Spec Iss 2016

National Academy of Sciences of Ukraine
Palladin Institute of Biochemistry
The UKRAINIAN
BIOCHEMICAL
JOURnal
Volume 88, Special Issue, 2016
Bridges in Life Sciences 11th Annual

Scientific Conference Prague, Czech
Republic
April 7 – 10, 2016

Table of Contents
Organizing Committee 3
Agenda of the Bridges in Life Sciences 10th Conference 4
RECOOP HST Association 17
Croatian Medical Journal 20
Abstracts 21
Common Mechanism of Diseases 22
Cardiovascular Research in Progress 31
Nanomedicine Research 37
RECOOP Research in Progress 56
Infections and Inflammation 64
RECOOP Research in Progress 70
Experimental Works of RECOOP Members (Ukraine) 78
RECOOP Visegrad Scholarship Program 114
Conference Participants 116

Organizing Committee
USA - Cedars-Sinai Medical Center, Los Angeles, California
Edward Prunchunas
Senior Vice President for Finance and Chief Financial Officer, Cedars-Sinai Medical Center
& Chairman of the Supervisory Board of the RECOOP HST Association
Shlomo Melmed, M.D.

Senior Vice President, Academic Affairs, Dean of the Medical Faculty, Cedars-Sinai Medical
Center
Sandor G. Vari, MD
Director, International Research and Innovation Management Program, Cedars-Sinai Medical
Center & President of the RECOOP HST Association
RECOOP HST Association
Veronika Puska, Grant and Project Manager, RECOOP HST Association
Czech Republic
Jan Pitha, Head of Laboratory for Atherosclerosis Research, Centre for Experimental
Research, Institute for Clinical and Experimental Medicine (IKEM)
Editor: Sandor G. Vari, M.D.

Agenda
April 8, 2016 (Friday)

Congress Hall
8:30 – 9:00 Message from the President of RECOOP

Sandor G. Vari, Director, International Research and Innovation Management
Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA & President of
Message from the 35th President of the United States on Friday, January 20, 1961
“My fellow Americans, ask not what your country can do for you, ask what you can do for
your country.”
Message from the President of RECOOP HST Association on April 8, 2016

My fellow RECOOP members, ask not what RECOOP can do for you, ask what you can do
for RECOOP.
9:00 – Keynote lecture:
9:00 – 9:15 RECOOP Project Common Mechanism of Diseases

Diet induced obesity and impaired glucose metabolism in elderly rats
treated with Liraglutide (Victoza) and Metformin.
Robert Gaspar, Department of Pharmacodynamics and Biopharmacy, Faculty
of Pharmacy, University of Szeged, Hungary
9:15 – 9:25 Young Scietists Contribution – Electronic Interactive Poster Presentation

Five-minute power point presentation
Glucose and insulin tolerance test in obese, elderly rats with impaired
glucose metabolism treated with Liraglutide (Victoza) and Metformin
Kalman Szucs, Department of Pharmacodynamics and Biopharmacy, Faculty
of Pharmacy, University of Szeged, Hungary
Plenary Session: Common Mechanism of Disease

Oral presentation is max 15 minutes with all tolerance after 15 minutes no tolerance.
Electronic Interactive Poster Presentation EIPP = 5-minute power point presentation and 5-
minute discussion.
Posters in Conference Room 5
Chairs:
Ines Drenjančević, Josip Juraj Strossmayer University of Osijek, Croatia
Jan Pitha, IKEM, Prague, Czech Republic
Elizabeta Has-Schön, Josip Juraj Strossmayer University of Osijek, Croatia
9:25 – 9:40 Alteration of inflammatory markers and SSAO activity in diet

induced diabetic, obese elderly rats treated with Liraglutide (Victoza) and Metformin.
Tamás Tábi
Department of Pharmacodynamics, Faculty of Pharmacy, Semmelweis University, Budapest,
Hungary
9:40 – 9:55 Changes in leptin, adiponectin, insulin and gonadal steroid
receptors’ response in the brain of diet induced diabetic, obese elderly rats’ brain
treated with Liraglutide (Victoza) and Metformin.
Marija Heffer & Senka Blazetic
Department of Medical Biology, School of Medicine, Josip Juraj Strossmayer University of
Osijek, Croatia
9:55 – 10:00 Degenerative changes in the brain of diet induce diabetic, obese elderly
rats treated with Liraglutide (Victoza) and Metformin.
Srećko Gajović
Professor of Histology and Embryology, University of Zagreb School of Medicine, Croatia
10:00 – 10:15 Markers of Blood Oxidative Stress and Antioxidative Enzymes Activity in
Obese Diabetic Elderly Rats Treated with Metformin or Liraglutide
Ines Drenjančević
Department of Physiology and Immunology, Faculty of Medicine, University J. J.
Strossmayer Osijek, Croatia
10:15 – 10:30 Fatty acid changes in plasma, visceral and subcutaneous adipose tissue in
diabetic, obese elderly rats treated with Liraglutide (Victoza) and Metformin.
Eva Nagy
Doctorate School of Animal Husbandry, Faculty of Agriculture and Nutritional Sciences,
University of Debrecen, Hungary
10:30 – 10:45 Renal pathology alterations in diet induced diabetic, obese elderly rats
treated with Liraglutide(Victoza) and Metformin.
Peter Boor
Institute of Pathology and Department of Nephrology, RWTH University Aachen, Germany
10:45 – 11:00 Changes in leptin and adiponectin receptors and myocytes’ contractility
of uterus in diabetic, obese elderly rats treated with Liraglutide (Victoza) and
Metformin.
Robert Gaspar, Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy,
University of Szeged, Hungary
Bone density change in diabetic, obese elderly rats treated with Liraglutide (Victoza)
and Metformin. (only read in the abstract book)
Zora Krivosikova, Department of Clinical and Experimental Pharmacotherapy, Slovak
Medical University, Medical Faculty, Bratislava, Slovak Republic
11:00 – 11:30 Coffee Break Conference Room 3

Arts and Sciences Exhibits’ Opening Ceremony
Sandor G. Vari, Cedars – Sinai Medical Center, Los Angeles, CA, USA
Julia Mallasz, Semmelweis Publishing & Multimedia Studio, Budapest, Hungary
11:30 – 11:45 Panel discussion Common Mechanism of Disease

Moderators:
Robert Gaspar, Faculty of Pharmacy, University of Szeged, Hungary
Tamás Tábi, Semmelweis University, Budapest, Hungary
11:45 – 13:00 Cardiovascular Research in Progress
Chairs:
Eva Szoko, Semmelweis University, Budapest, Hungary
Oksana Zayachkivska, Danylo Halytsky Lviv National Medical University, Ukraine
Oral Presentation
11:45 – 12:00 Cardiovascular risk factors and their impact on vascular parameters in
women are modified by reproductive status and smoking, longitudinal study.
Jan Pitha,
Center for Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague,
Czech Republic
12:00 – 12:15 Shared decision making in life style and nutrition for intervention in woman
with risk factors in cardiovascular health
Slavica Juric Petricevic
Department of Emergency Medicine in Split, Department of Family Medicine, University of
Split School of Medicine, Split, Croatia
Electronic Interactive Poster Presentation

12:15– 12:25 Influence of acute and intermittent hyperbaric oxygenation on the

reactivity of the blood vessels in healthy Sprague-Dawley rats
Zrinka Mihaljevic
Department of Physiology and Immunology, Faculty of Medicine Josip Juraj Strossmayer,
University of Osijek, Croatia.
12:25 – 12:35 Comprehensive analysis of ischemic conditioning techniques in a porcine

model of acute myocardial infarction
Andras Makkos
Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest,
Hungary
12:35 – 12:45 The Role of Oxidative Stress in Development of Impaired Vascular

Response Under the Influence of High Salt Intake in Sprague-Dawley Rats
Anita Cosic
Department of Physiology and Immunology, Faculty of Medicine University of J.J.
Strossmayer in Osijek, Osijek, Croatia
12:45 – 13:00 Evaluation of putative cannabinoid ligands as modulators of

neurodegeneration
Yulia Senkiv
Department of Regulation Cell Proliferation and Apoptosis, Institute of Cell Biology NAS of
Ukraine, Lviv, Ukraine
13:00 – 14:00 Lunch Break

14:00 – 16:00 Nanobiomedicine Research
Chairs:
Rostyslav Stoika, Institute of Cell Biology, NASU, Lviv, Ukraine
Tatiana Borisova, Palladin Institute of Biochemistry NASU, Kyiv, Ukraine
Artur Podhorodecki, Wroclaw University of Technology, Poland Wroclaw
Srecko Gajovic, University of Zagreb School of Medicine, Croatia
Oral presentations
14:00 – 14:15 Research activity at Institute of Cell Biology, NAS of Ukraine:
achievements, problems and perspectives of development within RECOOP-HST
Association.
Rostyslav R. Stoika
Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology
(ICB), NAS of Ukraine, Lviv, Ukraine
14:15 – 14:30 Synthesis and surface functionalization of inorganic nanocrystals for their
biomedical applications
Artus Podhorodecki
Department of Experimental Physics, Wroclaw University of Technology, Wrocław, Poland
14:30 – 14:45 Biocompatibility and stem cell labelling properties of maghemite

nanoparticles
Srecko Gajovic
Croatian Institute for Brain Research, University of Zagreb School of Medicine, Zagreb,
Croatia
14:45 – 15:00 S, N - containing carbon dots: fluorescent properties and application in

cells experiments
Mariia Dekaliuk
Lab. of Nanobiotechnologies, Palladin Institute of Biochemistry of the NAS of Ukraine, Kiev
EIPP – Electronic Interactive Poster Presentations

15:00 – 15:10 Neurotoxic properties of carbon dots synthesized from sulphur-containing

carbohydrate precursor
Maryna Dudarenko
Department of Neurochemistry and Lab.of Nanobiotechnology, Palladin Institute of
Biochemistry, National Academy of Sciences of Ukraine
15:00 – 15:20 CdSe/CdS quantum dots functionalization by d-penicillamine

Anna Lesiak
15:20 – 15:30 Synthesis of Cd-based core shell nanocrystals for bio-imaging

Maciej Chrzanowski
15:30 – 16:00 Panel Discussion: Future of RECOOP Nanobiomedicine Research
Moderators:
Tatiana Borisova, Palladin Institute of Biochemistry NASU, Kyiv, Ukraine
Artur Podhorodecki, Wroclaw University of Technology, Poland Wroclaw
16:00 – 17:00 Coffee Break

Arts and Sciences Exhibit Conference Room 3
RECOOP Play Room Conference Room 2
Each artist – scientist has to introduce the artwork presented at the Conference
17:00 – 19:30 RECOOP Research in Progress – Young Scientists Forum
Chairs
Robert Gaspar, Faculty of Pharmacy, University of Szeged, Hungary
Tamás Tábi, Semmelweis University, Budapest, Hungary
Marija Heffer, Josip Juraj Strossmayer University of Osijek, Croatia
Tibor Ertl, University of Pecs, Hungary
Oral presentation
17:00 – 17:15 Sex-specific oxidative-antioxidative status in the liver of the chronically

and acutely stressed rats
Elizabeta Has-Schön for Rosemary Vuković
Department of Biology, University J. J. Strossmayer of Osijek, Osijek, Croatia;
17:15 – 17:30 Search for New Antifibrotic Agents among (amino)iminothiazolidinones

Danylo Kaminskyy
Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv
National Medical University, Ukraine;
17:30 – 17:40 Birth outcome yearly dustrubution at Department of Gynecology and

Obstetrics, University Hospital Center Osijek
Andrijana Müller
Department of Gynecology and Obstetrics, University Hospital Center Osijek, Faculty of
Medicine, J.J. Strossmayer University of Osijek, Croatia
Young Scientists Forum
EIPP – Electronic Interactive Poster Presentations

17:40 – 17:50 Metformin may influence neurodegeneration and neuroinflammation in

Sprague Dawley rats on high fat/sugar diet in a sex specific manner
Marta Balog and Milorad Zjalic
Faculty of Medicine, J.J. Strossmayer University of Osijek, Croatia
17:50 – 18:00 The importance of autophagy in the antiapoptotic effect of resveratrol

Fruzsina Bagamery
Department of Pharmacodynamics, Faculty of Pharmacy, Semmelweis University, Budapest,
Hungary
18:00 – 18:10 Application of THY1 – YFP neural stem cells in regenerative neuroscience
Ivan Alic
Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine,
University of Zagreb, Zagreb, Croatia
18:10 – 18:20 ARHGAP25 Rac-GAP has an important role in autoantibody-induced

model of rheumatoid arthritis
Tim F. Svanya
Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary
18:20 – 18:30 The use of formalin-fixed paraffin-embedded samples in early diagnosis of

diseases
Boglarka Donczo
Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen,
Hungary
18:30 – 18:40 The role of gamma aminobutyric acid (GABA) in the regulation of
endocervical epithelium secretory activity
Marta Skelin
Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Croatia
18:40 – 18:50 Analyses of impact circadian dysfunctions on the occurrence of

disautotonia in medical student population
Maryana Zvir
Physiology Department, Lviv National Medical University, Lviv, Ukraine
18:50 – 19:00 Plasma membrane Ca2+-pump selective regulators – calix[4]arene C-90

and imidazo[1,2-a]azepine – change parameters of uterus smooth muscles contraction
Iuliia Mazur
Department of Muscle Biochemistry, Palladin Institute of Biochemistry of the National
Academy of Sciences of Ukraine, Kyiv, Ukraine
19:00 – 19:10 Does endogen PACAP have any role in the development of retinopathy of
prematurity?
Timea Kvarik
Departments of Anatomy, Pacap-Lendulet Research Group and Obstetrics and Gynecology,
University of Pecs, Hungary
19:10 – 19:20 The comparison of hydrogen sulfide NSAID derivates in the modulation of
gastro-esophageal integrity.
Irena Pshyk-Titko
Department of Physiology, Lviv National Medical University, Ukraine
20:00 – 22:00 Dinner

April 9, 2016 (Saturday)
Congress Hall
8:30 – 9:00 Summary of the Conference’s Progress

Sandor G. Vari
9:00 – 11:00 Annual Review of the Research Activities in RECOOP

Session I
Chairs
Session I
9:00 – 9:15 Antioxidants Selenomethionine and D-Pantethine Decrease Negative Side
Effects of Doxorubicin in Nk/Ly Lymphoma-Bearing Mice
Rostyslav R. Panchuk
National Academy of Sciences Ukraine, Lviv, Ukraine
9:15 – 9:30 Ovariectomy and chronic stress lead toward leptin resistance in satiety centers
and insulin resistance in hippocampus of Sprague-Dawley rat
Vedrana Ivic
Josip Juraj Strossmayer University of Osijek, Faculty of Medicine Osijek, Department of
Medical Biology and Genetics, Osijek, Croatia
9:30 – 9:45 Diurnal variation in cholesterol 7α-hydroxylase activity is determined by

the -203A>C polymorphism of the CYP7A1 gene
Tereza Blahová
Laboratory for Atherosclerosis Research, Institute for Clinical and Experimental Medicine,
Czech Republic
9:45- 10:00 Putative anticancer potential of novel 4-thiazolidinone derivatives: toxicity

towards rat C6 glioma cells and correlation of general toxicity with balance of free
radical oxidation in rats
Lesya Коbylinska
Department of Biochemistry, Danylo Halytsky Lviv National Medical University, Ukraine
10:00 – 10:15 The effects of estrogen on the α2-adrenergic receptor subtypes in late
pregnant uterine function in vitro
Judit Hajagos-Toth
Department of Pharmacodynamics and Biopharmacy, University of Szeged, Szeged, Hungary
10:15 – 10:30 H. pylori in sedentary males is linked to higher heart rate, sympathetic
activity and insulin resistance but not inflammation or oxidative stress
Mykhailo Pliatsko
Department of Internal Medicine №1, Danylo Halytskyi Lviv National Medical University,
Lviv, Ukraine
10:30 – 10:45 Morphological and pathological response in primary systemic therapy of
patients with breast cancer and the prediction of disease free survival
Gyöngyver Szentmartoni
Semmelweis University, Department of Clinical Oncology, Budapest, Hungary

Each artist – scientist has to introduce the artwork presented
11:30 – 12:00 Panel Discussions Annual Review of the Research Activities in RECOOP
Moderators:
12:00 – 13:00 Young Scientist Forum - Infection and Inflammation
Chairs
Pavel Bostik, Faculty of Military Health Sciences, Univ. of Defence, Czech Republic
Shubhada Bopegamage, Slovak Medical University, Bratislava, Slovakia
12:00 – 12:10 Induction of cytokines by CVB2-Ohio and its mutants

Brigita Benkoova
Enterovirus Laboratory, Faculty of Medicine, Slovak Medical University in Bratislava,
Slovakia
12:10 – 12:20 Possible mechanisms involved in the development of drug resistance in

Ruk/CIN85-overexpressing MCF-7 cells
Ganna Pasichnyk
Laboratory of Cell Signaling, Palladin Institute of Biochemistry, National Academy of
Sciences of Ukraine, Kiev, Ukraine
12:20 – 12:30 QSAR Studies of 4-thiazolidinone derivatives showing antitrypanosomal

activity
Anna Kryshchyshyn
National Medical University, Lviv, Ukraine;
12:30 – 12:40 Plasmid-mediated fluoroquinolone resistance in Enterobacteriaceae

Orsolya Szabó
Institute of Microbiology, Semmelweis University, Budapest, Hungary
12:40 – 12:50 Selected concepts and investigations among thiazolo[4,5-b]pyridines as

perspective anticancer and anti-inflammatory agents
Andrii Lozynskyi
National Medical University, Lviv, Ukraine
13:00 – 14:00 Lunch Break
14:00 – 16:00 Annual Review of the Research Activities in RECOOP

Session II
Chairs
Charles F. Simmons, Cedars – Sinai Medical Center, Los Angeles, CA, USA
Iuliana Ceausu, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
Peter Boor, RWTH University Aachen, Germany
Session II
14:00 – 14:15 Relationship of soluble receptors for advanced glycation end products and
metabolic syndrome in adolescents from Bratislava region
Radana Gurecka
Institute of Molecular Biomedicine, Faculty of Medicine and Institute of Medical Physics,
Biophysics, Informatics and Telemedicine, Comenius University, Bratislava, Slovakia
14:15 – 14:30 The effects of anthocyanin-rich wheat diet on the oxidative status and
behavior of rats
Katarina Jansakova
Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava,
Slovakia
14:30 – 14:45 Interleukin 6/Wnt interactions in rheumatoid arthritis: Interleukin 6

inhibits Wnt signaling in synovial fibroblasts and osteoblasts
Khrystyna Malysheva
Institute of Cell Biology National Academy of Sciences Ukraine, Lviv, Ukraine
14:45 – 15:00 Exploiting Hydrogen Sulfide of Novel 4-Thiazolidinone Derivatives in

