhosis, 56 with decompensated uninfected and 19 with compensated uninfected cirrhosis at inclusion. Endocan, PCT, CRP, IL-6 and TNF-α were assayed in serum samples by ELISA analyses.

Results. Serum levels of endocan, PCT, CRP and TNF-α were significantly higher in cirrhotic patients with clinically overt infections. Endocan levels were correlated to neither PCT levels nor IL-6 levels in each group of patients with cirrhosis. CRP and TNF-α levels and ChildPugh score correlated only in the infected group of patients with endocan levels, while in the uninfected groups of cirrhotic patients no significant correlation could be detected. The diagnostic accuracy of endocan increased in advanced stage of the disease. Serum endocan levels ≥ 2.05 ng/ml had a sensitivity of 76.1% and specificity of 85% for the diagnosis bacterial infection in decompensated cirrhotic patients.

Conclusion. The endocan measured at admission is a good clinical parameter predicting the occurrence of infection in these patients.

6th Central European Congress of Life Sciences Eurobiotech 2017

212 Abstracts

P20.28 New model using pentraxin 3 to predict liver fibrosis in patients with chronic hepatitis C Joanna Górka-Dynysiewicz1, Monika Pazgan-Simon2, Jolanta Zuwala-Jagiello1 1 Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Poland; 2Clinic of Infectious Diseases, Liver Diseases and Acquired Immune Deficiency, Wroclaw Medical University, Poland

Background. The invasive nature of liver biopsy and a number of contraindications stimulate the introduction of alternative diagnostic methods which give an indirect, but substantial insight into liver tissue assessment. The aim of this study was to investigate the relevance of an index/ model, based on serum pentraxin 3 levels, to the degree of liver fibrosis in hepatitis C virus (HCV)-positive patients.

Methods. A total of 76 HCV-positive patients were assessed based on the values of a new model based on serum pentraxin 3 (PTX3) and its activators (TNF-α, Il-1β). The model findings were used to investigate the relationship with the degree of liver fibrosis.

Results. The PTX3 showed an association with the stage of fibrosis. TNF-α, Il-1β were not useful parameters in the construction of noninvasive model assessing the advancement of hepatic fibrosis in HCV patients. The new model showed a higher correlation related to liver function than Forns and the FIB-4 did. In addition, the model showed a higher AUROC value (0.945) than that of Forns (0.916) and the FIB-4 (0.905) for prediction of liver fibrosis.

Conclusion. The novel noninvasive model, based on serum pentraxin 3 levels, is shown to relate to the degree of liver fibrosis in HCV-positive patients.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 213

Art in Biotechnology Lectures

L21.2

L21.1

The significance of interdisciplinary research in the field of landscape architecture and environmental biotechnology. A case study of the green space revitalization in the City of Lodz (Poland)

Art in the Society of Knowledge. Twisting Symmetries Joanna Hoffmann-Dietrich University of Arts in Poznan, Faculty of Art Education, Poland The title refers to the profound shift of the civilisation paradigm related to the global revolution of knowledge that triggers substantial social, economic, political and cultural changes. The term “New Renaissance” is often used to describe our times because of the impact of scientific research on intellectual and cultural movements resulting from interdisciplinary initiatives and activities in knowledge transfer and sharing.

Biotechnology forms a vital part of this revolution and thus deserves more widespread public awareness, understanding and engagement.

Although it is hard to predict the ultimate outcome and cultural consequences of today’s revolution of knowledge, it brings radical shift of values and norms and changes our way of thinking.

The two questions asked in my presentation are as follows: Where is the society of knowledge heading and how is it shaping its culture?

What is the place and role of art in the society of knowledge?

I believe that art can function as a great platform for interdisciplinary, inter-cultural communication and knowledge exchange, leading to better quality of life and helping to understand and manage the complexities of the world that we generate.

My presentation will be based on my own investigation and experience as professor of the University of Arts, Chair of the Club for Science and Art in Poznan, Leader of Art & Science Node in Berlin and as an artist collaborating with scientists from research institutes in Poland, Germany and India.

Długoński Andrzej1, Siewiera Paulina2, Bernat Przemysław2, Słaba Mirosława2, Długoński Jerzy2 1 Cardinal Stefan Wyszyński University in Warsaw, Department of Environmental Engineering, Cardinal Stefan Wyszyński University in Warsaw, Poland; 2 University of Lodz, Department of Industrial Microbiology and Biotechnology, University of Lodz, Poland

In the context of the revitalization law in Poland (2016), the term “revitalization” defined as a long-term process aimed at improving the urban conditions as well as the living conditions of inhabitants, including users of green areas, which serve as places for everyday rest of citizens.

The case study are two selected pocket parks located in downtown of the City of Łódź in Poland, included in the Municipal Revitalization Program (GPR) of the town centre. The PAHs and heavy metals analyses by GC-MS/MS and AAS (respectively) and microbial studies of both sites revealed presence in soil contaminates characteristic for coal furnace dust and road transport and negligible biodegradation abilities of the urban park soil microcosm and vegetation. Moreover, pollutants typical for mammals urine were noticed in one of parks area. The conducted analyses indicate that investigated urban green spaces are not sufficiently safe and healthy for the daily users.

Therefore, the additional revitalization approach should be done for their reconstruction and adaptation to contemporary needs of users including introduction of selected plant species capable of efficient xenobiotics elimination in cooperation with soil microorganism as well as fixation of dog runways, park toilets, leisure facilities, monitoring system, etc. The presented case emphasizes also the value of complementary studies in the field of landscape architecture and environmental biotechnology for men’s health and rest.