Cytoprotection of Small Intestine Under Indometacin-Induced Injury
Iryna Ilkiv
Department of Biochemistry, Danylo Halytsky Lviv National Medical University, Lviv,
Ukraine
15:00 – 15:15 Application of novel polymeric nanocarrier for temozolomide delivery to

glioblastoma cells
Nataliya Finiuk
National Academy of Sciences Ukraine, Lviv, Ukraine
15:15 – 15:30 Mapping of Residues of Fibrinogen Cleaved by Protease II of Bacillus thuringiensis

var. israelensis IMV B-7465
Volodymyr Chernyshenko
Palladin Institute of Biochemistry National Academy of Sciences Ukraine, Kyiv
15:30 – 15:45 Synthesis of potential antiviral agents among chromeno [4’,3’:4,5]
thiopyrano[2,3-d][1,3]thiazole-5-carboxylates
Nataliya Zelisko
15:45 – 16:00 Panel Discussions Annual Review of the Research Activities in RECOOP
Moderators:
Charles F. Simmons, Cedars – Sinai Medical Center, Los Angeles, CA, USA
Iuliana Ceausu, “Carol Davila” University of Medicine and Pharmacy,
Bucharest, Romania

Each artist – scientist has to introduce the artwork presented
17:00 – 18:00 Breakaway Sessions
Session 1 Common Mechanism of Diseases – Low grade inflammation

Congress Hall
Chairs:
Éva Szoko, Semmelweis University, Budapest, Hungary
Oksana Zayachkivska, Danylo Halytsky Lviv National Medical University, Ukraine
Oleksandr Korchynskyy, Institute of Cell Biology, NASU, Lviv, Ukraine
Tamas Tabi, Semmelweis University, Budapest, Hungary
Volodymyr Chernyshenko, Palladin Institute of Biochemistry NASU, Kyiv, Ukraine
Participants:
Anita Ćosic, Josip Juraj Strossmayer University of Osijek, Croatia
Zrinka Mihaljevic, Josip Juraj Strossmayer University of Osijek, Croatia
Marta Balog, Josip Juraj Strossmayer University of Osijek, Croatia
Vedrana Ivic, Josip Juraj Strossmayer University of Osijek, Croatia
Milorad Zjalic, Josip Juraj Strossmayer University of Osijek, Croatia
Marta Skelin, University of Zagreb School of Medicine. Croatia
Slavica Jurić Petričević, University of Split School of Medicine, Croatia
Tereza Blahová, IKEM, Prague, Czech Republic
Fruzsina Bagamery, Semmelweis University, Budapest, Hungary
Judit Hajagos-Tóth, Faculty of Pharmacy, University of Szeged, Hungary
Anita Sztojkov-Ivanov, Faculty of Pharmacy, University of Szeged, Hungary
Kálmán Szucs, Faculty of Pharmacy, University of Szeged, Hungary
Eva Nagy, University of Debrecen, Hungary
Boglarka Donczo, University of Debrecen, Hungary
Máté Szarka, University of Debrecen, Hungary
Timea Kvarik, Medical School, University of Pécs, Hungary
Radana Gurecka, Comenius University, Bratislava, Slovakia
Nazar Bula, Danylo Halytsky Lviv National Medical University, Ukraine
Maryana Zvir, Danylo Halytsky Lviv National Medical University, Ukraine
Jaroslav Pavlovskyi, Danylo Halytsky Lviv National Medical University, Ukraine
Irena Pshyk-Titko, Danylo Halytsky Lviv National Medical University, Ukraine
Khrystyna Malysheva, Institute of Cell Biology, NASU, Lviv, Ukraine
Arsenii Borysov, Palladin Institute of Biochemistry NASU, Kyiv, Ukraine
Iuliia Mazur, Palladin Institute of Biochemistry NASU, Kyiv, Ukraine
Session 2 Nanobiomedicine – Drug conjugation

Conference room 1
Chairs:
Danylo Kaminskyy, Danylo Halytsky Lviv National Medical University, Ukraine
Rostyslav Panchuk, Institute of Cell Biology, NASU, Lviv, Ukraine
Ivan Alić, University of Zagreb School of Medicine, Croatia
Lesya Kobylinska, Danylo Halytsky Lviv National Medical University, Ukraine
Participants:
Beata Zasonska, Institute of Macromolecular Chemistry AS CR, Prague, Czech Republic
Maksym Moskvin, Institute of Macromolecular Chemistry AS CR, Prague, Czech Republic
Maciej Chrzanowski, Wroclaw University of Technology, Poland Wroclaw
Anna Lesiak, Wroclaw University of Technology, Poland Wroclaw
Nataliya Finiuk, Institute of Cell Biology, NASU, Lviv, Ukraine
Julia Senkiv, Institute of Cell Biology, NASU, Lviv, Ukraine
Maryna Dudarenko, Palladin Institute of Biochemistry NASU, Kyiv, Ukraine
Mariia Dekaliuk, Palladin Institute of Biochemistry NASU, Kyiv, Ukraine
Ganna Pasichnyk, Palladin Institute of Biochemistry NASU, Kyiv, Ukraine
Session 3 Contribution of perinatal viral infections to adverse outcomes of

pregnancy in Central and Eastern Europe
Conference room 6
Chair
Participants:
Pavel Bostik, Faculty of Military Health Sciences, University of Defense, Hradec Kralove,
Czech Republic
Brigita Benkoova, Slovak Medical University, Bratislava, Slovakia
Andrijana Müller, Josip Juraj Strossmayer University of Osijek, Croatia
Linn Defensor, Cedars – Sinai Medical Center, Los Angeles, CA, USA
Veronika Puska, RECOOP HST Association, Budapest, Hungary
Session 4 RECOOP HST Association General Assembly Conference room 4
USA
Cedars-Sinai Medical Center, Los Angeles, USA
Edward Prunchunas, CSMC represented by Charles F. Simmons, Jr., MD and Sandor G.
Vari, CSMC - RECOOP
Croatia
Josip Juraj Strossmayer University of Osijek, School of Medicine, Osijek, Croatia
Ines Drenjancevic
Josip Juraj Strossmayer University of Osijek, Department of Biology, Osijek, Croatia
Elizabeta Has-Schon
University of Zagreb School of Medicine, Croatia
Srecko Gajovic
University of Split School of Medicine, Department of Family Medicine, Split, Croatia
Ivancica Pavlicevic
Czech Republic
IKEM - Institute for Clinical and Experimental Medicine, Prague, Czech Republic
Jan Pitha
Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech
Republic
Pavel Bostik
Hungary
Absent
Tibor Ertl
Robert Gaspar
Wroclaw University of Technology, Institute of Physics, Wroclaw, Poland

Absent
Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

Iuliana Ceausu
Slovak Medical University, Bratislava, Slovak Republic

Authorized representative Shubhada Bopegamage
Ukraine
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kiev,
Ukraine
Tatiana Borisova
Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine
Rostyslav Stoika
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Oleh Pinyazhko
18:00 – 18:40 Summary of the Breakaway Sessions 10 minutes for each session
Congress Hall
Session 1 Common Mechanism of Diseases – Low grade inflammation

Session 2 Nanobiomedicine – Drug conjugation

Session 3 Contribution of perinatal viral infections to adverse outcomes of

pregnancy in Central and Eastern Europe
Session 4 RECOOP HST Association General Assembly

Sandor G. Vari, CSMC - RECOOP
18:40 – 19:00 Summary of the Young Scientists Competition

Sandor G. Vari, CSMC - RECOOP
Veronika Puska, RECOOP HST Association, Budapest, Hungary
19:00 – 19:30 Closing Remarks

Sandor G. Vari, Director, International Research and Innovation Management
Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA & President of
19:30 Dinner
April 10, 2016 Departure

The Association for Regional Cooperation in the (RECOOP) Fields of Health, Science
and Technology (HST)
Short Name: RECOOP HST Association
The Regional Cooperation for Health, Science and Technology (RECOOP HST) Consortium,
led by Cedars-Sinai was formed in 2006 and transformed into Association in 2012 includes 17
universities and academic organizations from nine countries in Central and Eastern Europe
(Croatia, Czech Republic, Hungary, Poland, Romania, Slovakia and Ukraine), Denmark and
USA. The Association is a structured, functional and working research organization. According
to its mission statement: "The RECOOP HST Association explores and enhances LOCAL
scientific outputs of the partner organizations, creates critical mass of scientifically sound
innovative research at REGIONAL level and exploits the research outcomes at GLOBAL level
to improve the prevention and treatment of major public health problems."™
RECOOP builds multinational, multidisciplinary collaborations, and assists, coordinates the

research activities of the eighteen research groups are the Cedars-RECOOP Research Centers
(CRRC). Implementations of RECOOP’s strategic goals enable diverse talents geared towards
integration of new knowledge derived from multispecialties to investigate Gender Differences
(GD) in Common Mechanism of Diseases (CMD). In the CRRCs, researchers study genetic
preconditions and the common mechanisms in molecular biology provide information on up-
regulation or activation and down regulation or suppression of genetic codes by risk factors in
nutrition, lifestyle (smoking, and alcohol, drug, mental and physical abuse), acute and chronic
stress.
The scientific quality of the CSRRC’s research reflected in the RECOOP’s Annual Scientific
Review Journals Biopolymers & Cell and Croatian Medical Journal.
Biopolym Cell. 2014;30(5 supplementary):1-4.

http://www.biopolymers.org.ua/pdf/30/5suppl/001/biopolym.cell-2014-30-5suppl-001-en.pdf.
Biopolym Cell. 2014;30(5 supplementary):5-44.
http://www.biopolymers.org.ua/pdf/30/5suppl/005/biopolym.cell-2014-30-5suppl-005-en.pdf.
Biopolym Cell. 2013;29. http://www.biopolymers.org.ua/content/29/.
Croat Med J. 2014 Jun,55(3):181-286. http://www.cmj.hr/default.aspx?id=12345&issue=yes
Croat Med J. 2015 Apr,56(2):75-176. http://www.cmj.hr/default.aspx?id=12482&issue=yes.
Croat Med J. 2016 Apr, 57 (2) 87 -214 http://www.cmj.hr/default.aspx?id=12631&issue=yes
The RECOOP HST Association organizes annually the Bridges in Life Sciences Conferences
to review the scientific progress in the Association. During the Bridges in Life Sciences Annual
Meeting the Scientific Advisory Board selects the top ten young scientists.
The top ten young scientists selected during the Bridges in Life Sciences Annual Conferences
have the opportunity to apply for International Visegrad Fund (IVF) Scholarship and
receive the RECOOP Young Scientists Matching Fund. Also for the Bohdan Malaniak
CSMC - RECOOP Young Scientists Research Award. The Visegrad Scholarship is the
Visegrad Four European Macro-Region’s Fulbright Program. Therefore, it could be important
to link the Visegrad Scholarship and the Fulbright Foreign Student Program. CSMC –
RECOOP Research Centers (CRRC) are the Center of Excellences of the RECOOP HST
Association. They host young scientists, Ph.D. students with CSMC – RECOOP (IVF – CSMC
- RECOOP) Scholarship. The RECOOP HST Association Scientific Advisory Board selects
the young scientists who could apply for IVF – CSMC - RECOOP Scholarship. The selected
young scientists (preferably Ph.D. students) will spend maximum four semesters receiving:
€2,300 / semester and the corresponding host universities/institutes will receive
€1,500/semester/scholar. The host CRRC will get €1,000 for laboratory expense and
consumables from CSMC – RECOOP HST Association.
The Bohdan Malaniak CSMC - RECOOP Young Scientists Research Award for
preclinical and clinical studies.
The host and appointing organizations can be basic or translational research organizations
nevertheless they should have an assisting clinical organization with clinical practice. The
research project and the performed lab tests and measurements must have clinical relevance.
The maximum requested amount is 1,500 USD but it will only be paid if the applicant provides
and proves matching found for the same amount. The submission date July 27, 2016, the
application should be sent to Sandor G. Vari, MD. Please follow strictly the word counts in the
template!
RECOOP would like to provide more opportunities for young scientists, therefore agreed
with Korányi Frigyes Science Dormitory, Semmelweis University Budapest to select the
top abstracts were submitted to the call of Korányi Frigyes Scientific XXI Forum of
University Semmelweis, Budapest, Hungary.
The Forum was on March 10 -11, 2016, and aimed to provide opportunities for young
researchers, clinicians, pharmacists and students involved in Students’ Scientific Association,
while promoting a scientific contact between various disciplines within our university.
The submitted 22 English abstracts were reviewed by RECOOP Review Board for the Bridges
in Life Sciences 11th Annual Conference, Prague, Czech Republic. This peer –review is
considered by RECOOP as a pre-selection, and will be finalized after the outcome of the
Koranyi Science Forum 2015. All abstracts were reviewed by minimum 2 RECOOP experts.
Those who had no ECA - Ethical Committee or IACUC – Institutional Animal Care and Use
Committee approvals and could not or did not provide the approval were excluded from the
final selection.
Only those received RECOOP invitation for the Bridges Conference in Prague who were
already selected by RECOOP Review Board and at the same time ranked first or second in
their session during the 2016 Koranyi Science Forum.
Andras Makkos
Medical Student VI
Department of Pharmacology and Pharmacotherapy, Semmelweis University,
Budapest, Hungary
Tim Frederik Svanya

Medical Student V.
Department of Physiology, Faculty of Medicine, Semmelweis Universtiy
Orsolya Szabo
Medical Student V.
Institute of Microbiology, Faculty of Medicine, Semmelweis University
The Head of the Korányi Frigyes Scientific XXI Forum of Organizing Committee Klára Alíz
Stark, 3rd year Medical Student, Faculty of Medicine, Semmelweis University, Budapest,
Hungary received an invitation as an appreciation of her hard work she is displaying her art
work in the Arts and Sciences Exhibit.
The RECOOP HST Association with Korányi Frigyes College for Advanced Studies,
Semmelweis University, Budapest, Hungary (http://harsfa.semmelweis.hu/recoop-fkcas-
lsiiaw.php ) announced the first annual Art and Sciences Competition. The main goal of the
RECOOP Annual Art and Sciences Competition is to initiate a creative working relation
between RECOOP young scientists and artists to create artworks from life science and
medical images and communicate the beauty of the living organism.
Dr. Sandor G. Vari, MD

Director, International Research and Innovation Management Program
Cedars-Sinai Medical Center &
President of the RECOOP HST Association
6420 Wilshire Blvd., Ste. 300, Los Angeles,
CA 90048-5502, USA
Office 1: 1 323-866-8122; 1 323-866 -6824
Office 2: 1 818 904 1954
Mob: 1 818 398-2642
E-mail: vari@cshs.org
The Croatian Medical Journal (April 2016, www.cmj.hr)
published now the third RECOOP thematic issue, this time
dedicated to the RECOOP Bridges 11th Annual Scientific
Conference in Prague, Czech Republic.
The mission of Croatian Medical Journal (CMJ) was from its very beginning to support
the biomedical research in emerging countries. CMJ first aimed to help Croatian research
community to publish their high quality papers in a friendlier way, but very soon, the scope
was extended to the neighboring countries, Central East Europe, Asia and Africa. Currently,
CMJ is orientated to all researchers having low visibility either due to their geographical
location or due to their professional status.
The major tool that CMJ is offering to this group of emerging researchers is a specific
Author-Helpful Policy. The submitted articles were judged according to their potential and the
dedicated team of professionals is there to assist the authors. The team consisted of the
members of CMJ editorial group and CMJ associates were engaged to help authors to present
their research according to the high standards of international publishing. Such policy had
many benefits. The first were for the Journal itself, as it helped to establish a core of knowledge
under CMJ umbrella which although offered internationally was indeed a national asset.
Croatian experts in statistics, manuscript editing, scientific language, and manuscript
production were established. The criteria of manuscript quality subsequently raised and the
CMJ Impact Factor increased to its maximum of 1.8, which made it to rank among first 10
general medical journals.
The effects on global community were as well visible. Many young researchers were
able to publish their first contributions in CMJ, many other national journals followed the suite
and the strength of the previously unrecognized research became obvious and accessible to the
global scientific community.
CMJ has published the second thematic issue made by scientists from RECOOP
institutions. We can say that there is already a small tradition to offer and share CMJ virtues
with the RECOOP scientists and to collaborate successfully.
We at CMJ strongly believe that RECOOP and CMJ share their basic mission. By
increasing the across border collaboration in the Central and Eastern Europe the undiscovered
opportunities start to shine and bring benefits to the national, regional and global community.
Indeed, the scientific community should continuously discover multinational and
multidisciplinary collaborations, and should be nourished, developed and properly presented.
The whole venture to create this thematic issue was an excellent international team
effort, were many small pieces were arranged together to identify, develop and present the
excellence growing under RECOOP umbrella. Therefore, we wish that the published papers in
the thematic issues are a clear sign of growing common RECOOP activities, and a long-term
establishment of regional excellence, excellence that CMJ wants to be part of.
Prof. Srecko Gajovic

Editor-in-Chief
Croatian Medical Journal
CMJ is an international peer-reviewed Diamond Open Access journal published six times per
year. The CMJ uses the Diamond Open Access model. This means that there are NO author
processing fees and NO fees to access the published papers. The free access is available on our
web page, www.cmj.hr, also on PubMed Central. Impact factor of CMJ for 2013 is 1.373.
The RECOOP thematic issue is as well fully accessible at journal web page www.cmj.hr
Abstracts
Abstracts of Common Mechanism of Diseases
Diet induced obesity and impaired glucose metabolism in elderly rats treated with
Liraglutide (Victoza) and Metformin.
1Gaspar R, 1Ducza E, 1Seres AB, 1Szűcs KF, 1Hajagos-Tóth J, 1Sztojkov-Ivanov A, 1Tiszai Z
1Minorics R, 1Schelz Zs, 1Bóta J, 1Csányi A, 2Vari SG
1Department of Pharmacodynamics and Biopharmacy, University of Szeged, Hungary
2International Research and Innovation in Medicine Program, Cedars - Sinai Medical Center,
Los Angeles, CA, USA and Regional Cooperation for Health, Science and Technology
(RECOOP HST) Association, Debrecen, Hungary
Corresponding author: Robert Gaspar gaspar@pharm.u-szeged.hu
Key words: high calorie diet, obesity, glucose tolerance, elderly rat, liraglutide, metformin
Introduction: Obesity is a major public health concern. Our aim was to develop an animal
model for obesity in aging rats by diet. We intended to measure -among others- the obesity-
induced changes in body weight, glucose metabolism, cognitive and renal functions and uterine
contraction.
Methods: Male and female rats (44-weeks-old) were put in 4 groups containing 8 male and 8
female rats each: 1. Standard diet (SD); 2. High fat-high sucrose diet (HFHSD); 3. HFHSD+
metformin (50 mg/kg/day); 4. HFHSD+liraglutide (0.3 mg/kg/day) from 51 weeks for 14
weeks of ages. Obesity was induced by HFHSD by from 45 weeks of age for 20 weeks.
Animals were sacrificed at their 64 weeks of age. Obesity was evaluated by body weight and
waist circumference; glucose metabolism was measured by glucose tolerance and insulin
sensitivity tests, cognitive function was tested by water maze, renal function was measured by
urine tests, uterine contractility was measured in organ bath in vitro.
Results: HFHSD induced obesity. The gain in weight was significantly reduced by liraglutide
in both sexes, while metformin slightly reduced the bodyweight only in males. Obesity induced
an impaired glucose metabolism in both sexes that was reduced by metformin and liraglutide.
A slight reduction in cognitive function was detected in male rats. The majority of the urine
tests shown inconsistency, slight increases in microalbuminuria (HFHSD males) and glucose
(HFHSD females) were detected.
Discussion and Conclusion: Long-term HFHSD induced obesity and impaired glucose
metabolism in elderly rats and affected cognitive and renal functions. Liraglutide was more
effective to restore the consequences of obesity. Our animal model seems to be a highly
identical experimental system to induce the process and consequences of human obesity.
Sources of Funding: This work has been supported by RECOOP HST grant.
Acknowledgement: The study was supported by Cedars Sinai Medical Center’s International
Research and Innovation in Medicine Program, the Association for Regional Cooperation in
the Fields of Health, Science and Technology (RECOOP HST Association) and the
participating Cedars – Sinai Medical Center - RECOOP Research Centers (CRRC).
Ethical Committee Approval: All experiments were carried out with the approval of the
Hungarian Ethical Committee for Animal Research (registration number: IV/3796/2015).
Glucose and insulin tolerance test in obese, elderly rats with impaired glucose
metabolism treated with Liraglutide (Victoza) and Metformin
1Szűcs KF, 1Ducza E, 1Seres AB, 1Tiszai Z, 2Vari SG, 1Gaspar R,
1Departmentof Pharmacodynamics and Biopharmacy, University of Szeged, Hungary
2International
Research and Innovation in Medicine Program, Cedars - Sinai Medical Center,
Key words: obesity, glucose tolerance, elderly rat, liraglutide, metformin
Introduction: The increasing prevalence of obesity is a major public health concern. Obesity
is responsible for the development of diabetes mellitus, arteriosclerosis, hypertension and
several other diseases. Our aim was to develop an animal model for obesity in aging rats by
diet. We intended to measure the obesity-induced changes in glucose metabolism and insulin
sensitivity.
Methods: Male and female rats (44-weeks-old) were put in 4 groups containing 8 male and 8
female rats each: 1. Standard diet (SD); 2. High fat-high sucrose diet (HFHSD); 3. HFHSD+
metformin (50 mg/kg/day); 4. HFHSD+liraglutide (0.3 mg/kg/day) from 51 weeks for 14
weeks of ages. Obesity was induced by HFHSD by from 45 weeks of age for 20 weeks. Glucose
tolerance was measured at 44, 50, 57 and 63 weeks of age, while insulin sensitivity test was
carried out at 64 weeks of age with OneTouch® UltraMini® Glucose Meter (Milpitas, CA,
USA).
Results: HFHSD induced impaired glucose tolerance after 5 weeks of diet that were increased
gradually every month. In females, the aging itself induced worsening of glucose tolerance.
Liraglutide restored the worsening glucose tolerance after 7 weeks of treatments while
metformin was able to moderate the process, only. However, the glucose tolerance increasing
effect of the liraglutide was disappeared on 13th week of treatment while metformin maintained
its moderating effect especially in females. The insulin sensitivity test before the sacrifice
revealed that liraglutide slightly increased the sensitivity in males, while metformin was
effective in females.
Discussion and Conclusion: Long-term HFHSD induced impaired glucose tolerance in
elderly rats. Liraglutide is able to restore the glucose tolerance while Metformin is less effective
in 7-week therapy. However, 13-week treatment caused benefit cease of liraglutide therapy.
Sources of Funding
This work has been supported by RECOOP HST grant.
Acknowledgement:
The study was supported by Cedars Sinai Medical Center’s International Research and
Innovation in Medicine Program, the Association for Regional Cooperation in the Fields of
Health, Science and Technology (RECOOP HST Association) and the participating Cedars –
Sinai Medical Center - RECOOP Research Centers (CRRC).
Alteration of inflammatory markers and SSAO activity in diet induced diabetic, obese
elderly rats treated with Liraglutide (Victoza) and Metformin
Tábi T1, Szökő É1, Gáspár R2, Heffer M3, Vári SG4…
1Department of Pharmacodynamics, Semmelweis University, Hungary
3Department of Medical Biology, University of Osijek, Osijek, Croatia
Los Angeles, CA, USA

Corresponding author: Tamas Tabi abi.tamas@pharma.semmelweis-univ.hu
Keywords: obesity, diabetes, low grade inflammation, inflammatory cytokines
Introduction: The unhealthy lifestyle is one of the major risk factors of metabolic disorders
such as obesity, insulin resistance and type 2 diabetes. Accumulation of excess adipose tissue
especially in the visceral fat depots accompanies to a low grade inflammatory state and
increased production of proinflammatory cytokines and adipokines. These mediators in turn
can interfere with insulin signaling triggering insulin resistance.
Aims: In the present study we aimed at characterizing the metabolic parameters and adipose
tissue inflammation in a high fat, high sugar diet induced model of obesity in elderly rats. Effect
of gender and antidiabetic therapies was also evaluated.
Results: Diet induced obesity was accompanied by fasting hyperinsulinemia reaching
significance only in case of male animals due to the high variance in the obese group. These
animals can be divided to two groups; ones with normal or slightly elevated insulin levels and
ones with considerable hyperinsulinemia suggesting the importance of genetic predisposition
factors. Plasma leptin levels showed a similar profile with significantly higher leptin level in
females. Adiponectin levels were unaltered.
Inflammatory cytokines (TNFα, IL-1β, IL-6) concentrations were altered mainly in the visceral
fat. Their levels were generally increased in obese male animals while females showed elevated
concentration inherently with no further increase in the obese animals. SSAO activity in the
visceral fat was unaltered and reduced in obese males and females, respectively suggesting the
enzyme plays no significant role in adipose tissue inflammation.
Antidiabetic therapies had only minor effects on the examined parameters. Liraglutide
generally showed more favorable effects compared to metformin.
Conclusion: High fat, high sugar diet induced metabolic changes characteristic to type 2
diabetes but a portion of animals showed resistance to the effect of the diet. Inflammation in
the adipose tissue of elderly females was more pronounced while unhealthy diet has higher
effect in males that is in accordance with the higher risk of postmenopausal females.
Ethical approval: All experiments were carried out with the approval of the Hungarian Ethical
Committee for Animal Research (registration number: IV/3796/2015).
Acknowledgement: This study was supported by Cedars Sinai Medical Center’s International
the Fields of Health, Science and Technology (RECOOP HST Association).
Leptin and insulin receptor expression in the brain of diet induced diabetic, obese
elderly rats/ treated with Liraglutide (Victoza) and Metformin
Senka Blažetić1, Irena Labak1, Marija Heffer2, Rober Gaspar3, Sandor Vari4
1 Department of Biology, University J. J. Strossmayer of Osijek, Osijek, Croatia;
2 Department of Medical Biology, School of Medicine, University J. J. Strossmayer of
Osijek, Osijek, Croatia;

3 Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, University of
Szeged, Eötvös u. 6. H-6720 Szeged, Hungary,

4 International Research and Innovation in Medicine Program, Cedars-Sinai Medical Center,
Los Angeles, CA, United States of America or just USA and Regional Cooperation for
Health, Science and Technology (RECOOP HST) Association, Debrecen, Hungary
Corresponding author e-mail address: mheffer@mefos.hr
Key words: obesity, diabetes, insuline resistance, leptin resistance, Liraglutide, Metformin
Introduction: Advancing age and obesity are the major risk factors for development of
diabetes type 2. Occurrence of insulin resistance in brain is named ‘diabetes type 3’ and
probably contributes to cognitive defect in Alzheimer. Anti-diabetic drugs, like Metformin and
Liraglutide, may have different capacity to treat central insulin resistance.
Method: Twenty week old male and female SD rats were divided in: control group and 3
groups fed high fat-high sugar diet (HSHFD). From fifty first week one HSHFD group was
treated with Metformin (50 mg/kg/day s.c.) and another one with Liraglutide (0.3 mg/kg/day
s.c). All groups were sacrificed at 65 weeks of age. Brain was fixed and immunostained for
insulin (IR) and leptin (ObR) receptor.
Results: Compared with control group, male rat groups on HSHFD had significantly higher
expression of IR in hippocampus, while female group had decreased level of IR (P = 0.04).
HSHFD generally reduced expression of ObR in hypothalamus. Both drugs, Metformin and
Liraglutide increased hypothalamic ObR expression during HSHFD. Liraglutide had stronger
effect in female group.
Discussion – conclusion: It is well known that insulin resistance is related to obesity. Results
indicate that there are gender differences in insulin signaling pathway. IR in brain supports
cognitive functions, including learning and memory and it decreases in aging. Based on idea
that reduced IR results with synapse loss it is possible that female group on HSHFD, more
likely than male group, develop cognitive defect. Low levels of ObR in hypothalamus in male
and female groups accompanies obesity. According to our results the HSHFD affects IR and
ObR distribution in the brain. Also, Metformin and Liraglutide had sex specific capacity to
treat central insulin resistance.
Sources of Funding: This work has been supported in part by VIF-MEFOS-1 (Faculty of
Medicine Osijek, Croatia) and by RECOOP HST grant.
Ethical Approval: All experimental procedures are conformed to the European Guidelines for
the Care and Use of Laboratory Animals (directive 86/609). They were approved by the by
Hungarian Ethical Committee for Animal Research (IV/3796/2015).
Markers of Blood Oxidative Stress and Antioxidative Enzymes Activity in Obese
Diabetic Elderly Rats Treated with Metformin or Liraglutide
Drenjancevic, Ines1; Cosic, Anita1; Vuković, Rosemary2; Kljajić, Julijana2; Heffer, Marija 3;
Gaspar, Robert 4; Vari, G Sandor 5
1- University Josip Juraj Strossmayer Osijek, Faculty of Medicine, Department of Physiology and
Immunology, Cara Hadrijana 10e, 31000 Osijek, Croatia, Osijek, Croatia,
2- University Josip Juraj Strossmayer Osijek, Department of Biology, Osijek, Croatia,
3- University Josip Juraj Strossmayer of Osijek, Faculty of Medicine Osijek, Department of Medical
Biology and Genetics, Cara Hadrijana 10e, 31000 Osijek, Croatia,
4- Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, University of Szeged,
Eötvös u. 6. H-6720 Szeged, Hungary,
5-, International Research and Innovation in Medicine Program, Cedars-Sinai Medical Center, Los
Angeles, CA, United States of America or just USA and Regional Cooperation for Health, Science
and Technology (RECOOP HST) Association, Debrecen, Hungary
The corresponding author: Ines Drenjancevic; ines.drenjancevic@mefos.hr
Key words: antioxidative enzymes activity, TBARS, FRAP, diabetes, obesity,
Introduction: Diabetes mellitus and obesity present with increased oxidative stress. Metformin
increases insulin sensitivity and influences glucose metabolism in liver and in peripheral tissues.
Liraglutide is a glucagon-like peptide-1 receptor agonist that stimulates insulin secretion.This study
aimed to determine the markers of oxidative stress in diabetic obese elderly Sprague-Dawley (SD) rats
treated with metformin or liraglutide.
Methods: Male and female SD rats were divided in: 1) control group; 2)group fed high fat-high sucrose
diet from 20 to 65 weeks of age (HSHFD); 3) HSHFD+Metformin (50 mg/kg/day s.c.); and 4)
HSHFD+Liraglutide (0.3 mg/kg/day s.c). Drugs were given concomitantly to HSHFD from 51-65
weeks of age when rats were sacrificed. Plasma lipid peroxidation products were assessed by TBARS
and antioxidative capacity by FRAP. SOD, catalase and gluthatione peroxidase activity was assessed
by spectrophotometry.
Results: Females have no difference in TBARS among groups. FRAP was significantly higher in
HFHSD+Liraglutid compared to HFHSD+Metformin and HSHFD. Catalase activity was higher in
control compared to other female groups. TBARS and FRAP were similar among male groups. Control
group had higher catalase activity compared to other male groups; higher GPx activity compared to
HSHFD+metformin and HSHFD+liraglutide and higher SOD activity compared to
HSHFD+metformin.
Comparing Female vs. Male showed female control rats had significantly higher TBARS compared to
male control.Catalase activity was significantly decreased in all diet groups of both sexes compared to
respective control groups and decreased in female groups overall, compared to male.
Discussion: Metformin significantly decreased all antioxidative enzymes activity compared to control
in male. Treatment with liraglutide increased antioxidant capacity compared to HFHSD+metformin and
HFHSD groups in female to control levels, but oxidative stress was not significantly changed.
Conclusion: There are sex-related differences in the level of oxidative stress and antioxidative
enzymes activity. Antidiabetic drugs may modify antioxidative capacity more in female than male.
Metformin and liraglutide decrease activity of antioxidative enzymes in male.
Sources of Funding This work has been supported in part by VIF-MEFOS-15 (Faculty of Medicine
Osijek, Croatia) and by RECOOP HST grant.
Ethical Approval All experimental procedures are conformed to the European Guidelines for the Care
and Use of Laboratory Animals (directive 86/609). They were approved by the by Hungarian Ethical
Committee for Animal Research (IV/3796/2015).
Acknowledgement: The study was supported by Cedars Sinai Medical Center’s International Research
and Innovation in Medicine Program, the Association for Regional Cooperation in the Fields of Health,
Science and Technology (RECOOP HST Association) and the participating Cedars – Sinai Medical
Center - RECOOP Research Centers (CRRC).
Fatty acid changes in plasma, visceral and subcutaneous adipose tissue in diabetic,
obese elderly rats treated with Liraglutide (Victoza) and Metformin.
In memoriam of Janos Harangi, University of Debrecen, Hungary
Nagy E1, Krivosikova Z2, Gajdos M2, Kebis A3, Vari SG4, Tábi T5
.
1Doctorate School of Animal Husbandry, Faculty of Agriculture and Nutritional Sciences,

2Department of Clinical and Experimental Pharmacotherapy, Slovak Medical University,
Medical Faculty, Bratislava, Slovakia

3Department of Toxicology, Faculty of Public Health, Slovak Medical University, Bratislava,
Slovakia
4International Research and Innovation in Medicine Program, Cedars - Sinai Medical
Center, Los Angeles, CA, USA

5Department of Pharmacodynamics, Faculty of Pharmacy, Semmelweis University, Hungary
Corresponding author: Dr. Tamás Tábi, PhD tabi.tamas@pharma.semmelweis-univ.hu
Keywords: fatty acid composition, high fat diet, physical exercise
Introduction: Adipose tissue contains triglycerides of various fatty acids. Among them the
most abundant saturated and monounsaturated ones serve mainly as energy storage while the
polyunsaturated ones are the precursors of a range of mediators such as prostanoids and
leukotrienes are potent inflammatory mediators. Significant evidence has been accumulated to
support the cardiovascular protective role of n-3 fatty acids compared to n-6 ones.
Aims: In this study we aim to develop a sample preparation method to analyzing fatty acid
composition in visceral and subcutaneous adipose tissue of rats in response to diet and physical
activity.
Methods: High-fat diet was used to induce obesity in rats. A subgroup of rats was forced to
running to mimic physical exercise while another subgroup was also fed by antioxidant
selenized cookie. In the sample preparation step we tried more acid catalyzed and base
catalyzed transesterification method before we developed the appropriate one. Fatty acid
composition of white adipose tissue was measured by gas chromatography.
Results: The first results showed that high-fat diet significantly increased the saturated and
monounsaturated fatty acid content, while the polyunsaturated fatty acid content decreased
significantly in both tissues contrary its relatively high intake. These fatty acids are included in
very low concentration in the tissue and in some cases their amounts were under the detection
limit. The method development shows that we need a higher concentration step to be able to
determine and compare these essential fatty acids.
Conclusion: Our data shows that we need acid catalyzed derivatization followed by a strong
concentration step in the sample preparation. In this way we will be able to follow up the diet-
induced changes in the body.
Ethical approval: The proposal was approved by the Ethical Committee for Animal
Experiment of the Slovak Medical University and by the State Veterinary and Food Authority
of the Slovak Republic (number of approval: Ro-1651/11-221b, 7.10.2011).
Acknowledgement: This study was supported by Cedars Sinai Medical Center's International
the Fields of Health, Science and Technology (RECOOP HST Association) and "Center of
excellence of environmental health" project, ITMS No.26240120033, based on the supporting
operational Research and development program financed from the European Regional
Development Fund."
Renal pathological alterations in diet induced diabetic, obese elderly rats treated with
Liraglutide and Metformin
B.M. Klinkhammer1, P. Boor1 R Gaspar2, S. G. Vari3