6th Central European Congress of Life Sciences Eurobiotech 2017

214 Abstracts

L21.3

Posters

Influence of selected factors for permeate and absorption of prolactin by small intestine of suckling piglets

P21.1

Barbara Dolińska1,2, Barbara Gajda3, Florian Ryszka2, Zdzisław Smorąg3

Aneta Ostróżka-Cieślik1, Barbara Dolińska1,2, Florian Ryszka2

1 Department of Applied Pharmacy, School of Pharmacy and the Division of Laboratory Medicine, Medical University of Silesia, Poland; 2Pharmaceutical research and Production Plant „Biochefa”, Sosnowiec; 3Department of Animal Reproduction Biotechnology, National Research Institute of Animal Production, Balice/Kraków, Poland

1 Department of Pharmaceutical Technology, Chair of Applied Pharmacy, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Sosnowiec, Poland; 2Pharmaceutical Research and Production Plant „Biochefa”, Sosnowiec

Prolactin (PRL) is one of multifunction protein hormone. PRL i salso qualitatively non-specific it suggests possibility of its wider application in medicine and veterinary. This is why it is so important the way of application.

One of the most important ways is oral especially for newborn individuals. High purified porcine prolactin (F.Z.N.P. Biochefa, Sosnowiec) was used as a sterile solution of 1 ml in newborn piglets. They have examined permeate and absorption prolactin by natural mambrane – small intestine section from one day suckling piglets. Experiment performer in diffusion cells by Frantz. Pure hormone 96% dissolved in 0,1 M hydrochloric acid and examined influence hormone concentraition, pH value and auxiliary substances concentraition (mannitol, trehalose) for PRL permeate and absorption percentage. They observed that the largest permeate PRL (~31,5%) occurs from solution of composition : hormone concentration – 0,25 mg/ml; pH value of solution – 3,5; trehalose concentration – 18 mg/ ml; mannitole – 6 mg/ml. The largest absorption PRL (~ 17,6%) by intestine observed from solution: hormone concentration – 0,25 mg/ml; pH value of solution – 2,5 without auxiliary substances. Obtained results show possibility of oral administration PRL to sucking piglets in earlier stages after their birth.

Directions for optimizing organ preservation solution

Organ damage during ischemia is one of the clinical problems of today’s transplantation. It occurs during warm ischemia (WIT) when the flow of blood is stopped and during cold ischemia when the graft is chilled in situ until the circulation is restored in the recipient. Fast cooling of the organ slows metabolism and activates intracellular enzymes, which minimizes the effects of warm ischemia.

Unfortunately, during hypothermia also occurs: inhibition ATP synthesis and cell swelling and intracellular acidosis, so research is continuing to develop new and optimize the composition of existing preservation solutions that allow longer and more effective storage of grafts and restoration of optimal functions after transplantation. Present information will be provided on the world-wide preservation solutions and modification directions of preservation solutions with hormones and bioelements.

6th Central European Congress of Life Sciences Eurobiotech 2017

Abstracts 215

Bioethics Lectures

L22.2

L22.1

Science and medicine in an era of populism: a case of stem cell “therapies”

Eye of beholder: risk perception and precautionary principle in decision making in biotechnology Tomasz Zimny Institute of Law Studies, Polish Academy of Sciences, Poland Precautionary principle (PP) constitutes basis of current legislation in biotechnology. It is a decision making directive that mandates undertaking precautionary measures in situations of scientific uncertainty as to the outcomes of an action, when prima facie there may be risks associated with such action.

Application of PP is costly and time consuming. It should be applied in situations, where there is indeed scientific uncertainty or lack of data as to a planned action. Consequently, risk perception influences the decision whether to apply PP at all, as well, as decisions about precautionary measures taken during its execution. Perception of particular risks (and their moral acceptability) depends among others, on the features of the beholder (also a collective one) – their professional and social background, interests at stake or experiences. Even a decision to apply PP may influence risk perception of a particular action.

While risk perceptions of various stakeholders may have different scientific significance, it does not presuppose that this significance is proportional to the moral or political significance of such perceptions. I analyse and compare decisions of selected stakeholders and European authorities (e. g. EFSA, governments) in the field of green biotechnology, to determine whose and which risk perceptions influence PP application the most. As it turns out, not necessarily most scientifically sound perceptions prevail in this decision making.

Józef Dulak Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; Kardio-Med Silesia, Zabrze, email: jozef.dulak@uj.edu.pl The believe in the existence of miracle panacea has been always associated with medicine. Looking back even into the recent, XX- century history one can find numerous examples of a strange mixture of medicine with magic and religious beliefs. And although our times are characterized by the enormous dependence on science and evidence-based medicine, still, however, numerous conditions are not treatable and the new ones arise as the epidemies due to extension of human longevity and treatment of the previously mostly deadly conditions. As a result a number of patients with heart failure, Alzheimer or Parkinson diseases is constantly increasing raising expectations of patients and physicians to find the effective and permanent cure.

Since 20 years the scientists, physicians and the patients are exploring the possibility of stem cells as the treatment for previously untreatable diseases. The common belief and expectations is that poorly or improperly defined “stem cells” are panacea for every condition, the faith strengthen additionally by claimed “naturality” of such therapies, in contrast to “artificial, chemical” drugs.

This promise finds the widespread acceptance which is propagated by the media, particularly internet. The generated hype is amplified by the expectations and lack of basic scientist knowledge among not only the patients but also the physicians. Accordingly, fulfilling the populistic wishes the physicians are naming some cell types the “stem cells” and promise that their injections into any organs will do what is expected: cure the heart, restore vision or even treat the autism. Such claims create also the environment for some scientists propagating the miracle properties of special type of “stem” cells, generating faked impression of scientific solidity of the unproven treatments. At the same time such unconfirmed or even falsified theories fertilize numerous charlatan offers, eg. deer horns “stem cells” or plant “stem cells”.

As always it is extremely difficult to deal with irrational thinking. This danger is increased additionally due to the involvement in hype propagation of physicians and scientists not aware of the complexity of real stem cells.

However, warning on such problems is crucial due to the risk for the patients who can be severely harmed by the unproven “therapies” and the danger for stem cell science which reputation can suffer from serious side effects of applied treatments.