1
Institute for Pathology, University hospital RWTH Aachen, Germany
2
Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, University of Szeged,
Eötvös u. 6. H-6720 Szeged, Hungary,
3
International Research and Innovation in Medicine Program, Cedars-Sinai Medical Center, Los
Angeles, CA, USA and Regional Cooperation for Health, Science and Technology (RECOOP HST)
Association, Debrecen, Hungary
Corresponding Author: Peter Boor pboor@ukaachen.de
Key words: metabolic syndrome; kidneys, histology,
Introduction The metabolic syndrome is a major world-wide public-health problem associated with
lifestyle. Metabolic syndrome promotes development of co-morbidities like diabetes, cardiovascular
diseases and chronic kidney disease. In this experimental study we analyze the effects of a glucagon-
like peptide-1 (GLP-1) mimetic Liraglutide and the classical anti-diabetic drug Metformin on obesity-
induced kidney disease.
Methods Nine month ole female and male rats were kept on normal diet (control group) or fed with a
high fat high sugar diet (HFHSD) for 19 weeks. Starting at week 6 of HFHSD diet, the chow was
supplemented either with Liraglutide (0.3mg/kg) or Metformin (50mg/kg). Control animals received
pure HFHSD or continued to receive normal rat chow. Body weight was measured weekly and blood
glucose tolerance was measured monthly. At the end of experiment, plasma and urine was collected
and waist circumference and kidney weights were recorded. Kidneys were harvested for histological,
immunohistochemical and ultrastructural analyses.
Results No difference in kidney weights was observed. Histologically, kidneys showed focal
pathological glomerular changes, e.g. thickening of basement membrane with subendothelial exudates,
mesangial expansion and sclerotic lesions. Additionally, activation of parietal epithelial cells and
tubular cells was observed in CD44 staining, a marker of injury-associated epithelial cell activation in
kidneys. PAS staining showed focal tubular injury and atrophy, including loss of brush-border, tubular
dilation and cellular simplification, and thickening of tubular basement membranes. Associated to these
changes interstitial mononuclear inflammation was observed. Immunohistochemistry showed increased
number of interstitial aSMA positive myofibroblasts, a marker of activated fibroblasts, and increased
deposition of collagen type I confirming the presence of tubulointerstital fibrosis. No obvious changes
between the different groups on HFHSD were observed, requiring further detailed histomorphological
analyses and quantification.
Discussion Kidneys from elderly rats with HSHFD showed obvious morphological renal changes.
Future plan To evaluate differences between the treatment groups, we will perform histomorphological
quantification of the observed kidney damage, i.e. tubulointerstitial injury and glomerular sclerosis (in
PAS), activation of parietal epithelial cells (CD44), podocyte damage (Desmin), interstitial and
glomerular inflammation (CD68, CD44), interstitial fibrosis and glomerular sclerosis (Collagen I, IV),
interstitial myofibroblasts (aSMA). Ultrastructural changes, in particular of the glomeruli, will be
analyzed with transmission electron microscopy. Kidney function, e.g. serum creatinine and BUN, and
proteinuria/albuminuria and tubular injury, e.g. using urinary KIM-1 or NGAL ELISA, should be
assessed in blood and urine samples.
Ethical Approval: All experimental procedures are conformed to the European Guidelines for the Care
and Use of Laboratory Animals (directive 86/609). They were approved by the by Hungarian Ethical
Committee for Animal Research (IV/3796/2015).
Altered uterine contractility and response to adipokines after Liraglutide (Victoza) and
Metformin treatment in obese, elderly rats
1Gaspar R, 1Hajagos-Tóth J, 1Ducza E, 1Tiszai Z, 1Bóta J, 1Csányi A 2Vari SG
Key words: high calorie diet, obesity, elderly rat, liraglutide, metformin, uterine contractility
Introduction: Obesity in pregnancy is associated with failure in uterine contractions. Other

studies reported that maternal overweight and obesity during pregnancy were associated with
increased risks of preterm delivery. Some adipokines that have increased levels in obesity
reduce the contractility of human or rat myometria. Our aim was to investigate how obesity
alters the non-pregnant uterine contractility and its response to leptin and adiponectin.
Methods: Female rats (44-weeks-old) were put in 4 groups containing 8 rats each: 1. Standard
diet (SD); 2. High fat-high sucrose diet (HFHSD); 3. HFHSD+ metformin (50 mg/kg/day); 4.
HFHSD+liraglutide (0.3 mg/kg/day) from 51 weeks for 14 weeks of ages. Obesity was induced
by HFHSD by from 45 weeks of age for 20 weeks. Animals were sacrificed at their 64 weeks
of age. Spontaneous and KC-induced uterine contractility was measured in organ bath in the
presence of leptin (10-7M) or adiponectin (10-8M). The mRNA expressions of uterine leptin
and adiponectin receptors were detected by RT-PCR.
Results: Neither obesity nor metformin or liraglutide treatment changed the spontaneous
contractions. The KCl-induced contractions were reduced by both metformin and liraglutide
treatment. Leptin and adiponectin increased the non-obese, while reduced the obese
spontaneous contractions. The relaxation was enhanced by metformin treatment. Leptin
slightly reduced the KCl-induced contraction bot in non-obese and obese uteri. Liraglutide
reversed the action of leptin to contraction. Adiponectin had no effect on KCl-induced
contraction, while metformin or liraglutide treatment enhanced its action to relaxation.
Discussion and Conclusion: Obesity alters the non-pregnant uterine contractility, metformin
and liraglutide treatment can modify the action of leptin and adiponectin on contraction of
obese rat uterus.
Sources of Funding
This work has been supported by RECOOP HST grant.
Acknowledgement:
The study was supported by Cedars Sinai Medical Center’s International Research and
Health, Science and Technology (RECOOP HST Association) and the participating Cedars –
Bone mineral density and content in elderly diabetic rats. The impact of Metformin and
Liraglutide treatment.
Krivošíková Z1*, Štefíková K1, Heffer M2, Gáspár R3,Vari SG4.
1
Department of Clinical and Experimental Pharmacotherapy, Medical Faculty, Slovak Medical
University, Bratislava, Slovakia
2
Department of Medical Biology, School of Medicine, University J. J. Strossmayer of Osijek, Osijek,
Croatia
3
Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, University of Szeged,
Eötvös u. 6. H-6720 Szeged, Hungary
4
International Research and Innovation in Medicine Program, Cedars-Sinai Medical Center, Los
Angeles, CA, United States of America or just USA and Regional Cooperation for Health, Science
and Technology (RECOOP HST) Association, Debrecen, Hungary
* zora.krivosikova@szu.sk
Key words: rat, diabetes, bone, Metformin, Liraglutide
Introduction: The effects of type 2 diabetes on bone are not clear yet. Whereas bone mineral density
(BMD) has been reported to be either unchanged or modestly increased, incidence of low-stress
fractures is almost doubled together with delayed fracture healing. In vitro data indicates that metformin
can directly stimulate the proliferation, differentiation and mineralization of osteoblasts and liraglutide
has anabolic effects in young diabetic rats. Our objective was therefore to determine the potential
positive effect of Metformin and Liraglutide treatmenton femoral and tibialBMD and bone mineral
content (BMC) in rats with diabetes induced by high fat-high sugar diet (HFHSD).
Methods: Twenty week old male and female SD rats were divided in: control group and 3 groups fed
HSHFD. From fifty first week one HSHFD group was treated with Metformin (Met; 50 mg/kg/day s.c.)
and another one with Liraglutide (LG; 0.3 mg/kg/day s.c). All groups were sacrificed at 65 weeks of
age. Standard biochemistry was measured by biochemical analyzer (Vitros 250, J&J, Rochester, USA).
Dual X-ray absorptiometry wascarried out by using a Lunar Prodigy Advance with Encore 2011
software version 13.60 (GE Medical Systems, Madison, WI, USA).
Results: The bone marrow adiposity was similar in both genders. The excess of adipocytes was found
in HFHSD group and LG groups if compared to SD group, and much less in Met group if compared to
HFHSD group. No significant differences were found in length, BMD, BMC and area of right femur
and right tibia in all experimental groups in both female and male gender. Strong correlation was
confirmed between bone lengths and BMC or area in female rats and between bone lengths and BMD,
BMC and area in male rats. No correlation was observed between densitometrical data and biochemical
parameters in both genders.
Discussion and conclusion:All the available literary data on the detrimental effects of obesity and
diabetes on bone, and the potential anabolic effects of antidiabetic agents have been described in
growing young and adult rats. In our experiment, the animals were subjected to HFHSD at an advanced
age with sufficient peak bone mass. We concluded that obesity and diabetes affect probably more bone
formation than bone resorption. Elderly rats have already been subdued bone formation and therefore
the impact of diabetes and antidiabetics treatment on bone mineral was not shown.
Sources of research support: This work has been supported by RECOOP HST Association research
grant and by the frame work of realization of the project "Center of excellence of environmental health",
ITMS No.26240120033, based on the supporting Operational Research and Development Program
financed from the European Regional Development Fund.
Ethical Committee Approval: All experiments were carried out with the approval of the Hungarian
Ethical Committee for Animal Research (registration number: IV/3796/2015).
Abstracts of Cardiovascular Research in Progress
ORAL PRESENTATIONS
Cardiovascular risk factors and their impact on vascular parameters in women are
modified by reproductive status and smoking, longitudinal study.
J. Pitha, I. Kralova Lesna, P. Wohlfahrt, O. Auzky, A. Sekerkova, M. Lejsková, P. Stavek, R.

Dembovská, J. Mrázková, R. Houdková; Center for Experimental Medicine, Institute for
Clinical and Experimental Medicine, Prague
Corresponding author: Jan Piťha japi@ikem.cz
Key words: reproductive status – cardiovascular risk factors – remnant lipoproteins - vascular
biomarkers
Introduction: In our previous studies we detected increased sensitivity for development and
progression of atherosclerosis in women in menopausal transition especially when smoking.
Additionally, we observed strong association between preclinical atherosclerosis and remnant
lipoprotein particle cholesterol (RLP-C). The main aim of this study was to analyze impact of
changes of cardiovascular risk factors under study on several established vascular parameters.
Methods: We analyzed changes of main cardiovascular risk factors including RLP-C (total
minus LDL minus HDL cholesterol in fasting status) in 10year longitudinal study and the effect
of these changes on vascular parameters including aortic stiffness, arterial stiffness, endothelial
dysfunction and circulating microparticles measured at the end of the study.
Results: In 361 women aged 59.9 ± 2.6 years we detected unfavorable changes of almost all
risk factors during the study period. In these changes were not substantial different between
women who changed their status to menopause and permanently menopausal women. RLP-C
were negatively associated with endothelial function in the whole population (r=-0.133, p
<0.05); in smoking women transformed to menopause they were negatively correlated with
endothelial dysfunction (r=-0.272, p <0.05) and with circulating microparticles (r=-0.115,
p <0.05 and r=-0.276, p <0.01, respectively).
Discussion: Vascular parameters were influenced almost exclusively by changes in remnant
particle cholesterol and almost entirely in women who changed their reproductive status to
menopause during the study.
Conclusion: This data support hypothesis that change of reproductive status is sensitive period
for atherosclerosis development and remnant lipoproteins could be one of them main
accelerators of this process, especially in smoking women.
Acknowledgement: This project was supported through the Internal Grant Agency of the
Ministry of Health, Czech Republic (NT 14008-3/2013) and under this number approved by
local Ethical Committee in 2013.
Shared decision making in life style and nutrition for intervention in woman with risk
factors in cardiovascular health
Jurić Petričević S1, Pavličević I2, Malički M3, Mrduljaš-Đujić N2, Ćurčić L2, Došen Janković
S4, Škrabić S5, Tolić-Biočina A5, Marušić M3, Marušić A3
1Department of Emergency Medicine in Split, Department of Family Medicine, University of
Split School of Medicine, Šoltanska 2, Split, Croatia
2Department of Family Medicine, University of Split School of Medicine, Šoltanska 2, Split,
Croatia
3Department of Research in Biomedicine and Health, University of Split School of Medicine,
Šoltanska 2, Split, Croatia

4Family practice office, Put Skalica 2, Split, Croatia
5Family practice office, Getaldićeva 27, Split, Croatia
The corresponding author: Slavica Jurić Petričević, slavica.juric01@gmail.com
Key words: woman, cardiovascular health, life style, nutrition
Introduction: In 2015 we conducted an interventional study to decrease CVD risk in women

between 40 and 65 years of age. After 6 months, 33% of women decrease their risk. In order
to determine their satisfaction with the intervention, and possible way of improving it, we
conducted a survey on all women participating in the trial.
Methods: Prospective, interventional study included 95 women (7 patients dropped out of
study) of different reproductive status and one or more of cardiovascular risk factors. We
divided patients in 3 groups in accordance with the results of cardiovascular disease risk (CVD)
change: those who decreased CVD risk, those with no improvement and those who increased
CVD risk, and analyzed their answers.
Results: Of the 95 women 81 (91%) reported attempting to change their life habits, and 28
(61%) tried to stop smoking (out of 46 who smoked). However, only 3 actually stopped
smoking. Allmost all (91) found the intervention to be usefull for them. The women who did
not change or who increased the CVD risk after the intervention had higher hope than those
who were successful. It is possible these less successful women would have benefited from the
prolonged and more intensive intervention. The two less successful groups had higher score in
desire for more education (P=0.0383) and their will for changing the life style (P=0.0467).
Discussion: Our individually-tailored intervention lowered the decisional conflict of women
on their necessary lifestyle changes as well as their CVD risks. However, the less successful
women neither lost their hope nor desire for help.
Conclusion: The study indicates that individual women's characteristics which affect the
compliance with intervention should be studied in detail, and utilized to additionally modify
the interventions to more significant reduction of the CVD risk.
Acknowledgements: We thank Dr. Sandor G. Vari, MD, Director of the International
Research and Innovation in Medicine Program, Cedars – Sinai Medical Center, Los Angeles,
CA, USA and President of the RECOOP HST Association for the initiation of the project and
helpful comments.
We also thank Dr. Ian Pitha, MD, PhD, Head of Laboratory for Atherosclerosis Research,
Centre for Experimental Research, Institute for Clinical and Experimental Medicine (IKEM),
Prague, Czech Republic for the critical review of our research plan
Influence of acute and intermittent hyperbaric oxygenation on the reactivity of the
blood vessels in healthy Sprague-Dawley rats
Mihaljević, Z.1, Drenjančević, I.1
1Department of Physiology and Immunology, Faculty of Medicine Josip Juraj Strossmayer,

University of Osijek, Croatia.
Corresponding author: ines.drenjancevic@mefos.hr
Key words: rat, hyperbaric oxygen therapy, vascular reactivity, oxidative stress
Introduction: The effect of hyperbaric oxygenation (HBO2) on vascular function has been
intensively studied in last decade and the understanding of the mechanisms of its action has
been emerging, particularly in the diabetes mellitus. Our results suggest that repeated exposure
to hyperbaric oxygen and the time between the two therapies can be viewed as intermittent
pseudohypoxia and it is possible to trigger other adaptive mechanisms than previously
observed. Oxygen partial pressure changes may lead to changes in the synthesis of metabolites
of arachidonic acid (AA). This preliminary study aimed to examine the HBO2 effects on the
vascular reactivity to acetylcholine (ACh) and hypoxia in aortic ring of healthy rats.
Methods: Healthy Sprague-Dawley 9-12 weeks old rats were divided in four experimental
groups (1. healthy control; 2. acute HBO2 exposure; 3. 24 hour after acute exposure; and 4. 4
subsequent days of HBO2 exposure). Isolated aortic rings reactivity to ACh and hypoxia was
studied on Isolated Organ Bath (Experimetria LTD). The indicators of oxidative stress were
measured by TBARS and FRAP methods.
Results: Acute exposure of healthy animals (group 2) to HBO2 leads to the impaired
vasorelaxation response to both ACh and hypoxia, not present in the groups 1., 3. And 4.
Discussion: Duration of HBO2exposure diversely affects the vascular reactivity to different

stimuli.
Conclusion: Results indicate increased oxidative stress in acute exposure to HBO2, lost in
other HBO2 treatments.
Acknowledgments: This is preclinical animal research, partially supported by grants of

Ministry of Science, Education and Sports, Croatia, # 219-2160133-2034 and # 219-0000000-
0328 . All experimental procedures conformed to the European Guidelines for the Care and
Use of Laboratory Animals (directive 86/609) and were approved by the local Ethical
Committee #2158/61-02-139/2-06.
Comprehensive analysis of ischemic conditioning techniques in a porcine model of acute
myocardial infarction
András Makkos
medical student VI., Semmelweis University,
Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest,
Hungary
Corresponding author: Andras Makkos makkosandras@gmail.com
Key words: schemic conditioning, Ichemic postconditioning, Remote ischemic

conditioning, MRI, T2-oedema reduction
Introduction: Ischemic pre-, post- and remote conditioning are cardioprotective in animal
models of acute myocardial infarction (AMI). However, their effect in clinical trials is still
inconclusive, which could be attributed to the difficulties of the evaluation of cardioprotection.
Our aim was to compare the cardioprotective effect of ischemic post- and remote conditioning
by histological staining and magnetic resonance imaging (MRI) after AMI in a clinically
relevant porcine model.
Methods: Animals were randomized into ischemic (Isch), pre- (IPreC), post- (IPostC) and
remote conditioned (RIC) groups. In general anesthesia, the anterior descending branch of the
left coronary artery (LAD) was occluded for 90 min with balloon catheter (index ischemia). In
IPreC group before the index ischemia 3×5-5 min, in IPostC group after the index ischemia
6×30-30 sec LAD occlusion/reperfusion cycle was applied. RIPerC performed by 4×5 min
compression of the hind limb.
Results: After 3 hours of reperfusion, myocardial necrosis was significantly decreased by
IPreC, but not by IPostC and RIC as assessed by histological staining (Isch: 38.12±6.05%,
IPreC: 12.15±2.94%*, IPostC: 38.14±4.89%, RIPerC: 32.43±3.19%; % of AAR). There was
no difference in the area at risk (AAR) among groups. As evaluated with cardiac MRI (late
gadolinium enhancement) after 3 days, myocardial necrosis was not changed among groups
(Isch: 14.34±2.39%, IPreC: 9.01±2.32%, IPostC: 12.47±2.0%, RIC: 14.33±1.75%; % of left
ventricle), while myocardial edema (T2-weighted MRI) was significantly decreased by IPostC
and RIC (Isch: 30.38±4.05%; IPostC: 18.52±1.80%*; RIPerC: 17.40±2.68%*; % of left
ventricle). Microvascular obstruction was also decreased by IPreC and IPostC (Isch:
1.25±0.62%; IPreC: 0.12±0.12%*; IPostC: 0.26±0.15%*; % of left ventricle). *p<0.05 vs. Isch.
Discussion and Conclusion: In our translational model the T2-edema reduction showed the
edema can’t define as area-at-risk. The T2-edema reduction with no changes in infarct size can
be an independent marker of cardioprotection, as microvascular obstruction reduction.
Research support and grants: OTKA ANN 107803; TAMOP 4.2.4.A/1-11-1-2012-0001;

TAMOP 4.2.4.A/2-11-1-2012-0001
Acknowledgement: this study supervised by Tamas Baranyai M.D., Ph.D. student and Zoltan
Giricz, Pharm.D., Ph.D., senior research associate, Department of Pharmacology and
Pharmacotherapy, Semmelweis University, Budapest, Hungary
Institutional Animal Care and Use Committee Approval Kaposvár University SOI/31/26-
11/2014
The Role of Oxidative Stress in Development of Impaired Vascular Response Under
The Influence Of High Salt Intake in Sprague-Dawley Rats
Cosic A, Drenjancevic
Department of Physiology and Immunology, Faculty of Medicine University of J.J.
Strossmayer in Osijek, Osijek, Croatia
The corresponding author: Ines Drenjancevic; ines.drenjancevic@mefos.hr
Keywords: antioxidative enzymes, flow-induced dilation, high salt diet, middle cerebral
artery, oxidative stress
Introduction: This study aimed to determine the in vitro flow-induced dilatation (FID)
mechanisms of middle cerebral artery (MCA), the expression of antioxidative genes, genes
involved in dilatation pathways, level of leukocyte activation and the role of oxidative stress in
Sprague-Dawley (SD) rats on a high salt (HS) diet.
Methods: 11-weeks old healthy male SD rats were divided in low salt group (LS) given
standars rat chow (0.4%NaCl) and HS group given food containing 4%NaCl for 1 week
with/without superoxide dismutase (SOD) mimetic TEMPOL in vivo. MCA reactivity in
response to FID in the absence/presence of inhibitors L-NAME, INDO, MS-PPOH and
TEMPOL in vitro was examined. mRNA levels of enzymes from brain blood vessels were
determined with rtPCR. Leukocyte activation and level of oxidative stress in leukocytes from
blood and peripheral organs were mesured by flow cytometry. Blood pressure, lipid
peroxidation products and antioxidative capacity were also measured.
Results: FID was reduced in HS group compared to LS group. Inhibitors (independantly)
significantly reduced FID in LS group. FID in HS group was mediated by NO. TEMPOL
restored FID in HS group to basal level. Expression of GPx4 and iNOS in HS group was
significantly decreased. Antioxidant enzymes activity and BP were not affected by HS diet. HS
intake significantly increased basic reactive oxygen species (ROS) production in the leukocytes
of mesenteric lymph nodes and splenocytes, and intracellular production after stimulation in
peripheral lymph nodes.
Discussion: Mechanisms of FID are different in LS and HS groups. Low GPx4 expression,
increased superoxide production in leukocytes, together with decreased iNOS expression
underlies increased oxidative stress and reduced NO bioavailability in HS group, which caused
impairment of FID in HS group without changes in BP.
Conclusion: FID in HS diet is determined by synthesis of ROS in the vascular wall and affects
vascular reactivity mechanisms.
Sources of Funding: This work has been supported in part by Croatian Science Foundation
under the project # IP-2014-09-6380 and in part with VIF-MEFOS-15 (Faculty of Medicine
Osijek, Croatia).
Acknowledgements: I am grateful to my mentor professor Ines Drenjancevic and to all
colleagues from the Department of Physiology and Immunology for helpful advices and
assistance during the development of the study.
Ethical Approval: All experimental procedures are conformed to the European Guidelines for
the Care and Use of Laboratory Animals (directive 86/609). They were approved by the local
Ethical Committee (Faculty of Medicine, University of Osijek;
Class: 602-04/14-08/06; No.: 2158-61-07-14-04) and Ministry of Agriculture, Croatia (HR-
POK-005).
Electronic Interactive Poster Presentation- Neurodegenerative Diseases
Evaluation of putative cannabinoid ligands as modulators of neurodegeneration
Senkiv J.V.1, Golota S.M.2, Irving J.A.3, Lesyk R.B.2, Stoika R.S.1
1. Department of Regulation Cell Proliferation and Apoptosis, Institute of Cell Biology NAS
of Ukraine, Lviv, Ukraine
2. Department of Pharmacy, Organic and Bioorganic Chemistry, Danylo Halycky Lviv
3. School of Biomedical and Biomolecular Science, University College of Dublin, Dublin,
Ireland
Corresponding author Yulia Shenkiv Yu.senkiv@gmail.com
Key words (max 5): G-protein-coupled receptor (GPCR), putative cannabinoid ligands,
neuronal function
Introduction. Alzheimer's disease is a complex and progressive neurodegenerative disorder.
The available therapy is limited to the symptomatic treatment and its efficacy remains
unsatisfactory. The G-protein-coupled receptor (GPCR) superfamily of integral proteins is the
biggest family of signal transducers, comprised of 1000 members. Considering their prevalence
and functional importance, it is not surprising that 60 % of drugs target GPCRs. Cannabinoid
ligands have neuroprotective actions and may have utility in the treatment of
neurodegeneration. The major CNS targets for cannabinoids include the classical CB1
cannabinoid receptor and also GPR55. Although GPR55 is potently activated by the
endogenous lysophospholipid, L-alysophosphatidylinositol (LPI), it is sensitive to a number of
synthetic cannabinoid ligands.
Methods. In this study, we used a range of functional assays to compare the pharmacological
activity of selected cannabinoid ligands that were synthesized by the team of prof. R. Lesyk.
These are: Les-3105, Les-2769, Les-2615, Les-2659, Les-3836, and LPI (positive control). The
compounds were evaluated in cultured rat embryonic cortical neurons and immortalized cells
of HEK293 line engineered to stably express recombinant human GPR55. Stimulation of
protein kinase (ERK1/2) mitogen activated kinase MAP-kinases and of cAMP response
element binding protein (CREB) were measured by Western-blot analysis, while CREB
activation was additionally monitored using confocal imaging of nuclear phospho-CREB
labelling.
Results and Discussion. Striking effects of the compounds on pCREB activation in neurons
were observed. Current studies are directed at evaluating their effects on GPR55 and CB1
activity.
Conclusion. We have evaluated a new series of cannabinoid ligands which have clear effects
on neuronal function that might be beneficial with respect to neurodegeneration. In particular,
activation of CREB by phosphorylation has been implicated in cell survival. The molecular
precise targets for these ligands are currently under evaluation.
Acknowledgements. We thank the Physiological Society of the UK for travel grant awarded
to Julia Senkiv.
All animal experiments were conducted in accordance with S.I. No. 543/2012 - European
Union (Protection of Animals Used for Scientific Purposes) Regulations 2012. This Irish
legislation serves the purpose of giving effect to the European Union Directive 2010/63/EU.
The structure of this study and the experimental procedures conducted were approved by the
Health Products Regulatory Authority (HPRA)
Abstracts for Nanobiomedicine Research
ORAL PRESENTATIONS
Research activity at Institute of Cell Biology, NAS of Ukraine: achievements, problems

and perspectives of development within RECOOP-HST Association.
Stoika R.S.
(ICB), NAS of Ukraine, Drahomanov Str. 14/16, Lviv, 79005, Ukraine
Corresponding author: Rostyslav Stoika stoika@cellbiol.lviv.ua
In 2015-2016, the following projects have been executed by our team in collaboration with
RECOOP and none-RECOOP partners.
NanoBioTech research projects: 1) Imaging of Formalin-Fixed, Paraffin-Embedded
melanoma tissues using specific lectin-functionalized nanocrystals. Since 2016, this project is
performed at Lviv National Medical University. Future studies will be focused on detection of
functionalized nanocrystals using X-ray excitation for X-ray compatible detection systems
[10,11,12]. 2) Biomedical application of novel polymeric particles of nano- and micro-scale
with a reactive shell suitable for bio-functionalization (in collaboration with Lviv National
Polytechnic University) [14]. Enhanced anticancer activity and circumvention of drug
resistance mechanisms was demonstrated at applying these polymeric/phospholipidic
nanocarriers. 3) Targeting drug-resistant tumor cells in vitro and drug-resistant tumor in vivo
by Fullerene C60/anticancer drug nanoconjugates [6, 15]. 4) Novel oligoelectrolyte-based non-
viral gene delivery systems for genetic transformation of eukaryotic (yeast, plant, mammalian)
cells [1,9]. 5) Development of novel polymer-mineral nanocomposites of ZrO2-Gd2O3
functionalized with hyaluronic acid for regeneration of bone defects. 6) Development of novel
affinity sorbents based on functionalized nano- and microparticles [3,5,7]. 7) Evaluation of
eco- and bio-safety of metal-containing nanomaterials for the aquatic organisms, such as
mollusks, fish and amphibia [8]. Main problem is in a lack of real interest of RECOOP partners
to the developed drug and gene delivery systems.
Cancer research projects: 1) Pre-clinical study (in vitro and in vivo) of biological action of
novel anticancer drugs of natural (Landomycin antibiotics) and synthetic (4-thiazolidone
derivatives) origin (in collaboration with Lviv National Medical University). 2) Diminishing
of negative side effects of these drugs by their complexation with the developed novel
polymeric/phospholipidic nanocarriers [4]. 3) Evaluation of the role of antioxidants in
anticancer chemotherapy. A capability of antioxidants (selenomethionine and D-pantethine) to
affect doxorubicin action and decrease its negative side effects in tumor-bearing mice is under
study [16].
Immunity and Autoimmunity research projects: 1) Search for and characteristics of the
unique catalytic activities (hydrolysis of Histone H1 and sialidase action) of antibodies in
autoimmune patients and evaluation of their diagnostic and prognostic role [2,3,5,7]. 2) Search
for and identification of novel protein and peptide biomarkers of diagnostic and prognostic
value in blood serum of the autoimmune patients (rheumatoid arthritis, multiple sclerosis,
others) [13].
RECOOP partners: Institute of Macromolecular Chemistry in Prague (actual), Institute of
Biochemistry in Ukraine (actual), Lviv National Medical University in Ukraine (actual),
Slovak Medical University in Bratislava (potential), Wroclaw Technical University in Poland
(potential), Debrecen University in Hungary (potential), University of Zagreb in Croatia
(potential), University of J.J. Strossmayer in Croatia (potential), Cedars-Sinai Medical Center
in USA (supervising).
Non-RECOOP partners: Lviv National Polytechnic University in Ukraine (actual), Taras

Shevchenko Kyiv National University in Ukraine (actual), Ivan Franko Lviv National
University in Ukraine (actual), Institute for Cancer Research at Vienna Medical University in
Austria (actual), Ilmenau Technical University in Germany (actual), Institute of Food
Biotechnology and Genomics in Ukraine (actual), Institute of Hereditary Pathology in Ukraine
(actual), Institute of BioOrganic Chemistry in Belarus (actual), Nencki Institute of
Experimental Biology in Poland (actual).
Acknowledgements. Our activity was supported by Cedars Sinai Medical Center’s

International Research and Innovation in Medicine Program, the Association for Regional
Cooperation in the Fields of Health, Science and Technology (RECOOP HST Association),
and the participating Cedars – Sinai Medical Center - RECOOP Research Centers (CRRC).
Selected topic-focused international publications (2015-2016) of the ICB Team

(names of the ICB co-authors are printed in Bold)
1. Y. Filyak, N. Finiuk, N. Mitina, A. Zaichenko, R. Stoika // In: "Genetic transformation
systems in fungi". Chapter 8.1. Springer. 2015. P. 201-207.
2. Tomin A, Dumych T, Tolstyak Y, Kril I, Mahorivska I, Bila E, Stoika R, Herrmann M, Kit
Y, Bilyy R. // Clin Exp Immunol. 2015; 179(1):17-23. ІF= 3.037
3. Kit Y, Bilyy R, Korniy N, Tomin A, Chop'yak V, Tolstyak Y, Antonyuk V, Stoika R. //
Biomed. Chromatogr. 2015; 29(3):328-332. ІF= 1.723
4. Berdyshev AG, Kosiakova HV, Onopchenko OV, Panchuk RR, Stoika RS, Hula NM. //
Prostaglandins Other Lipid Mediat. 2015; 121(Pt A): 91-96. ІF= 2.377
5. Horak D., Plichta Z., Starykovych M., Myronovskij S., Kit Y., Chopyak V, Stoika R. //
RSC Advances. 2015, 5, 63050–63055. ІF= 3.84
6. Panchuk R.R., Prylutska S.V., Chumak V.V., Skorokhyd N.R., Lehka L.V., Evstigneev
M.P., Prylutskyy Yu.I., Berger W., Heffeter P., Scharff P., Ritter U., Stoika R.S. // J. Biomed.
Nanotechnol. – 2015. – V. 11, N.7. – P. 1139-1152. ІF= 5.338
7. Antonyuk V., Grama S., Plichta Z. , Magorivska I., Horak D., Stoika R. // Biomed.
Chromatography. – 2015. - V. 29, Issue 5. p. 783 – 787. ІF= 1.723
8. Falfushynska H, Gnatyshyna L, Fedoruk O, Mitina N, Zaichenko A, Stoliar O, Stoika R.
Comp Biochem Physiol. C Toxicol Pharmacol. 2015; 172-173:45-56. ІF= 2.301
9. Tanasienko IV, Yemets AI, Finiuk NS, Stoika RS, Blume YB. Cell Biol Int. 2015;
39(3):243-245. ІF= 1.933
10. Turcheniuk K, Turcheniuk V, Hage CH, Dumych T, Bilyy R, Bouckaert J, Héliot L, Zaitsev
V, Boukherroub R, Szunerits S. Chem Commun (Camb). 2015;51(91):16365-16368. IF=
6.834
11. Yan X, Sivignon A, Alcouffe P, Burdin B, Favre-Bonté S, Bilyy R, Barnich N, Fleury E,
Ganachaud F, Bernard J. Chem Commun (Camb). 2015;51(67):13193-13196. IF= 6.834
12. Bilyy R, Podhorodecki A. Nanomedicine (Lond). 2015;10(13):1997-2000. IF= 5.413
13. S. Myronovkij, N. Negrych, T. Nehrych, M. J. Redowicz, S. Souchelnytskyi, R. Stoika, Y.
Kit // Biochemical and Biophysical Reports (Accepted, 2.12.2015).
14. 14.A. Riabtseva, N. Mitina, I. Grytsyna; N. Boiko, R. Stoika, A. Zaichenko. // Eur. Polymer
Journal (Accepted, 2016). IF= 3.005
15. 15.Prylutska S.V. , Skivka L.M. , Didenko G.V. , Prylutskyy Yu.I., Evstigneev M.P.,
Potebnya G.P. , Panchuk R.R. , Stoika R.S. , Ritter U., Scharff P. // 2016. Nanoscale
Research Letters (Accepted, 2016). IF= 2,779
16. R.R. Panchuk, N.R. Skorokhyd, Y.S. Kozak, L.V. Lehka, V.V. Chumak, S.N.
Omelyanchik, V.A. Gurinovich, A.G. Moiseenok, R.S. Stoika // Croat. Med. J. 2016 (in
press). IF= 1.4
Synthesis and surface functionalization of inorganic nanocrystals for their biomedical
applications
A. Podhorodecki*, A.Lesiak, M.Chrzanowski, D.Kociolek, B.Sojka, M.Bański, A.Noculak

Department of Experimental Physics, Wroclaw University of Technology, Wybrzeże
Wyspiańskiego 27, 50-370 Wrocław
*Coressponding autor: artur.p.podhorodecki@pwr.edu.pl
Keywords:nanocrystals, quantumum dots, rare earth ions, ligand exchange, ligand

attraction
INTRODUCTION: Considerable interest has been focused on semiconducting quantum dots

(QDs) and lanthanide doped nanocrystals (LaNCs) due to their possible application in
biomedicine as optical markers. In contrast to conventionall organic dyes LaNCs can be
effectively excited in NIR thanks to up-conversion process. This is especially beneficial for
application in the living organism, because in NIR tissues posses the lowest absorbance.
Additionally, lower toxicity, reduced photobleaching and lack of autofluorescence pose
additional advantages. Placement of Gd3+ ions in crystal lattice opens new attractive application
paths for nanocrystals as multifunctional optical and magnetic markers. On the other hand,
semiconducting quantum dots can be designed as a small (1-5 nm) and very bright probes (up
to 90% QY) with a tunable emission from UV to IR spectral range.
AIMS: The aim of our work was the synthesis of several types of inorganic nanoparticles and
adaptation of their surface to water environment.
METHODS: NaGdF4:Yb3+, Er3+ nanocrystals have been synthesized by co-thermolysis

method. The PbS/CdS and CdSe/CdS QDs have been obtained by solvo-thermal techniques.
The structural properties of the products were characterized by XRD and TEM. The
luminescent properties were investigated by excitation, emission and absorption
measurements. In order to transfer NCs to aqueous solvent ligand attraction protocol has been
used. For QDs we used ligand exchange protocol.
RESULTS: For LaNCs the average size of obtained nanocrystals, determined from TEM is in
the range between 3 and 30 nm and for QDs between 2 and 5 nm, depending on synthesis
parameters. In all case the size distribution is very high, below few %. Both procedures of NCs
surface modification have been successful what allowed for the placement of as obtained
nanocrystals in different biological environments.
CONCLUSIONS: Using different nanostructures the emission spectrum, size and morphology
of NCs can be adjusted. What is more, successful NCs water transfer makes them applicable
in biological media
Biocompatibility and stem cell labelling properties of maghemite nanoparticles
Igor Pongrac1, Ivana Vinković Vrček2, Marina Dobrivojević1, Hrvoje Mlinarić1, Daniel
Horák3, Srećko Gajović1
1 Croatian Institute for Brain Research, University of Zagreb School of Medicine, Šalata 3,
10000 Zagreb, Croatia

2 Institute for Medical Research and Occupational Health, Ksaverska cesta 2, 10 000
Zagreb, Croatia
3 Institute of Macromolecular Chemistry, Academy of Sciences, Heyrovského Sq. 2, 16206
Prague 6, Czech Republic

Corresponding author: srecko.gajovic@hiim.hr
Key words: neural stem cells, maghemite, biocompatibility, brain
Introduction. Nanoparticles represent possible stem cell tracers, but they differ in cellular
uptake and side effects. Their properties can be modified by coating with different
biocompatible polymers. To test if a coating polymer, or nanoparticle type, can improve the
biocompatibility of nanoparticles applied to neural stem cells, differently coated maghemite
nanoparticles were prepared and characterized.
Methods. We evaluated their cellular uptake, mechanism of internalization, cytotoxicity,

viability and proliferation of neural stem cells, and compared them to the commercially
available dextran-coated nanomag®-D-spio nanoparticles.
Results. Light microscopy of Prussian blue staining revealed a concentration dependent

intracellular uptake of maghemite nanoparticles in neural stem cells. The methyl thiazolyl
tetrazolium assay and calcein acetoxymethyl ester/propidium iodide assay demonstrated that
differently coated maghemite nanoparticles scored better than nanomag®-D-spio in cell
labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked
cellular nanoparticle uptake indicating actin-dependent process, such as macropinocytosis to
be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry
analysis of neural stem cells after nanoparticle treatment showed that they preserve their neural
stem cell identity and their potential to differentiate into all three major neural cell types
(neurons, astrocytes and oligodendrocytes).
Discussion and conclusion. Improved biocompatibility and efficient cell labeling makes
poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem
cell in vivo tracking studies.
Ethical approval: HR-POK-006 from 2nd July 2015.
Acknowledgements. The study was supported EU FP7 grant GlowBrain (REGPOT–2012–