References Dulak J, et al,. Adult stem cells: hopes and hypes of regenerative medicine. Acta Biochim Polon, 2015, 62(3):329–37

6th Central European Congress of Life Sciences Eurobiotech 2017

EXHIBITION

218 Sponsors

Sponsors: GOLD SPONSOR Sartorius Sartorius is one of the world’s leading suppliers of laboratory equipment and industrial technology. Thanks to innovative products and high quality services, we support our customers worldwide in efficiently implementing complex processes in biopharmaceutical production and in laboratories. Our clients are biotech, pharmaceutical and food industry, and state research institutes and laboratories.

Sartorius has manufacturing facilities in Europe, Asia and America, and has more than 110 subsidiaries and subsidiaries in more than 110 countries.

In Poland, sales of Sartorius products are carried out within two companies: Sartorius Poland Sp. z o. o. and Sartorius Stedim Poland Sp. z o. o.

The company, heavily embedded in science and research, works with clients and business partners in their day-to-day operations with the slogan “Turning science into solutions”, so it easily adapts to new trends, particularly in the biopharmaceutical industry.

Sartorius Poland Sp. z o. o.

Product portfolio Sartorius Polska concentrates on high quality laboratory instruments such as laboratory scales, pipettes and laboratory water treatment systems. In addition, we offer the broadest range of consumables such as laboratory filters and pipette tips. In laboratory weighing technology, our company is the second largest equipment supplier in the world.

Sartorius products: - precision balances, analytical balances, microbalances, moisture analyzers, - mass standards E1, E2, F1, F2 (also with calibration certificates), - accessories, software for scales (also according to customer’s wishes), - microfiltration and ultrafiltration in the laboratory scale: kits and ready-made filter units, membrane filters, microbiological media, cross-flow systems, microbiological air purification equipment, ultrapure water purification systems, Liquid assortment (Liquid Handling, automatic pipettes, dispensers and burettes and tips) Service includes: - maintenance reviews, - preparation of scales for legalization, - calibration, Contact: Sartorius Poland Sp. z o. o. ul. Wrzesińska 70, 62-025 Kostrzyn Tel. 61 647 38 30, fax 61 647 38 39 E-mail: info.pl@sartorius.com

Sartorius Stedim Poland Sp. z o.o.

Sartorius Stedim is a supplier of fermentation, filtration, transportation and purification equipment for the biopharmaceutical, food industry and scientific institutions.

We provide single-use solutions that allow aseptic management of liquids from formulation and fermentation to finished product using special bags, connections, filters and full process automation.

High purity liquids are capable of achieving not only microfiltration but also ultrafiltration with cross flow and chromatographic techniques. A wide range of sterilization filters, as well as indented materials from a variety of materials on a variety of scale, allow us to deliver solutions even in the most demanding applications of fluids and gases.

We also provide technical advice and service at every stage of the process. The design of our fermenters, filters, bags and many other products allows us to scale up R & D units to the stage of industrial production.

Contact: Sartorius Stedim Poland Sp. z o.o. ul. Wrzesińska 70, 62-025 Kostrzyn Tel. +48 61 64 73 840, fax 61 879 25 04 E-mail: biuro.pl@sartorius.com

6th Central European Congress of Life Sciences Eurobiotech 2017

Sponsors 219

Gold Sponsor Polpharma Biologics Polpharma Biologics is a division of Polpharma Group – one of the largest pharmaceutical companies in Central and Eastern Europe. We offer a one-stop-shop approach – fully integrated solutions along the biopharmaceutical development and production value chain to serve todays and tomorrow`s global market needs. Our world class team of scientists and engineers provides more than just their long-term experience and know-how but solutions that bring products to the market faster. Even more, our unique modular one-stop-shop concept allows a customized selection of the services you need. With the integration of cell line developing specialist Bioceros in the Netherlands we are now a fully backward-integrated biopharmaceutical one-stop-shop CDMO. Our site in Gdansk is a state-of-the-art cell culture facility, one of the most modern in Europe. In order to produce and supply commercial quantities for global markets, we are building a new modular GMP facility in Duchnice, nearby Warsaw. This site will also house our aseptic fill and finish unit.

Contact: Polpharma Biologics Gdański Park Naukowo-Technologiczny ul. Trzy Lipy 3 80-172 Gdańsk office.biologics@polpharma.com

Silver Sponsors Bayer Bayer is a global enterprise with core competencies in the Life Science fields of health care and agriculture. Its products and services are designed to benefit people and improve their quality of life. At the same time, the Group aims to create value through innovation, growth and high earning power. Bayer is committed to the principles of sustainable development and to its social and ethical responsibilities as a corporate citizen. In fiscal 2016, the Group employed around 115,200 people and had sales of EUR 46.8 billion. Capital expenditures amounted to EUR 2.6 billion, R&D expenses to EUR 4.7 billion. These figures include those for the hightech polymers business, which was floated on the stock market as an independent company named Covestro on October 6, 2015.

For more information, go to www.bayer.com.pl Follow us on Facebook: http://www.facebook.com/Bayer Follow us on Twitter: https://twitter.com/BayerFollow us on YouTube: http://www.youtube.com/user/BayerPolska

Nanotemper Technologies NanoTemper Technologies is deeply committed to the best customer experience. Central to this is a strong focus on enabling researchers to easily, efficiently, and accurately perform protein characterization. With a broad offering of instrumentation, software and consumables for evaluating binding affinities and protein stability, scientists in pharmaceutical, biotech or academic labs will find an optimized workflow, quality results and responsive customer support, work with a deeply experienced and globally operating team, and realize the NanoTemper experience.

Bronze Sponsor Applikon Biotechnolgy Proud enabler of improved Quality of Life Applikon Biotechnology is a world leader in developing and supplying advanced bioreactor systems and is passionate to contribute to Life Science. Known for delivering new technologies, covering the whole market from laboratory, to pilot, to production scale.

Next to advanced cultivation systems, its core business is in developing own Process Control and Process Information Management systems. With continuous focus on ensuring bioprocess optimization. Large investments in R&D and in-house development & production, enables Applikon to be flexible and efficient in order to deliver the best solution for each individual customer.

Read more on www.applikon-biotechnology.com!