CT2012–316120) and by Cedars-Sinai Medical Center’s International Research and
Innovation Management Program, the Association for Regional Cooperation in the Fields of
Health, Science and Technology (RECOOP HST Association), and the participating Cedars-
Sinai Medical Center – RECOOP Research Centers (CRRC).
S, N - containing carbon dots: fluorescent properties and application in cells
experiments
Mariia Dekaliuk, KyryloPyrshev, Alexander Demchenko
Lab. of Nanobiotechnologies, Palladin Institute of Biochemistry of the NAS of Ukraine, Kiev
Corresponding author: Mariia Dekaliuk dekalyuk_m@mail.ru
Keywords: carbon nanoparticles, cell imaging, apoptosis, fixation
Introduction Inorganic carbon nanomaterials, also called carbon nanodots, exhibit a strong
photoluminescence with unusual properties and, thus, have been the focus of intense research.
Carbon nanodots (“C-dots”) due to their excellent fluorescence characteristics and
biocompatibility have ample opportunities for their use in imaging and functional
transformations in living cells. The focus of our research was to determine the possibility of
using C-dots as the easily available probes for apoptotic cells detection and visualization of
intact cells.
Methods The carbon nanoparticles were prepared from sulfur- and nitrogen containing
precursors: citric acid, urea, thiourea etc by hydrothermal treatment at 180 0C. The studies were
performed with adherent epithelial Vero and HeLa cell lines (ATCC).
Results The nanoparticles used in these studies have typical fluorescence characteristics, in
particular, the maximum of the excitation spectrum at 350 nm and the emission maximum at
445 nm. The quantum yield of synthesized nanoparticles was about 34%, which is comparable
to a number of widely used organic dyes. With these tools we demonstrate that both intact and
apoptotic cells can be easily visualized by CDots. Using the different methods of sample
preparation, they show the ability for labeling various structural compartments of the cell. For
living cells there are the intracellular vesicles and lysosomes. In contrast, in fixed cells the
nucleus is labeled preferentially. The fact that apoptotic cells accumulate strongly increased
amount of CDots can be efficiently used in flow cytometry for characterizing the cell
populations regarding the relative amount of apoptotic cells in different experimental
conditions. The application of such cheap and easily accessible nanoparticles, in combination
with previously showed possibility of using these fluorophores for super resolution method
SOFI [2], provides more opportunities to simplify the popular methods of cell labeling and
detection.
Discussion and Conclusion
Here, we present new results on simple
and efficient method of cell visualization
and detection of apoptosis. Its main
advantage is the use of easily
synthesized fluorescent nanoparticles,
which do not require further
modifications.
Confocal imaging of intact Vero cells
(left) and HeLa cells (right) incubated
with carbon nanoparticles during 1 hour (excitation 405 nm).
References
(1) Dekaliuk M. et al., Phys. Chem. Chem. Phys., 2014,16, 16075-16084
(2) Ghosh S. et al., Nano Lett., 2014, 14 (10), pp 5656–5661
(3) Borisova T. et al, Int. J. Biochem. Cell Biol., 2015 (59), 203-215
Neurotoxic properties of carbon dots synthesized from sulphur-containing carbohydrate
precursor
M. Dudarenko, A. Borysov, N. Pozdnyakova, A. Pastukhov, A. Nazarova, T. Borisova
Dept. Neurochemistry and Lab.of Nanobiotechnology, Palladin Institute of Biochemistry,
Keywords: carbon dots from thiourea; glutamate; GABA; Na+-dependent uptake; brain
nerve terminals
Aims: Recently, the authors of this study demonstrated neuromodulatory properties of newly
discovered carbon fluorescent nanoscale particles, carbon dots (CDs), synthesized from amino
acid β-alanine. It is known, however, that being prepared from different types of organic
precursors, CDs expose different atoms at their surface suggesting broad variation of functional
groups. This study is focused on synthesis, characterization and neurotoxicity assessment of
CDs synthesized from sulfur-containing precursor thiourea (TU-CDs) exposing the atoms of
sulfur on the nanoparticle structure.
Methods: preparative biochemistry, fluorescence and radiolabel assay.
Results: Neurotoxic properties of TU-CDs were assessed based on the analysis of their effects
on the key characteristics of glutamatergic and -aminobutryic acid (GABA)
neurotransmission in isolated rat brain nerve terminals. It was observed that TU-CDs (0.5 –
1 .0 mg/ml) attenuated the initial velocity of Na+-dependent transporter-mediated uptake and
accumulation of L-[14C]glutamate and [3H]GABA by nerve terminals in dose-dependent
manner and increased the ambient level of the neurotransmitters. Starting from concentration
of 0.2 mg/ml, TU-CDs evoked gradual dose-dependent depolarization of the plasma membrane
of nerve terminals measured with cationic potentiometric dye rhodamine 6G. Within the
concentration range 0.1-0.5 mg/ml, they caused an “unphysiological” step-like increase in
fluorescence intensity of a рН-sensitive fluorescent dye acridine orange preliminary
accumulated by synaptic vesicles. Fluorescence quenching of both dyes was observed at
concentrations higher than 1 mg/ml.
Discussion and Conclusion: Therefore, despite different surface properties and fluorescent
features of CDs prepared from different starting materials (thiourea and β-alanine), their
neuromodulatory effects are analogous but displayed on a different level of efficiency. From
one side, fluorescent and neuromodulatory features combined in CDs create the basis for their
potential usage for labeling and visualization of key processes in nerve terminals, and,
potentially, in theranostics. From the other side, uncontrolled presence of carbon-containing
nanoparticles in the air especially during natural disasters and in the human food chain poses
a risk of central nervous system toxicity.
Support: This work was supported by Grant # 6055 of Science and Technology Center in
Ukraine (STCU).
Experiments were carried out in accordance with the European Guidelines and International
Laws and Policies (Directive 86/609/EEC); the protocols were approved by the Animal Care
and Use Committee of the Palladin Institute of Biochemistry (Protocol # 2 from 19/09-2014).
20 animals were used in the study.
CdSe/CdS quantum dots functionalization by d-penicillamine
A.Lesiak, M.Chrzanowski, M.Bański, A. Podhorodecki*

Keywords: nanocrystals, quantumum dots, ligand exchange, functionalization
Introduction: Quantum dots (QDs) are interesting fluorescent tools for many applications in
the life science. Fluorescent QDs can be linked with bioactive moieties (e.g. antibodies,
receptor ligands). However, to be useful, they must meet several conditions: water solubility,
small size, chemical stability and high fluorescence quantum yield. Specific functionalization
of QDs by biocompatible compounds lets obtain wide range of applications. Long temporal
stability of nanoparticles in desired solvent is a fundamental challenge. For that purpose, the
ligand molecule on the surface can be exchanged by others that can possibly provide additional
properties or functionality to the particles. D-penicillamine (DPA) is commercially available
ligand, useful because of its functions: non-toxic, small size, zwitterionic and biocompatibility
(e.g. can works like cysteine).
Aims: The aim of our work was to functionlaize quantum dots surface with d-penicillamine.
Methods: Surface ligands have been exchanged from oleic acid to DPA at basic pH ~11 with
use of ligands exchange protocol. The structural properties of the products were characterized
by XRD and TEM. The luminescent properties were investigated by excitation, emission and
absorption measurements.
Results: Highly water soluble, and highly emissive CdSe/CdS-DPA nanocrystals have been
obtained with an average crystalline size of 3 nm.
Conclusions: Using ligand exchange approach highly hydrophilic CdSe/CdS QDs have been
obtained. This protocol can be extended easily to other semiconducting quantum dots like PbS
or ZnS which both types of quantum dots are synthesized in our laboratory.
Synthesis of Cd-based core shell nanocrystals for bio-imaging
M.Chrzanowski, M.Bański, A. Podhorodecki*

Keywords: nanocrystals, quantumum dots, synthesis
Introduction: Quantum dots (QDs) are interesting fluorescent tools for many applications in
the life science. Fluorescent QDs can be linked with bioactive moieties (e.g. antibodies,
receptor ligands). However, to be useful, they must meet several conditions: water solubility,
small size, chemical stability and high fluorescence quantum yield. Among listed expectation
high quantum yield is one of the most important and first one which must be achieved to go
further to fulfilling the other expectations. Highly emissive quantum dots can be used as probes
not only for macro imaging but also for a single dot tracking, enabling observation of complex
process in a single cell.
Aims: The aim of our work was to synthesise a highly emissive Cd-based quantum dots in
core-shell geometry with different emission properties, including different colors of their
emission from blue to red.
Methods: Cd-based QDs have been obtained by solvo-thermal technique. The structural
properties of the products were characterized by XRD and TEM. The luminescent properties
were investigated by excitation, emission and absorption measurements.
Results: Highly emissive Cd-based core shell nanocrystals have been obtained characterized
by an average size varied from 1 to 5 nm depending on synthesis parameters.
Conclusions: High quality and extremely stable Cd-based core-shell QDs have been obtained.
The emission from these QDs can be tuned from 450 up to 600 nm while the excitation for all
QDs can be kept the same i.e. as 405 nm. This enables to use these QDs as multicolor probes
for bioimaging.
Young Scientists Forum - Electronic Interactive Poster Presentation
Metformin may influence neurodegeneration and neuroinflammation in Sprague
Dawley rats on high fat/sugar diet in a sex specific manner
Milorad Z1, Balog M1, Senka M2, Labak I2, Fenrich M1, Vari SG3, Gaspar R4, Gajovic S5,
Heffer M1
1 Faculty of Medicine, J.J. Strossmayer University of Osijek, Croatia
2Department of Biology, J.J. Strossmayer University of Osijek, Croatia
3 International Research and Innovation in Medicine Program, Cedars-Sinai Medical Center,
Los Angeles, CA, USA

4 Department of Pharmacodynamics and Biopharmacy, University of Szeged, Hungary
5 School of Medicine, University of Zagreb
Corresponding author: prof. Marija Heffer, mheffer@gmail.com
Key words: high fat/sugar diet, aging, neurodegeneration, neuroinflammation, Metformin,

Liraglutide
Introduction High content of sugars and fat in diet promote long term metabolic changes,
which can lead to a variety of diseases starting with metabolic syndrome, specifically diabetes
type 2 which has lately been connected to central diabetes and Alzheimers disease. Aging puts
additional pressure on entire body, especially on maintenance of nervous system. Aim of this
study was to compare sex specific neurodegeneration and neuroinflammation in Sprague
Dawley rats on high fat/sugar diet (HFHSD) upon treatment with Metformin and Liraglutide.
Methods Male and female rats were divided in two control groups: one fed with standard
laboratory diet (SD) and second fed with standardized HFHSD. Two experimental groups were
fed with HFHSD with addition of therapy – Metformin (MF) (50 mg/kg) or Liraglutide (LG)
(0,3 mg/kg). Feeding with HFHSD or SD and two types of therapies had lasted for 6 months
starting at the age of 9 months. Brains were collected at the age of 15 months and
immunostained with inflammatory marker Iba1 and GFAP together with histological stainings
for neurodegeneration (Congo Red and Bielschowski stainings). Images were photographed
using Zeiss Axioskop 2 MOT microscope and analyzed in ImageJ software. Statistical analysis
was performed in SPSS software.
Results Inflammation measured with Iba1 antibody detection is reduced in male and female
groups treated with MF if compared with their SD controls (p=0,030 for males, p=0,017 for
females). However, inflammation is increasing in males and females in case of LG treatment
compared with their HFHSD controls (p=0,009 for males). Male LG group shows an increase
compared to all other male groups, without statistical significance. There is a statistically higher
Iba1 expression in males MF than female MF group (p=0,006). GFAP staining showed an
increased astrocytes volume in MF females compared to SD female control (p=0,049). We also
observed increase in MF female group compared with HFHSD female group (p=0,049).
Bielschowski and Congo Red stainings were analyzed in motor cortex and smaller number of
plaques was observed in control SD groups. All other male and female groups showed higher
number of plaques. The only distinction we observed was bigger plaques formation in case of
MF male group. In piriform cortex we observed no plaques in both control SD groups. Male
HFHSD group as well as MF and LG groups showed overall higher density of plaques than
female groups. MF male group again showed bigger plaques than other animal groups.
Bielschowski staining of motor cortex showed small number of plaques in control SD male
and female group and higher number of plaques in all other male and female groups. Piriform
cortex showed no visible plaque formation in male and female control SD groups and small
number of plaques was seen in all other male and female groups.
Conclusion HFHSD and aging increase risks for neurodegeneration and inflammation in the
brain. Inflammation and neurodegeneration are two major hallmarks of Alzheimers disease.
Both Metformin and Liraglutide treatments had stronger protective effects of inflammation in
the brain of female animals than in males. Using histological staining for plaque formation we
observed the presence of neurodegeneration in all animal groups except SD male and female
control groups. These results are inconclusive on extent of the brain damage and further
analyses are needed to confirm the profile of neurodegeneration.
Acknowledgements: This study was supported by Cedars Sinai Medical Center's International
the Fields of Health, Science and Technology (RECOOP HST Association) and internal
research grant from J. J. Strossmayer University of Osijek, Croatia.
The study was performed at the Department of Pharmacodynamics and Biopharmacy,
University of Szeged, Hungary during 2015.
The importance of autophagy in the antiapoptotic effect of resveratrol
Ulakcsai Zs, Bagaméry F, Szökő É, Tábi T
Department of Pharmacodynamics, Faculty of Pharmacy, Semmelweis University, Hungary
Introduction: Previously we have demonstrated that resveratrol dose dependently prevented

caspase 3 activation secondary to serum deprivation in non-transformed cells.
Aim: In the present work we aimed to assess the role of the probable molecular targets of
resvereatrol, i.e. estrogen or aromatic hydrocarbon receptors and autophagy in its
cytoprotective effect.
Methods: Apoptosis was induced by serum deprivation in primary mouse embryonic

fibroblasts. Caspase 3 activation was assayed by using its fluorogenic substrate. Reactive
oxygen species production and depolarization of the mitochondrial membrane were measured
by fluorescence methods. The involvement of the receptors and autophagy in the effect of
resveratrol were also analyzed using special agonists and antagonists.
Results: We found that neither aromatic hydrocarbon receptors nor estrogen receptors play an
important role in the antiapoptotic effect of resveratrol. An autophagy inhibitor, chloroquine
abolished the preventive effect of resveratrol, which indicates the importance of autophagy. In
the presence of serum deprivation resveratrol induced a highly significant depolarization in
mitochondrial membrane potential. Reactive oxygen species production was elevated by serum
deprivation and further increased after resveratrol treatment.
Conclusion: We have demonstrated that resveratrol can protect primary fibroblasts against
serum deprivation induced apoptosis by provoking mild intracellular stress and upregulating
autophagy.
Ethical approval: All animal procedures were approved by the ethics committee of the
Semmelweis University (22.1/1375/7/2010).
Acknowledgement:
Thank you for Cedars Sinai Medical Center’s International Research and Innovation in
Medicine Program, the Association for Regional Cooperation in the Fields of Health, Science
and Technology (RECOOP HST Association) for their support of our organization as
Application of THY1 – YFP neural stem cells in regenerative neuroscience
Alić I1,2, Kosi N2, Kapuralin K2, Gajović S2, Pochet R2, Mitrečić D2*
1Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine,

2Laboratory for Stem Cells, Croatian Institute for Brain Research, School of Medicine,
Corresponding author: dinko.mitrecic@mef.hr
Key words: neural stem cells, transgenic mice, stroke
Introduction: This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which
under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in all parts
of neurons. In this study, we have determined and compared expression pattern of: 1) Thy1 -
YFP positive cells during in vitro differentiation of neural stem cells (NSC), 2) during
embryonic development of Thy1 – YFP mouse embryo and 3) after transplantation of Thy1 –
YFP cells into the stroke-affected mouse brain.
Materials and Methods: Neural stem cells were isolated from the forebrain of 14.5 old mouse
embryos and cultured as neurospheres. Immunocytochemistry and RT-PCR were performed
after cells differentiation. Embryonic slices were prepared for immunohistochemistry. In third
part of this study, PKH26 labeled NSC were transplanted into the stroke-affected mouse brain
obtained by MCAO method.
Results: THY1 - YFP expression was described during differentiation of NSC and observed
in neural progenitors as well as in mature neurons. Neural progenitors were nestin positive
while mature cell were not. THY1 – YFP positive cells were positive for mature neuronal
markers (MAP2, β3-tubulin and NeuN) during the whole differentiation period, while
astrocytes which were GFAP positive were not expressing YFP. During embryonic
development THY1 – YFP positive cells were observed from E12.5 and the expression steadily
increased to the end of pregnancy. Positive cells were present in both, central and peripheral
nervous system. After transplantation, cells survived and incorporated into the stroke-affected
brain.
Conclusion: Our results showed that mature neurons as well as neuronal progenitor cells do
express THY1 – YFP construct in in vitro conditions, during embryonic development and after
transplantation into the stroke-affected mouse brain. This suggests that in addition to analyses
of neuronal differentiation in B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse, NSCs isolated from this
strain can be successfully used in studies where tracing of cells with neuronal fate is needed.
Source: The work has been supported by projects: YoungBrain (EU-ESF), the Croatian
National Foundation (02.05/40), Foundation Adris awarded to D.M. and by FP7 Glowbrain
project (REGPOT–2012–CT2012–316120) awarded to S.G.
Ethical approval: All experiments on animals described in this work received approval of
the Internal Review Board of the Ethical Committee of the School of Medicine, University of
Zagreb. Approval number (640-01/12-17/33) and date (22.05.2013.)
ARHGAP25 Rac-GAP has an important role in autoantibody-induced model of
rheumatoid arthritis
T. F. Svanya, P. Lévai
Semmelweis University, Faculty of Medicine, Department of Physiology, Budapest
Corresponding author: Tim F. Svanya timsvanya@gmail.com
Keywords: neutrophils, arthritis, ARHGAP25, inflammation, GAP
Introduction: Small G-proteins of the Rac family play a central role in regulation of numerous
cellular functions. ARHGAP25, a GTP-ase activating protein (GAP), is known to be
responsible for Rac inactivation. According to our previous results, it is mainly expressed in
leukocytes and plays a major role in the function of neutrophilic granulocytes, such as
transmigration through endothelium, production of reactive oxygen species (ROS) and
phagocytosis. In addition, it plays a regulatory role in actin cytoskeleton reorganization.
Methods: Arhgap25-/- (KO) mouse strain was purchased from the Knockout Mouse Project
(KOMP). Arthritis was induced in male KO and wild type mice by intraperitoneal injection of
150µl K/BxN mouse strain-derived serum. For control non-arthritogenic BxN serum was used.
For the following 10 days ankle thickness was measured and a clinical score, indicating the
severity of inflammation by subjective evaluation of edema and hyperemia, was determined.
The loss of function was investigated by placing the mice on a wire-grid measuring the latency
to fall upon turning over.
Results: Absence of ARHGAP25 caused a significant decrease in clinical scores, as well as in
ankle thickness compared to wild type mice. Similar results were observed in the functional
test; ARHGAP25-/- animals spent longer time on the grid. In mice treated with control serum
neither inflammation, nor loss of function could be observed, regardless of the genotype.
Discussion: Our previous work, investigating the effect of ARHGAP25 on neutrophil
responses in cell-based models, indicated enhanced effector functions in the absence of
ARHGAP25. However, the present study shows a less severe arthritis induced by
autoantibodies due to yet not elucidated mechanisms.
Conclusion: Our current results indicate, that beyond the elementary phagocyte functions,
ARHGAP25 is a crucial player of inflammation in a human disease-related complex model.
Acknowledgement
Supervisors: Dr. Roland Csépányi-Kömi, Prof. Dr. Erzsébet Ligeti
Research support: Hungarian Research Fund OTKA 108382
Institutional Animal Care and Use Committee Approval: 22.1/323/3/2011. Issuing date: 8th of
March 2011 by Pest Megyei Kormányhivatal Élelmiszerlánc-Biztonsági és Állategészségügyi
Igazgatósága
The use of formalin-fixed paraffin-embedded samples in early diagnosis of diseases
Reviewed and accepted but could not present
B. Döncző1, M. Szigeti1,2, Gy. Ostoros2,3, A. Gacs3, J. Tóvári3, A. Guttman1,2

1Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen,
Hungary
2MTA-PE Translational Glycomics Group, Pannon University, Veszprám, Hungary
3National Institute of Oncology, Department of Experimental Pharmacology, Budapest,
Hungary
Corresponding author: Andras Guttman guttman@mik.uni-pannon.hu
Key words: FFPE, glycosylation, capillary electrophoresis
Introduction: Formalin - fixed paraffin - embedded (FFPE) samples are generally used in
histology as well as to archive clinical and pathological samples. FFPE tissue collections, with
their accompanying clinical outcome information, are invaluable resources for translational
studies of cancer and other diseases. However, this huge sample collection was mainly used so
far for nucleic acid analysis. The objective of this work is to show that formalin fixed samples
hold a great promise in glycomics studies. Information from these samples could be useful in
early diagnosis of some diseases such as cancer.
Methods: The carbohydrate moiety of intact and formalin treated standard glycoproteins as
well as human serum was removed by endoglycosidase digestion. The released glycans were
labeled with a charged fluorophore and analyzed by capillary electrophoresis - laser induced
fluorescent detection (CE-LIF). Glycans from tumor biopsy samples and their FFPE
counterparts were extracted with radioimmunoprecipitation assay (RIPA) buffer followed by
the same glycan removal, labeling and analysis process was used as described above.
Results: and Discussion: Preliminary experiments with standard glycoproteins showed
identical glycosylation patterns before and after formalin treatment. Human serum was also
treated and analyzed to compare the control to formalin treated and formalin fixed paraffin
embedded formats. The results show practically no differences among the peak patterns. In
case of tumor tissues, the effect of formalin fixation and paraffin embedding was investigated
on the global N-glycosylation profile of mouse samples, also with very little changes observed.
Conclusion: Our results suggested that the sugar moiety of glycoproteins remained intact after
formalin treatment, thus, can be used to discover disease associated glycosylation changes at
the molecular, cellular and tissue levels. The ability to effectively profile N-glycans form FFPE
samples offers new opportunities to understand disease associated glycan profile changes, even
retrospectively from large hospital archives.
Ethical Committee Approval: All animal-model protocols were carried out in accordance with
the Guidelines for Animal Experiments and were approved by the Institutional Ethics
Committee at the National Institute of Oncology, Budapest, Hungary (permission number:
22.1/722/3/2010)
The role of gamma aminobutyric acid (GABA) in the regulation of endocervical
epithelium secretory activity
Skelin M, Gajović S, Ćurlin M

Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Croatia
Corresponding author: Marija Ćurlin (marija.curlin@mef.hr)
Key Words: endocervical, mucus, gaba, cell culture
Introduction: Endocervical secretory epithelium produces cervical mucus, a glycoprotein gel

that accepts, filters, prepares, stores and releases sperm for transport to the oviduct for
fertilization, as well as provides a barrier to the infection. The mechanisms of mucus secretion
in the cervix are poorly investigated. We have proposed a new model of regulation of mucus
secretion that involves action potential independent activity of GABA molecules.
Methods: To test this hypothesis, we analyzed mouse and human endocervical tissue for the
presence of GABA receptors by immunohistochemistry.
Reuslts: In accordance with our model, the results showed that GABAR-A is present in the
endocervical tissue of both human and mouse. These results present a scientific novelty and
show that our model is realistically set up.
Discussion: The main goal of our current efforts is to establish a 3D epithelial cell culture in
order to investigate the effect of estrogen on GABA signaling pathway action. We will test the
expression of estrogen receptors in cells that show positive signal with the markers of GABA
release. By applying inhibitors or activators of various receptors and by immunohistochemical
and electron microscopy analysis we will investigate the effect of estrogen on mucin
exocytosis.
Conclusion: Preliminary results support our hypothesis and further research will enable us to
explore the molecular mechanism of endocervical epithelium secretory activity.
Ethical approval: All experiments received approval of the Internal Review Board of the
Ethical Committee of the School of Medicine, University of Zagreb. All experiments were
carried out in accordance with the EU Directive 2010/63/EU and 2004/23/EC.
Source: The work has been supported by FP7 Glowbrain project (REGPOT–2012–CT2012–
316120), and Croatian Science Foundation Fellowship to M.S.
Analyses of impact circadian dysfunctions on the occurrence of disautotonia in medical
student population
Zvir M., Beliak A., Zayachkivska O.

Physiology Department, Lviv National Medical University, Lviv, Ukraine
Corresponding author: Maryana Zvir maryana.zv@gmail.com
Key words: chronic stress, disautotonia, circadian dysfunctions, medical students
Introduction. Chronic stress and modern lifestyle cause disautotonia that leads to health
problems. WHO develops the International Classification of Diseases, version 11 that includes
stress-spectrum diseases. Our studies showed that stress symptoms are more frequent among
medical students (MS) and it makes important search of new non-invasive diagnostic stress
markers.
Methods: 50 MS were interviewed using questionnaire that included stress perception,

physical activity, sleep quality and time spent on IT devices. Anthropometric data, heart rate
variability (HRV) and the saliva microcrystallization (SM) by dehydration of mix saliva were
analyzed.
Results: examinees tend to have body mass index with mean 21.7; overweight was in 4%.
Males had slightly lower muscle mass of 39.2% (normal value (N) above 40%) and higher total
fat content - 21.4% (N - up to 20.0%). 68% of subjects spent >7 hrs/day sitting and 22% were
physically active. The time of using gadgets >6 hrs/day was 63%. The good sleep quality was
in 54% vs poor in 46% participants. 67% of MS confirmed high stress level and in 30% was
reflected preference sympathetic activity by HRV. Type I SM was found in 16%; II and III -
70%; IV - 14% of students and it correlated with decrease in adaptive abilities.
Discussion: Modern MS are a special population group, characterized by enormous workload,

circadian dysfunctions, which increase stress level in their life. Such high prevalence of stress,
found in our study, is alarming, because even in mild form it leads to negative effects on health
and academic performance. Such studies may clarify the notion of modern lifestyle and it non-
pharmacological correction.
Conclusion. Circadian dysfunction, high level of stress and increased sitting time are the early
triggers for disautotonia in MS. Further studies are required to assess saliva secretotome by
proteinomic applying for evaluation early biomarkers of disautotonia.
Study was approved by local bioethics committee 15.02.2016 (N2)

Plasma membrane Ca2+-pump selective regulators – calix[4]arene C-90 and
imidazo[1,2-a]azepine – change parameters of uterus smooth muscles contraction
Mazur1 Iu.Iu., Veklich1 T.O., Shkrabak1 O.A., Gerashchenko2 I.V., Kosterin1 S.O.
1 Department of Muscle Biochemistry, Palladin Institute of Biochemistry of the National
Academy of Sciences of Ukraine, Kyiv, Ukraine
2 Institute of Pharmacology and Toxicology of the National Academy of Medical Sciences,
Kyiv, Ukraine
Corresponding author Iuliia Mazur yuliya.vorona@gmail.com
Key words: myometrium, calix[4]arene, contractility, imidazo[1,2-a]azepine, oxytocin
Introduction. Disfunction of plasma membrane Mg2+,ATP-dependent Ca2+-pump (PMCA)

activity is often observed in diseases of smooth muscle (SM), i.e., uterus SM: preterm labor,
atony, dysmenorrhea, hypo- and hypertonus, prolonged labour. Specific and target influence
on PMCA activity may help to improve those pathological conditions. For this purpose we use
calix[4]arene C-90 (5,11,17,23-tetra(trifluoro)methyl(phenyl-sulfonylimino)-methylamino-
25,26,27,28-tetrapropoxycalix[4]arene) and imidazo[1,2-a]azepine derivate (IFT-35).
Methods. Calix[4]arene C-90 was characterized with infrared spectroscopy and nuclear
magnetic resonance methods. PMCA enzymatic and Ca2+-transport activity was determined on
swine myometrium plasma membrane vesicle by measurements of Pi and by fluorescent probe
fluo-4 AM respectively. Contractile activity was induced by hyperpotassium solution or
oxytocin and studied isometrically. Institutional Animal Care protocols were approved by the
Animal Care and Use Committee of the Palladin Institute of Biochemistry (Protocol #4 from
21/10/2012).
Results. Calix[4]arene C-90 selectively inhibits enzymatic (by 75%) and transport activity of
PMCA (I0.5=20.5 µM). PMCA inhibition by different structural components of calix[4]arene
C-90 and its structural analogs was estimated. Thus, effectivness of its action depends on
localization and spatial orientation of 4 sulfonilamide groups; and on level of calix[4]arene
hydrophobity which can be changed by adding chemical groups to macrocycle lower ring. On
contrary, IFT-35 increase PMCA activity by 45% compering to control (А0.5=6,40 µM) and
slightly elevate its transport activity. Both compounds in oposite way changed amplitude and
frequency of myometrium oxytocin-induced contractions in vitro and in vivo: C-90 (100 µM)
increases the velocity in two-fold and IFT-35 (100 µM) decreases in two-fold. Also,
calix[4]arene C-90 changes the velocity of SM relaxation and contraction induced by
hyperpotassium solution or by oxytocin.
Conclusion. From obtained data it was concluded that selective influence on PMCA by
proposed compound of C-90 and IFT-35 may be used as a tool for manipulation with SM
contraction process and correction of SM pathological dysfunction.
Acknowledgements. We are grateful to our colleague from Institute of Pharmacology and

Toxicology and Institute of Organic Chemistry (prof. Kalchenko) for providing reagents and
scientific discussion of our results.
Does endogen PACAP have any role in the development of retinopathy of prematurity?
Kvarik T1,2, Mammel B1,2, Werling D1, Vaczy A1, Toth G4, Reglodi D1, Ertl T2, Gyarmati J2,
Atlasz T3
1 Dept. of Anatomy, Pacap-Lendulet Research Group; 2 Dept. of Obstetrics and Gyneacology,
3 Dept. of Sportbiology, University of Pecs, Pecs, Hungary; 4 Dept. of Medical Chemistry,
University of Szeged, Szeged, Hungary

Corresponding author’s email: kvarik.timi@gmail.com
Keywords: Retinopathy of prematurity (ROP); Pituitary adenylate cyclase-activating

polypeptide (PACAP)
Introduction Retinopathy of prematurity (ROP) is one of the leading cause of childhood

blindness characterized by vessel obliteration and new, abnormal vessel formation. The
oxygen-induced retinopathy (OIR) is a well-established model of ROP.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to have neuroprotective
effects. Several studies have revealed the presence of PACAP and its receptors in the retina.
Recent studies reported its retinoprotective effects in ischemic and diabetic lesions. Recently
we have shown that local PACAP treatment improve the retinal vascularization, decreases the
avascular area in the rat model of ROP.
In this study we aimed to examine the effect of endogen PACAP in the mouse model of ROP.
Methods OIR was generated by placing litters of PACAP knockout and wild-type mice in 75
% oxygen concentration from postnatal day 7 (P7) to P12, then returned them to room air.
Control mice of both types were kept in room air. On P17 retinas were isolated and the vessels
were visualized by isolectin staining. The percentages of avascular to whole retinal areas as
well as the number of vascular tufts were measured. Statistical analysis was done by using
Student’s t-test.
Results The retina of PACAP knockout OIR mice showed greater central avascular area than
that of the wild type (8.68% vs 3.52%; p=0.003). Retinal images of controls kept in room air
did not show vascular alterations. No changes in the number of vascular tufts were observed
between deficient and wild mice.
Discussion This is the first study to examine the endogenous effect of PACAP in ROP.
Previously we have shown the beneficial effect of exogenous local PACAP treatment on the
vessel formation and cytokine profile in the rat OIR model. Together with the present findings
we suppose that PACAP could be a novel retinoprotective agent in ROP research.
Ethical approval Animal housing, care and application of experimental procedures were in
accordance with institutional guidelines under approved protocols (No: BA02/2000-
15024/2011, University of Pecs following the European Community Council directive).
Funding MTA-Lendulet Program, Janos Bolyai Research Scholarship of the Hungarian
Academy of Sciences, OTKA K104984
Acknowledgement Thank you for Cedars Sinai Medical Center’s International Research and
Health, Science and Technology (RECOOP HST Association) for their support of our
organization as participating Cedars – Sinai Medical Center - RECOOP Research Centers
(CRRC).
The comparison of hydrogen sulfide NSAID derivates in the modulation of gastro-
esophageal integrity.
Pshyk-Titko I., Bula N., Pavlovskiy J., Zayachkivska O.

Department of Physiology, Lviv National Medical University
Corresponding author: Irena Pshyk-Titko irena.pshyk@gmail.com
Keywords: H2S, cytoprotection, aspirin, naproxene, NSAID
Introduction: An important role in the secretion of in gastrointestinal tract (GI) is played by

hydrogen sulfide (H2S). It modulates gastro-oesophageal vagal afferents as well as motor
activity. In our previous investigations we have confirmed a cytoprotective, anti-inflammatory
and vasodilatatory effects of H2S as a critical mediator of esophageal mucosa. It was shown
that non-steroid anti-inflammatory drugs (NSAIDs) could be beneficial in chemoprotective
strategy for Barrett’s esophagus, esophagus- adenocarcinoma progression or tumors associated
with esophagogastric junctions but also may contribute to the occurrence of non-reflux
esophagitis.
Method: We determined the effect of 1 day and 10 days pretreatment of H2S-related aspirin
(ATB 340, 17,5 mg/kg) on gastro-esophageal mucosal integrity in the normally and H2S-
restricted states (suppression of H2S synthesizing enzyme cystathionine γ-lyase (CSE) activity
by inhibitor L-propargylglycine (PAG, 25 mg/kg), cystathionine β-synthase (CBS) by O-
carboxymethylhydroxylamine; CHH, 20 mg/kg) in comparison with the effect of similar
duration of aspirin-pretreatment (10 mg/kg) without and with stress-induced injury by Takagi,
1964. We carried out the qualitative changes in the content pro-inflammatory cytokines against
the background of modification of the cytoprotection. The histological damage index was used
for the estimation and scoring of the gastro-esophageal mucosa.
Results: Inhibition of COX-1 and COX-2 activity with single dose ATB 340 vs aspirin-induced
damage significantly diminished NSAIDs-related gastro-esophageal injury. ATB 340
attenuated severity of NSAIDs-induced gastro-esophageal lesions vs aspirin-induced mucosal
lesions and significantly decreased sign of inflammation, edema and vascular changes. The use
of pretreatment with PAG and CHH reduced the mentioned effects.
Discussion and conclusion: The cytoprotective effect of H2S against NSAIDs injury of
gastro-esophageal lesions depends on CSE and CBS activity status. H2S-related aspirin exerts
gastro-esophageal protection against stress-induced lesions by expressing of microcirculation
and vasotropic effects.
Acknowledgements: Cedars Sinai Medical Center’s International Research and Innovation in

Medicine Program, the Association for Regional Cooperation in the Fields of Health, Science
and Technology (RECOOP HST Association) for their support.
prof. J.L. Wallace, Family Digestive Health Institute, McMaster University, Antibe
Therapeutics Inc., Toronto, Ontario.
Study was approved by Lviv Medical University bioethics committee (2016).?