6th Central European Congress of Life Sciences Eurobiotech 2017

220 Partners

Substantive Partner The National Centre for Research and Development The National Centre for Research and Development (NCBR) is an executive agency of the Minister of Science and Higher Education. It finances innovative projects of entrepreneurs and scientists with market potential as well as university projects to enhance the competences of students and teaching staff. The NCBR supports innovators, connects science with business and helps at all stages of project implementation – from idea to industry. By co-financing R&D works, the NCBR “insures” from risks associated with their implementation.

NCBR activities are financed by the state treasury and EU funds. In the 2014-2020 EU Financial Perspective, the NCBR acts as an Intermediate Body in the Operational Programmes Smart Growth and Knowledge Education Development, under which it currently carries out more than 30 programmes dedicated to specific customer groups: entrepreneurs, investors, science units, science-industry consortia as well as universities. The NCBR offer includes horizontal, international and sectoral programmes and international programs, such as Fast Track, BRIdge Alfa, INNONEUROPHARM and ERA CoBioTech.

Partners Innovative Małopolska The Małopolska Region is a strong economic centre and, without a doubt, a perfect place to develop business – it can be confidently said that we are one of the most dynamically developing regions in Poland and Central Europe. Thanks to a massive support to entrepreneurship as well as optimal use of European Funds and other public resources, Małopolska – as the first Polish region – has been awarded by the Committee of Regions with the title of the European Entrepreneurial Region 2016.

Innovations in regional economies is a basic condition for building competitive advantage. Therefore innovations determine strengths and potential of enterprises as well as local and national economy. Supporting innovativeness of companies is one of the most important activities of regional authorities in Małopolska. The region strengthens its economic potential by creating conditions for developing its smart specializations corresponding to priority sectors – such as ICT, chemical branch, sustainable energy, metalworking, electrotechnologies, creative industries, biotechnology and life sciences.

Thanks to dynamic progress in those specialities, we build our competitive advantage over our competitors both on the national and international market.

Evestra Onkologia Sp. z o.o Evestra Onkologia Sp. z o.o is a Polish biopharmaceutical start-up company located in Lodz launched by a distinguished team with an impressive record in developing and commercializing innovative oncology and other proliferative diseases products.

The key mission of Evestra Onkologia is to develop highly innovative oncology and antiproliferative assets such as endometriosis and fibroids in Poland, and the EU. To accomplish its core mission, the Company has and will continue to establish a variety of fruitful collaborations with leading Polish research institutions and investigators and for-profit CRO companies in Poland, to enhance development and commercialization of its innovative assets. Evestra Onkologia managing board is led by two executives of the pharmaceutical industry with extensive in drug development and managing companies with multinational divisions.

Evestra Onkologia portfolio includes issued composition of matter patents which describes EC317 and EC313 with applications in oncology and endometriosis.

Evestra Onkologia is interested in establishing cooperation with academic research organization in the field of oncology. We are currently seeking for preclinical and clinical partners in triple negative breast cancer, however our interest is not limited to this scope.

Evestra Onkologia conducts project POIR.01.01.01-00-0123/16 “Development of selective treatment of endometriosis based on mesoprogestins” funded from the funds of Operational Programme Smart Development.

6th Central European Congress of Life Sciences Eurobiotech 2017

Exhibitors 221

EXHIBITORS

ABL&E-JASCO Polska Sp. z o.o.

SPECTROSCOPY UV/VIS/NIR spectrophotometers, Microscopic UV/VIS-(NIR) spectrophotometers FT-IR, FT-Raman, FT-IR microscopes, IR spectroscopy accessories, Raman spectrometers Circular Dichroism Spectrometers (ECD and VCD), CPL, ORD, Polarimeters, Spectrofluorometers Surface Plasmon Resonance Spectrometers (SPR) CHROMATOGRAPHY HPLC, UHPLC systems and modules Columns. Flash Chromatography. BIOORGRANICS microwave reactors and accessories, peptide synthesizers, SPE, Dry Evaporator Concentration System. Represented Companies: JASCO, BIOTAGE, HANSON RESEARCH, COBALT LIGHT SYSTEMS, TEKNOKROMA, REICHERT, LABLOGIC SYSTEMS, ATHENA, METERTECH Contact: ul. Wadowicka 12, 30-415 Kraków tel. +48 12 267 71 87, e-mail: ablepol@ablelab.com, www.ablelab.com

Accela ACCELA became a leading supplier of high-tech innovative Life-Science technologies in the territory of Central and Eastern Europe. Ranging from the field of Molecular and Cellular biology, covering Cytometry methods, being experts in Preclinical imaging, our mission is to add value for our customers by providing progressive and cutting edge instrumentation, which can help to answer their scientific questions. We work with a great deal of force to always follow new trends and technology development in order to offer relevant advices.

Our commitment to supporting our customers drives everything we do. Accelerate your science with us!

AGEMA/INFORS BHZ ‘AGEMA’ EWA & MICHAL MATCZAK S.C. It was a result of the transformation of the Biuro Handlu Zagranicznego ‘AGEMA’, which has been operating on the Polish market since 1991.

Our offer includes the highest quality research and laboratory equipment of the world’s leading manufacturers. We are the exclusive representative in Poland of the Swiss company INFORS AG, the leading manufacturer of shakers, incubation shakers and bioreactors.

Other companies we cooperate with are: Ziegra, Labnet, Froilabo, Fryka and Leec.

Our company has experienced and trained service. For more than twenty-five years we are close to our customers.

BioIndustry e.V. The BioIndustry e.V. is a german registered association which main purpose is to activate, to concentrate and to develop biotechnological competences in the Ruhr region and Westphalia. The focus of BioIndustry’s activities is the promotion of biotechnology and related technologies in the whole range of application.

As a networked service cluster of 55 companies, research and training institutes, technology centres, biotechnological service providers and communal business development organizations the associations objective is to generate product- and process-innovations. This objective will be met by supporting the active transfer from ideas to market.

Four technology centres provide support and infrastructure for small and medium biotechnology-companies: • BioMedizinZentrum Bochum (www.tgr-bmz.de) • BioMedizinZentrumDortmund (www.bmz-do.de) • Kompetenzzentrum Bio-Security (www.bio-security.de) • Zahnmedizinisch-Biowissenschaftliche Forschungs- und Entwicklungszentrum Witten (ZBZ) (www.zbz-witten.de) 6th Central European Congress of Life Sciences Eurobiotech 2017

222 Exhibitors

Bionanopark Bionanopark is a science and technology park with an attractive research, investment and incubation offer addressed to companies and institutions operating in the field of advanced technology.

Our offer contains: Research services – we focus mostly on bio- and nanotechnology and personalized medicine; Incubator – we suport young and innovative enterpreuners by creating for them favourable conditions to run and develop their business; Conference rooms – in our conference center, you can organize an event for even 300 attendants; Investment areas – the company can build its own headquarters on our beautiful green areas.

The offer is particularly attractive to those companies that do not wish to invest in their own R&D units while they may outsource the research of their products to our laboratories on favorable conditions.

For more information visit: www.bionanopark.pl

Centre for Technology Transfer CITTRU, Jagiellonian University CTT CITTRU is responsible for commercialization processes regarding intellectual property developed at the Jagiellonian University in Kraków. Its activities cover areas such as identification of innovative research results, IP protection and evaluation, preparation of technology offers, promotion and brokering, negotiation, preparation and execution of the commercialization agreements.

Commercialization of research results is organized through licensing, assignment of IP rights or spin-out companies. Secondly, CTT CITTRU coordinates contract research which includes 200 offers of research teams from different fields and gives a possibility to organize a research team for resolving specific problems. Last but not least, CTT CITTRU organizes work of the academic entrepreneurship incubator.

Embassy of the Argentine Republic, Poland Argentina is the third largest producing country of genetically modified organisms (GMOs) in the world. The development of GMO crops in Argentina was a key milestone towards the generation of benefits not only for the agricultural sector but also for the country’s development, both in relation to domestic production and consumption as well as for Argentina’s participation in the international markets. GMO use results in benefits for farmers, consumers and the environment. It also constitutes an important means to address food security.

Eppendorf Poland Eppendorf is a leading life science company that develops and sells instruments, consumables and services for liquid, sample and cell handling in laboratories worldwide. Its product range includes pipettes and automated pipetting systems, dispensers, centrifuges, mixers, spectrometers and DNA amplification equipment as well as ultra-low temperature freezers, fermentors, bioreactors, CO2 incubators, shakers and cell manipulation systems. Associated consumables like pipette tips, test tubes, microliter plates and disposable bioreactors complement the instruments for highest quality workflow solutions.

Eppendorf products are most broadly used in academic and commercial research laboratories, e.g., in companies from the pharmaceutical and biotechnological as well as the chemical and food industries. They are also aimed at clinical and environmental analysis laboratories, forensics and at industrial laboratories performing process analysis, production and quality assurance.

6th Central European Congress of Life Sciences Eurobiotech 2017

Exhibitors 223

EURx EURx is a privately held biotechnology company located in Gdańsk, Poland. EURx delivers high quality reagents and services to the molecular biology research community, offers a full line of DNA ladders, isolation & purification kits, PCR products, modifying enzymes, restriction endonucleases, nucleic acid labeling products, and other ancillary molecular reagents.

With over 200 products spanning our product line, EURx is dedicated to meeting the needs of the research community. EURx has considerable expertise in the areas of fermentation, cloning and protein engineering, purification, nucleic acid amplification, and microplate detection technologies. We apply aggressive research and development in these technologies to sustain our innovative stance in the molecular biology arena.

Fundacja Małopolskie Centrum Transferu Technologii Technology Transfer Center Foundation in Malopolska Founders are are entrepreneurs with more than 20 years of experience in the area of broadly understood business and financial consulting, they are also private investors and business angels willing to share their skills, knowledge and experience.

MCTT offers assistance and cooperation in the implementation of innovative projects, including: R & D project planning, search for a scientific partner, research team, technology, search for a business partner, develop a business model for a commercialized solution, preparation of application for co-financing, project management.

Contact: www. mctt.pl biuro@mctt.pl GSM + 48 602 393 119

Genomed S.A. Genomed S.A. is an innovative company, with headquaters in Warsaw, Poland, that offers molecular diagnostics based on DNA sequencing, with a special focus on NGS-based methods. The main goal of Genomed is to further improve prophylactic and clinical whole genome analysis with an exhaustive diagnostic and medical report to provide a basis for prevention and personalised treatment.

Our offer also includes the whole range of DNA sequencing services, oligonucleotide and probe synthesis as well as bioinformatic analysis of data.

Owing to the technology transfer performed in June 2015, Genomed carries out non-invasive prenatal genetic testing (NIFTYTM) in its own, new, high-throughput laboratory. The network of over 300 collaborating clinics covers the entire Poland and Genomed is the most important NIPT provider in the country.

Genore chromatografia dr Jacek Malinowski “Genore chromatografia” is a distributor of modern GC and GCMS gas chromatographs, analytical and preparative liquid HPLC/ UHPLC, LCMS and Flash chromatographs, mass spectrometers, GC, HPLC and HPLC columns, autosamplers, aminoanalysers, gas generators, chromatographic software, chemical standards and general laboratory equipment. In our offer range you will find top quality and budget solutions from Young Lin, SYKAM, SRI, Cinel, ASI, ChromSword, DataApex, Bionacom, Nacalai Cosmosil, AMT, YMC, Nomura, Jordi, Shinwa and many more.

We are active chromatographers and we support our customers at a sale stage and with real work with our equipment.

Contact: Inżynierska 3 lok.3, 20-484 Lublin tel. 22 40 107 34, 22 40 107 35, fax: 22 40 107 36 www.genore.pl, e-mail: info@genore.pl

6th Central European Congress of Life Sciences Eurobiotech 2017

224 Exhibitors

Genos GENOS s.c. trading company is a distributor of high quality laboratory media, reagents and equipment with emphasis on tissue culture products.

We are sole distributor in Poland of cell culture media of Biological Industries: classical media, FBS, supplements, antibiotics, special media for MSC, iPS, hESC (defined, xeno-free, serum-free system), cytogenetic products, etc.

We offer also tissue culture plastics of TPP (www.tpp.ch): flasks, dishes, plates, bioreactors, serological pipettes, cryotubes, falcons, racks etc.

In our portfolio you will find also kits for the detection of viruses using ISH of PanPath, semen analysers of MES and medical devices of MRC. GENOS Non-Public Outpatient Clinic and Genetic Laboratory provide specialist tests and counselling in clinical genetics, including prenatal diagnostics, reproduction failure, marital infertility, high risk pregnancy management and genetic prophylactics and diagnostics of predispositions to selected neoplastic diseases.

Irtech Irtech Ltd. offers following groups of equipment dedicated to research and industrial applications: Spectrometers for molecular and elemental aanalysis including Raman, FT-IR, XRF, LIBS, GC/MS, NIR as portable and on-line devices; Systems and accessories for crystallography studies: SAXS/WAXS/BioSAXS, X-ray sources (incl. MetalJet), spectrometers, mirrors, detectors, power sources etc.; Imaging and analytical devices for nanomaterials verification: AFM, MFM, STM, hybrid systems like Raman + AFM/SEM/ IR/XRF, 2xAFM, 4xAFM, BioAFM, mK AFM, aqua AFM, SEEC microscopes and lab station; Detectors and spectrometers for alpha, beta, gamma and x-rays including HPGe, CZT and standard scintiallators.

We offer tests at customer site, verification of customer samples in suppliers’ facilities, in-time delivery, installation, training and service of our equipment.

More info at our web page: www.irtech.pl

KAWA.SKA KAWA.SKA Ltd. is present on the Polish market since September 1999.

Company mission “Teach. Advise. Support”, sets the path of our actions. That’s why we constantly support customers in making decisions concerning purchase of laboratory equipment, until the moment, when we are sure our clients will reach the level of “Total Customer Satisfaction”.

Company product portfolio is focused on devices used in biology, biotechnology, molecular diagnostics and histopathology to achieve our target “ from the sample to the results”.

We operate in Poland and Central and Eastern Europe.

At present we represent in Poland companies: Leica Microsystems, Leica Biosystems, Andor, Agena Bioscience, Picoquant.

We organize trainings, seminars and workshops.

More information about company on: www.kawaska.pl

Lab-JOT Lab-JOT was funded in 1993 and for over 20 years supplies scientists and research institutes with reagents and laboratory equipment.

We represent manufacturers that provide innovative solutions and high quality products for genomics, biochemistry, cell and molecular biology research.

Our offer includes: Antibodies and related products, Restriction and modifying enzymes, molecular cloning kits, NGS, qPCR, RTPCR, Sample collection and preservation kits, DNA, RNA and protein isolation and purification kits, Kits for measuring oxidative stress, protein carbonylation, thiol status, antioxidant activity, Novel range of kits for in vitro diagnostics based on next generation sequencing.

We offer reliable, fast and professional service to support and facilitate your research goals. Our priority is customer satisfaction and we do our best to meet customers’ expectations.

6th Central European Congress of Life Sciences Eurobiotech 2017

Exhibitors 225

Labnatek

Labnatek for over 10 years is supplying cutting edge scientific equipment on Polish market. We focus on nano and biotechnology applications. Our portfolio includes advanced microscopy (holotomographic, light sheet, super-resolution, fluorescence, atomic force etc.), particle analysis, hyperspectral imaging, nanoindentation, profilometry, XPS, ESCA as well as wide variety of tools and accessories.

For more information visit our website www.labnatek.pl.

Lonza Cellab Lonza is one of the world’s leading and most trusted suppliers to the pharmaceutical, biotech and bioscience markets.

It offers:animal and human stem cells from healthy donors; animal and human primary cells from healthy and diseased donors; cell culture media, including dedicated to stem/primary cells culture; serum-free cell culture media; buffers and other reagents; cells assays; a system for efficient cells transfection scalable up to clinical applications • a system for easy 3D culture setup • a system for remote live cell monitoring.

• LAL and rFc tests for endotoxin detection.

For Poland, contact distributor: CELLLAB, tel. 781120300, kontakt@celllab.pl, www.celllab.pl

LPP Equipment LPP Equipment Warsaw is a part of the LPP Group Switzerland. Joining LPP Group (LPP stands for Lab, Pilot and Process) we extended our expertise into the fields of Pilot and Process equipment, which is also reflected in our product portfolio. The aim of the company is to guarantee the highest global standards of offered equipment, which enables its users to reach the best possible results. Besides we provide advice and support in choice of equipment, we run training courses on operation and maintenance of equipment and also provide warranty and non-warranty service. Regular trainings by manufacturers allow our sales representatives and service engineers to guarantee quick and complex assistance, even in the most complicated projects or repairs.

Nencki Institute Nencki Institute is the largest non-university biological research center in Poland. High quality of research, excellent publication record, and strong international links place the Nencki among the leading biological institutions of Central and Eastern Europe. The R&D goals of the Nencki are to challenge sophisticated and intellectually satisfying problems of human wellbeing. The main focus of research relates to novel therapies and diagnostic methods in neurological disorders, oncology, diabetes and others diseases. We also provide a wide range of services including preclinical trials, dermocosmetology research support, genetic engineering, transgenic animals production and biological imaging from microscopy to MRI levels. We appreciate the existing collaborations and we are open to new cooperation with industrial entities to bring novel products to market.

PerkinElmer, Inc. PerkinElmer is a global leader committed to innovating for a healthier world. Our dedicated team of 9,000 employees worldwide are passionate about providing customers with an unmatched experience as they help solve critical issues especially impacting the diagnostics, discovery and analytical solutions markets. Our innovative detection, imaging, informatics and service capabilities, combined with deep market knowledge and expertise, help customers gain earlier and more accurate insights to improve lives and the world around us. PerkinElmer technologies and expertise were instrumental in the development of 22 novel therapeutic drugs.

For more information please visit our website: www.perkinelmer.com 6th Central European Congress of Life Sciences Eurobiotech 2017

226 Exhibitors

Promega

With more than 1.400 employees and a portfolio of more than 3.500 products Promega belongs to one of the largest global acting Life Science research companies. Founded in 1978 in Madison, USA, Promega manufactures products covering the fields of genomics, protein and cellular analysis, drug discovery and genetic identity. Promega is a global leader in providing innovative solutions support to scientists in academic, industrial and government settings. Since 1997 the Promega GmbH in Germany, as a subsidiary of the Promega Corp. is responsible for the distribution of Promega products in Germany, Austria, Poland and Eastern Europe. A direct distribution of Promega products in Poland guarantees customers on-site support in Polish language, individual solutions and attractive prices.

Research in Germany

“Research in Germany” is an international research marketing campaign, funded by the German Federal Ministry of Education and Research (BMBF), which seeks to strengthen and expand academic R&D collaboration between Germany and international partners. The organisations involved in the campaign, the Alexander von Humboldt Foundation, the German Academic Exchange Service (DAAD), the German Research Foundation (DFG), the Fraunhofer-Gesellschaft and the BMBF International Bureau, organise joint communication activities and events which present German innovation and research in key international markets.

Contact: Internet: www.research-in-germany.org Email: research-in-germany@daad.de Newsletter: www.research-in-germany.org/newsletter

Sarstedt Sp. z o.o.

SARSTEDT Sp. z o.o.- an affiliated company of the German manufacturer of medical and research equipment. Its product portfolio includes: syringe tubes for blood collection, tubes, containers for biological material, pipette tips, Petri dishes, micro tubes, cell culture products, laboratory devices.

Contact: Blizne Łaszczyńskiego ul. Warszawska 25 05-082 Stare Babice Telefon: 022/ 722-05-43, 722-01-48, 722-95-11 Fax. 022/ 722-07-95 e-mail: info.pl@sarstedt.com www.sarstedt.com

SuperBIO SuperBIO is a Horizon2020 innovation project that aims to develop and support innovative, cross-border and cross-sectorial industrial value chains in the bio-based economy. The project is business oriented: SuperBIO selects promising value chain ideas which are submitted by companies or other industrial stakeholders and is committed to support SMEs in constructing their value chain. The project offers 10 innovation services to SMEs that are part of validated value chains.

Contact: Polish Technology Platform of Bioeconomy (PPT BG), Support and partnership in the bio-based economy (SuperBIO) Ewa Gromek, Maria Woźniak-Malczewska ewa.gromek@p.lodz.pl; maria.wozniak@p.lodz.pl Stefanowskiego 4/10, 90-924 Lodz, Poland; 4842 631 39 09 www.h2020-superbio.eu 6th Central European Congress of Life Sciences Eurobiotech 2017

Exhibitors 227

Sysmex Polska

Sysmex Corporation (Japan) has been producing and selling innovative diagnostic equipment as well as IT technology and automation for medical laboratories for more than 40 years. With its products in hematology, coagulation, urinalysis and laboratory software, Sysmex is one of the leading in-vitro diagnostic companies. The company mission is: “Shaping the advancement of healthcare”. Sysmex is investing in life science technologies, flow cytometry and molecular oncology. Since many years SYSMEX brand is synonymous with innovation and reliability of the products and services. www.sysmex.pl

Thermo Fisher Thermo Fisher is the world leader in serving science. Our mission is to enable our customers to make the world healthier, cleaner and safer. We help our customers accelerate life sciences research, solve complex analytical challenges, improve patient diagnostics and increase laboratory productivity. Through our premier brands – Thermo Scientific, Applied Biosystems, Invitrogen, Fisher Scientific and Unity Lab Services – we offer an unmatched combination of innovative technologies, purchasing convenience and comprehensive support. For more information, please visit www.thermofisher.com

Tigret TIGRET Sp. z o.o. is a distributor of cytotoxicity tests (single- and multi-parameter) LDHe, NR, SRB, XTT and its combination, genotoxicity tests UmuC Easy, mutagenicity tests Ames MPF, Ames II, endocrine disruptors tests XenoScreen YES/YAS and XenoScreen XL YES/YAS. We also distribute fresh or frozen human skin with accessories and alternative skin test Corrositex. There are many ecotoxicity tests in our offer (Toxkit and Microtox), based on bacteria, protozoa, crustacean, rotifers, algae, duckweed, seeds.

Contact: TIGRET Sp. z o.o.

Ul. Warszawska 27, 02-495 Warszawa www.tigret.eu

TK BIOTECH TK BIOTECH company as a distributor of high quality laboratory equipment and reagents for research centers actively participates in development of biotechnology leading to new discoveries in research and diagnostics. We provide modern equipment and systems for researchers that significantly increase research capabilities while reducing costs.

We supply laboratory equipment and reagents to most important scientific centers in Poland.

Our position was built on collaboration and reliability. Our team consists of specialists in the field of molecular biology, biotechnology, biochemistry and biology, so we are able to provide our customers with professional consulting, support and assistance in choosing optimal solutions.

We provide: consulting and specialist support for our clients, application trainings, professional technical service.

6th Central European Congress of Life Sciences Eurobiotech 2017

228 Exhibitors

TriMen Chemicals TriMen Chemicals â&#x20AC;&#x201C; 20 years of experience in medicinal chemistry at your service.

REAGENTS We offer not only chemical and biological reagents but also silica gels, TLC plates, NMR solvents and many more.

CUSTOM SYNTHESIS If the desired compound is not available we can try to synthesize it in our labs.

COOPERATION If you do the research, look for new pharmaceuticals or you need active compounds with unique structures, we encourage you to cooperate with us in the field of synthesis or joint research.

COMMERCIALIZAITON We can evaluate your ideas and products, and try to find the best way to succeed.

Contact: phone (+48) 733-874-636 sprzedaz@trimen.pl

WTS Rzecznicy Patentowi WTS Patent Attorneys The WTS partnership (www.wtspatent.pl) is a patent attorneys practice whose goal is to provide aid in all matters connected with the protection of intellectual property rights. We have specialised mainly in the protection of inventions in the areas of biotechnology, chemistry, pharmacology and medicine, conducting prior art and patentability searches, and trade mark protection. We are licensed to conduct IP cases before the Polish Patent Office and Polish courts and their counterparts in the European Union, i.e. EPO, EUIPO, WIPO.

6th Central European Congress of Life Sciences Eurobiotech 2017

YOUR MODULAR

ONE-STOP-SHOP FOR BIOPHARMACEUTICALS

Drug substance and Fill&Finish • Own CHOBC® platform • mABS • New AB formats

CELL LINE DEVELOPMENT (SHAKE FLASKS)

• Proteins ANALYTICAL DEVELOPMENT

LAB-SCALE PROCESS DEVELOPMENT USP

Meet us at: CPHI Frankfurt, 24-26 October

LAB-SCALE PROCESS DEVELOPMENT DSP

FORMULATION DEVELOPMENT

Bio Europe Berlin, 6-8 November

PILOT MANUFACTURING DEVELOPMENT (50-1000L)

GMP MANUFACTURING (2000L)

www.polpharmabiologics.com onestopshop@polpharma.com

COMMERCIAL GMP MANUFACTURING

(up to 12×2000l)

AgroBioTop – nagroda dla młodych uczonych, których uznane dokonania z zakresu biotechnologii przyczyniły się do rozwoju nauk rolniczych i wnoszą wybitny wkład w rozwój rolnictwa.

Po raz pierwszy zostanie przyznana w 2017.

Więcej informacji i regulamin na stronie Komitetu Biotechnologii Polskiej Akademii Nauk.

AgroBioTop – an award for young scientists whose recognised achievements from the scope of biotechnology gave rise to the development of agricultural sciences and significantly contribute to the agricultural development.

The prize will be awarded for the first time in 2017.

More information and regulations can be found at the website of the Committee of Biotechnology of the Polish Academy of Sciences.

www.kbiotech.pan.pl

When proteins matter.

Quickly and accurately analyze binding affinity, protein stability and sample quality with just a tiny amount of sample.

nanotempertech.com

Narodowe Centrum Badań i Rozwoju to agencja wykonawcza Ministra Nauki i Szkolnictwa Wyższego finansująca nowatorskie projekty przedsiębiorców i naukowców o potencjale rynkowym oraz projekty uczelni służące podnoszeniu kompetencji studentów i kadry dydaktycznej.

Centrum wspiera innowatorów, łączy naukę z biznesem, pomagając „od pomysłu do przemysłu” na wszystkich etapach realizacji projektów.

Współfinansując prace B+R „ubezpiecza” ryzyko związane z ich realizacją.

NCBR

Działalność NCBR finansowana jest ze środków skarbu państwa oraz funduszy Unii Europejskiej.

W perspektywie finansowej 2014-2020 NCBR pełni funkcję Instytucji Pośredniczącej w programach operacyjnych Inteligentny Rozwój oraz Wiedza Edukacja Rozwój, w ramach których realizuje obecnie ponad 30 programów dedykowanych konkretnym grupom odbiorców: przedsiębiorcom, uczelniom, jednostkom naukowym, konsorcjom naukowo-przemysłowym czy inwestorom.

W ofercie NCBR znajdują się programy horyzontalne, programy sektorowe oraz programy międzynarodowe, m.in. „szybka ścieżka”, BRIdge Alfa, INNONEUROPHARM i ERA CoBioTech.

Congress for 4 000 participants?

International Exhibition and Convention Centre EXPO Krakow

Targi w Krakowie PCO&DMC provides full service in organizing events.

Office Targi w Krakowie Ltd. Galicyjska 9 Street, 31-586 Krakow tel. +48 12 644 59 32, 644 81 65 biuro@targi.krakow.pl

www.targi.krakow.pl

â&#x201C;Ś

www.expokrakow.com

by

Targi w Krakowie Ltd. professional conference organizer operating in Krakow and throughout Poland 20 years’ experience in the meetings industry

Professional Congress Organizer congress budget and programme planning comprehensive office management coordination of the scientific programme negotiations & supplier contracting rental and arrangement of conference venues hotel reservations transfers and information points catering services running correspondence and financial settlements with congress participants accompanying exhibitions preparing and printing promotional materials accompanying tourist and cultural programme

Targi w Krakowie Ltd. / EXPO Kraków ul. Galicyjska 9, 31-586 Kraków tel. +48 12 651 90 49, kongresy@targi.krakow.pl

www.targi.krakow.pl expokrakow.com

236

List of Exhibitors

NAME OF THE COPMANY

STAND

ABL&E-JASCO Polska

34

Accela s.r.o.

5

AGEMA/INFORS

30a

Applikon Biotechnology

28

BioIndustry

3

Bionanopark

25

Centre for Technology Transfer CITTRU, Jagiellonian University

19

Embassy of the Argentine Republic

17b

Eppendorf Poland

11

EURx

12

Fundacja Małopolskie Centrum Transferu Technologii

21

Genomed

6

Genore chromatografia

17a

Genos

13

IRTECH

4

KAWA.SKA

10

Lab-JOT

2

Labnatek

31

Lonza Celllab

7

LPP Equipment

15

Innovative Małopolska

32

Nanotemper Technologies

1

Nencki Institute

22

PerkinElmer

33

Polpharma Biologics

27

Promega

29

Research in Germany

26

Sarstedt

9

Sartorius Stedim Poland

8

SuperBIO

23

Sysmex Polska

16

The National Centre for Research and Development

18

Thermo Fisher

14

Tigret

17

TK Biotech

30

TriMen Chemicals

20

WTS Rzecznicy Patentowi

24

6th Central European Congress of Life Sciences Eurobiotech 2017

Exhibition plan

eurobiotech@targi.krakow.pl Targi w Krakowie Sp. z o.o.

31-586 Krakรณw, ul. Galicyjska 9 tel. +48 12 644 59 32, 644 81 65 fax 12 644 61 41