Abstracts for Annual Review of the Research Activities in RECOOP
Session I
ORAL PRESENTATIONS
Antioxidants Selenomethionine and D-Pantethine Decrease Negative Side Effects of

Doxorubicin in Nk/Ly Lymphoma-Bearing Mice
Rostyslav R. Panchuk1*, Nadia R. Skorokhyd1, Yuliya S. Kozak1,2, Liliya V. Lehka1, Vira V.
Chumak1, Sofya N. Omelyanchik3, Valery A. Gurinovich3, Andrey G. Moiseenok3, Rostyslav
S. Stoika1,2
1
Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology NAS of
Ukraine, 79005, Lviv, Drahomanov Str 14/16, Ukraine
2
Department of Biochemistry, Biological Faculty, Ivan Franko Lviv National University, Hrushevsky
Str. 4, Lviv, 79005, Ukraine
3
Laboratory of Metabolomics, Institute of Biochemistry of biologically active compounds,
Leninskogo Komsomola Boulevard 50, Grodno, Belarus
Corresponding author: Rostyslav Panchuk rpanchuk@ukr.net
Keywords: antioxidants, selenomethionine, D-panthetine, doxorubicin, NK/Ly lymphoma, anticancer

drugs
Aim was to investigate potential tissue-protective effects of antioxidants selenomethionine and D-

pantethine applied together with doxorubicin (Dx) towards NK/Ly lymphoma bearing-mice. Impact of
this chemotherapy scheme on animal survival, blood cell profile, hepatotoxicity, glutathione level and
activity of glutathione-converting enzymes in liver was compared with action of Dx applied alone.
Methods Hematological profile of animals was studied by analysis of blood smears under Evolution
300 Trino microscope. Hepatotoxicity of studied drugs was evaluated measuring activity of ALT/AST
enzymes, De Ritis ratio, and coenzyme A fractions by McDougal assay. Glutathione level in animal
tissues was measured with Ellman reagent, and activity of glutathione reductase, transferase and
peroxidase was measured using standard biochemical assays.
Results D-pantethine (500 mg/kg) and, to lower extent, selenomethionine (600 µg/kg) partially reduced
negative side effects (leukocytopenia and erythropenia) of Dx (5 mg/kg) in NK/Ly lymphoma bearing
animals at 14th and 21st day of their treatment. That increased animal survival time from 47-48 to 60+
days and improved quality of their life. This ability of D-pantethine and selenomethionine was realized
via hepatoprotective and immunomodulating activities. D-panthetine also restored levels of acid-
soluble and free CoA in liver of tumor-bearing animals, while selenomethionine caused recovery of
glutathione peroxidase levels in liver that was significantly diminished under Dx treatment. Both
compounds decreased GSH level in liver considerably induced by Dx.
Conclusions Antioxidants selenomethionine and D-pantethine partially reversed negative side effects
of Dx in NK/Ly lymphoma-bearing mice and significantly increased therapeutic efficiency of this drug
in tumor treatment.
Support of the State Funds of Fundamental Research of Ukraine and Belarus (bilateral project
#F54/185-2013),
Acknowledgement: Thank you for Cedars Sinai Medical Center’s International Research and
Innovation in Medicine Program, the Association for Regional Cooperation in the Fields of Health,
Science and Technology (RECOOP HST Association) for their support of our organization as
Ethics Committee Approval: Protocol № 9/2015 from 1.09.2015 of the BioEthics committee of the
Institute of Cell Biology, NAS of Ukraine).
Ovariectomy and chronic stress lead toward leptin resistance in satiety centers and
insulin resistance in hippocampus of Sprague-Dawley rat
Vedrana Ivić1, Senka Blažetić2, Irena Labak2, Marta Balog1, Luka Vondrak2, Sandor G. Vari3, Marija
Heffer1*
1
Josip Juraj Strossmayer University of Osijek, Faculty of Medicine Osijek, Department of Medical
Biology and Genetics, Cara Hadrijana 10/E, HR-31000 Osijek, Croatia
2
Josip Juraj Strossmayer University of Osijek, Department of Biology, Cara Hadrijana 8/A, HR-
31000 Osijek, Croatia
3
International Research and Innovation in Medicine Program, Cedars–Sinai Medical Center, Los
Angeles, CA, USA
*Corresponding author: Marija Heffer, MD, PhD (mheffer@mefos.hr)
Key words: Brain, chronic stress, ovariectomy, leptin receptor, insulin receptor, gonadal
steroid receptors
Introduction: The aim of this study was to evaluate changes in the expression level of gonadal
steroid, insulin and leptin receptors in the brain of adult Sprague-Dawley female rats due to
ovariectomy and/or chronic stress.
Methods: Sixteen-week-old ovariectomized and non-ovariectomized female Sprague-Dawley
rats were divided in two groups and submitted to three 10-day-sessions of sham or chronic
stress. After the last stress session the brains were collected and free-floating
immunohistochemical staining was performed using androgen (AR), progesterone (PR),
estrogen-β (ER-β), insulin (IR-α) and leptin receptor (ObR) antibodies. The level of receptors
expression was analyzed in hypothalamic (HTH), cortical (CTX), dopaminergic (VTA/SNC)
and in hippocampal regions (HIPP).
Results and discussion: Ovariectomy alone downregulated levels of all gonadal steroid
receptors in hippocampal satiety centers. General effect of chronic stress was upregulation of
AR in same regions. When combined, ovariectomy and chronic stress upregulated levels of
PR. Also, combination of stressors pushed hypothalamic satiety centers toward rise of ObR
and susceptibility to leptin resistance particularly in nucleus arcuatus. Exposed to combined
stressors hippocampal regions, SCN and piriform cortex, upregulated expression of IR-α and
possibility develops insulin resistance.
Conclusion: We discussed possibility of stress induced metabolic changes under condition of
hormone deprivation. Ovariectomy exacerbates effect of chronic stress by preventing gonadal
receptor specific stress response reflected in upregulation of AR in satiety regions and
upregulation of AR and ER-β in hippocampal regions. Final outcome of inadequate stress
response is reflected in upregulation of ObR in satiety centers and IR-α in regions which are
susceptible to early neurodegeneration. The most significant finding of our study is possible
link between chronic stress response (amplified by ovariectomy) and development of insulin
resistance in hippocampus and other brain regions susceptible to early neurodegeneration.
Ethical approval: This study was approved by the Ethical Committee of the Faculty of Medicine Osijek, approval
number: 2158-61-07-11-51.
Source(s) of research support: This work has been supported in part by the Croatian Science Foundation under
the project number IP-09-2014-2324 and funded by internal research grant from Faculty of Medicine of Josip
Juraj Strossmayer University of Osijek (VIF2015-MEFOS-1).
Acknowledgement: This study was supported by Cedars Sinai Medical Center’s International Research and
Innovation in Medicine Program, the Association for Regional Cooperation in the Fields of Health, Science and
Technology (RECOOP HST Association) and the participating Cedars – Sinai Medical Center - RECOOP
Research Centers (CRRC).
Diurnal variation in cholesterol 7α-hydroxylase activity is determined by the -203A>C
polymorphism of the CYP7A1 gene
Tereza Blahová1, Miluše Vlachová1, Martin Leníček2, Věra Lánská1, Libor Vítek2, Jan Kovář1
1 Laboratory for Atherosclerosis Research, Institute for Clinical and Experimental Medicine,
Czech Republic
2 Institute of Medical Biochemistry and Laboratory Diagnostics, Charles University, First
Faculty of Medicine, Czech Republic
Corresponding autor: Tereza Blahová, tereza.blahova@ikem.cz
Aim To determine whether the promoter polymorphism -203A>C of cholesterol-7α-

hydroxylase encoding gene (CYP7A1) affects diurnal variation in CYP7A1 enzyme activity.
Methods The study included 16 healthy male volunteers – 8 homozygous for -203A and 8
homozygous for the -203C allele of CYP7A1. Three 15-hour examinations (from 7am to
10pm) were carried out for each of the participants: after one-day treatment with
cholestyramine; after one-day treatment with chenodeoxycholic acid (CDCA); and a control
examination without any treatment. The plasma concentration of 7α-hydroxy-4-cholesten-3-
one (C4), a marker of CYP7A1 activity, was determined in all the experiments at 90-min
intervals.
Results CYP7A1 activity was upregulated after treatment with cholestyramine and
suppressed after treatment with CDCA. There were no differences between -203A and -
203C allele carriers in the response of enzyme activity to both drugs. In the control
experiment, -203A allele carriers displayed diurnal variation in enzyme activity whereas
CYP7A1 activity did not change in those carrying -203C allele.
These results were confirmed by modelling the dynamics of C4 using polynomial regression.
Conclusion The promoter polymorphism of the CYP7A1 gene has a pronounced impact on
diurnal variation in CYP7A1 activity.
Keywords
cholesterol 7α-hydroxylase, genetic polymorphism, diurnal variation, bile acid sequestrant,
chenodeoxycholic acid
Supported by grant No. NT 13151-4/2012 from IGA MH CR.

Ethical Committee Approval: G 12-06-34, 27. 6. 2012
Putative anticancer potential of novel 4-thiazolidinone derivatives: toxicity towards rat
C6 glioma cells and correlation of general toxicity with balance of free radical oxidation
in rats
Lesya Коbylinska 1, Rostyslav Panchuk 2, Nataliya Boiko 2, Iryna Grytsyna 2, Olga Klyuchivska 2,
Liliya Biletska 1, Roman Lesyk 3, Borys Zіmenkovsky 3, Rostyslav Stoika 2
1 – Department of Biochemistry, Danylo Halytsky Lviv National Medical University, Ukraine
2 – Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, NAS of
Ukraine, Lviv, Ukraine
3 – Department of Pharmaceutical Chemistry, Danylo Halytsky Lviv National Medical University,
Ukraine
Corresponding author: Lasya Kobylinska lesya8@gmail.com
Key words: 4-thiazolidinone, reactive oxygen species, antioxidant system, general toxicity
Introduction. Novel 4-thiazolidinone derivatives (compounds denoted as 3288, 3882 and 3833) have
been recently synthesized by our team3. Their antineoplastic action towards 60 human tumor cell lines
was studied at National Cancer Institute (USA). Here we addressed such action of these derivatives
towards rat glioma cells of C6 line. This was necessary before moving to next step – experiments in
tumor-bearing animals. We compared the effects of compounds 3288, 3882 and 3833 and doxorubicin
(positive control) on a balance of free radical oxidation and antioxidant activity (AOA). Such analysis
was done for evaluating the role of the reactive oxygen species (ROS) in general toxicity effects of
studied compounds in the experimental rats.
Methods and Materials. Rat glioma C6 cells were treated with compounds 3882, 3288, 3833 and
doxorubicin, and their cytotoxicity was studied using MTT assay and Trypan blue exclusion test, light
and fluorescent microscopy, and flow cytometric study of cell cycling and apoptosis. These compounds
were also injected in rat’s vein, and the levels of superoxide radical, hydrogene peroxide, hydroxyl
radical, malonic dialdehyde (MDA), hydrogen sulfide were measured in blood serum of treated rats.
Additionally, the activity of superoxide dismutase (SOD), catalase (Cat), and glutathione peroxydase
(GPO) was determined in rat’s blood serum.
Results. Among different 4-thiazolidinone derivatives under study, compound 3288 demonstrated the
highest toxicity towards C6 glioma cells in all used tests, while the compound 3882 was relatively not
toxic for these cells. All applied derivatives were generally less active, comparing to doxorubicin, in
inducing ROS-related indicators in blood serum of rats. Similar behavior was observed in activity of
the enzymatic indicators of AOA processes. While doxorubicin inhibited the activity of SOD, GPO and
Cat, such effects of 4-thiazolidinone derivatives were less prominent.
Discussion and Conclusion. We have demonstrated that increased level of ROS and decreased AOA
correlated with the level of general toxicity of compounds 3288, 3882 and 3833 comparing with
doxorubicin in rats. Thus, a search of optimal balance between anticancer activity of novel and existing
drugs and their effects towards content of ROS and the AOA is required. Proper modulation of the level
of induced ROS and AOA might be a useful strategy to decrease negative consequences of toxic effects
of most anticancer drugs in treated organism.
Acknowledgements. This study was supported by Cedars Sinai Medical Center’s International
Research and Innovation in Medicine Program, the Association for Regional Cooperation in the Fields
of Health, Science and Technology (RECOOP HST Association) and the participating Cedars-Sinai
Medical Center - RECOOP Research Centers (CRRC).
BioEthics Committee Approval. All animal experiments were conducted in accordance with the
European Convention on Protection of Vertebrate Animals (Strasbourg, 1986) and corresponding Law
of Ukraine (N944, 14.12.2009). Structure of this study and experimental procedures were approved by
the Ethical Committee of Lviv National Medical University (N2, 16.02.2015).
The effects of estrogen on the α2-adrenergic receptor subtypes in late pregnant uterine
function in vitro
Judit Hajagos-Tóth1, Judit Bóta1, Eszter Ducza1, Reza Samavati2, Sándor Benyhe2, Anna
Borsodi2, Róbert Gáspár1
1 Department of Pharmacodynamics and Biopharmacy, University of Szeged, Szeged, Hungary
2 Institute of Biochemistry, Biological Research Centre, Szeged, Hungary
Corresponding author: Róbert Gáspár, gaspar@pharm.u-szeged.hu
Key words: estrogen, pregnancy, rat, uterus, α2-adrenergic receptor subtypes
Introduction: The disorders of myometrial contractility may lead to preterm birth or delayed
delivery. The α2A- and α2C-adrenergic receptors (ARs) decrease the myometrial response to (-
)-noradrenaline, while the α2B-ARs mediate contractions. Our aims were to clarify the change
in function and expression of the α2-AR subtypes after 17β-estradiol pretreatment in late
pregnancy.
Methods: SPRD rats from pregnancy day 18 were treated with 17β-estradiol for 4 days. The
myometrial expressions of the α2-AR subtypes were determined by RT-PCR and Western blot
analysis. In vitro contractions were stimulated with (-)-noradrenaline, and its effect was
modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C) and spiroxatrine
(α2A). The accumulation of cAMP was also measured. The activated G-protein level was
investigated by GTP binding assay.
Results: 17β-estradiol pretreatment decreased the contractile effect of (-)-noradrenaline via the
α2-ARs, and abolished the myometrial contraction increasing effect through the α2B-ARs. All
of the α2-AR subtypes mRNA was decreased. 17β-estradiol pretreatment increased the
myometrial cAMP level in the presence of BRL 44408 (P=0.001), ARC 239 (P=0.007) and
spiroxatrine (P=0.045), but did not modify it in case of spiroxatrine + BRL 44408 combination.
17β-estradiol pretreatment inhibited the G-protein-activating effect of (-)-noradrenaline by
25% in the presence of BRL 44408 – spiroxatrine combination.
Conclusions: The expression of the α2-AR subtypes is 17β-estradiol sensitive. It decreases the
contractile response of (-)-noradrenaline through the α2B-AR subtype, and might cause changes
in G-protein signaling pathway. Estrogen dysregulation may be responsible for preterm labour
or uterine inertia via the α2-ARs.
Acknowledgements: The study was supported by Cedars Sinai Medical Center’s International
participating Cedars – Sinai Medical Center - RECOOP Research Centers (CRRC). The study
was also supported by a grant from the National Research, Development and Innovation Office
(NKFI), Budapest, Hungary; grant identifier is OTKA-108518.
Ethical Committee Approval: All experiments involving animal subjects were carried out
with the approval of the Hungarian Ethical Committee for Animal Research (permission
number: IV/198/2013).
H. pylori in sedentary males is linked to higher heart rate, sympathetic activity and
insulin resistance but not inflammation or oxidative stress
Cherkas, A.1, Eckl, P.2, Guéraud, F.3, Abrahamovych, O.1, Serhiyenko, V.4, Yatskevych, O.1,
Pliatsko, M.1, and Golota, S.5
1Department of Internal Medicine №1, Danylo Halytskyi Lviv National Medical University,
Lviv, Ukraine
2Department of Cell Biology, University of Salzburg, Salzburg, Austria
3Research Center in Food Toxicology, INRA, INP, UPS, Team 9 "Prevention, Promotion of
Carcinogenesis by Food”, Toulouse, France

4Department of Endocrinology, Danylo Halytskyi Lviv National Medical University, Lviv,
Ukraine
5Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytskyi Lviv

Corresponding author: Andriy Cherkas cherkasandriy@yahoo.com
Key words: Helicobacter pylori, sedentary lifestyle, heart rate variability, insulin resistance,
autonomic imbalance
Introduction. The rate of Helicobacter pylori (H.pylori) contamination in healthy subjects

without any gastrointestinal complaints vary between 10 and 60 percent in different countries.
Contamination by this bacterium may promote the atherosclerosis and hypertension while
sedentary lifestyle may potentiate these effects.
Aim. To compare anthropometric parameters, body composition, hormonal and inflammatory
profiles, oxidative stress indices, and heart rate variability (HRV), in H.pylori positive and
negative healthy sedentary participants.
Methods. Among 30 randomly recruited apparently healthy male participants (age between 20
and 40) enrolled in this cross-sectional study, 18 were H.pylori negative and 12 were positive
(stool antigen test). Participants underwent routine physical examination and body composition
determination. The following biochemical parameters were determined in blood: fasting whole
blood glucose, glycated hemoglobin, insulin, C-peptide, cortisol, aldosterone, testosterone,
thyroid stimulating hormone, C-reactive protein, interleukins 6 and 10, tumor necrosis factor-
α, and the urinary level of 1,4-dihydroxynonane mercapturic acid. For HRV evaluation,
electrocardiogram in supine position and in orthostatic test was performed.
Results. H.pylori contamination was not associated with any changes in anthropometric
parameters, body composition, blood pressure, fasting glucose, or glycated hemoglobin levels.
No significant difference was found for inflammatory markers as well as 1,4-dihydroxynonane
mercapturic acid. H.pylori-positive participants, however, had significantly higher heart rate
(P=0.009), sympathetic/parasympathetic balance in orthostatic test (P=0.029), fasting insulin
level (P=0.037), and HOMA-index (P=0.047).
Conclusions. H.pylori contamination is linked to a significantly higher heart rate, sympathetic
activation, and increased insulin resistance, while inflammatory and oxidative stress markers
remain unaffected in healthy sedentary male subjects.
Acknowledgements (funding). The project was supported by State Agency of Science,

Innovations and Informatization of Ukraine contracts #M512-2011, #M473-2012, OEAD
Project UA 03/2011.
The Ethics Committee of Danylo Halytskyi Lviv National Medical University approved the
design and protocol of the study, protocol №5, May 17th 2010.
Morphological and pathological response in primary systemic therapy of patients with
breast cancer and the prediction of disease free survival
Gyöngyvér Szentmártoni1, Anna-Mária Tőkés2, Timea Tőkés1, Krisztián Somlai3, Attila

Marcell Szász2, László Torgyík1, Janina Kulka2, Magdolna Dank1*
[1] Semmelweis University, Department of Clinical Oncology, Budapest, Hungary

[2] 2nd Department of Pathology, Budapest, Hungary
[3] Surgical Division of St. Margit Hospital, Budapest, Hungary
Corresponding author: Magdolna Dank magdolna.dank@gmail.com
Keywords: breast cancer subtypes, primary systemic therapy, ultrasound, physical

examination
Introduction: This single center observational study aimed to identify breast cancer subtypes
likely to respond to primary systemic therapy (PST) and to assess the accuracy of physical
examination (PE) and ultrasonography (US) in evaluating and predicting residual size of breast
carcinoma following PST.
Methods: A prospectively collected clinical database was analyzed. 116 patients were included
who received PST between 1998 and 2009. Radiological assessment was done by
mammography and US (PET/CT and MRI were only available in the second part of the
analyzed period therefore not considered hereby). Prior to PST, core biopsy (NCB) and/or fine-
needle aspiration based immunohistochemical profiles of NCB subclassified the tumors.
Pathological response rates were assessed following the surgeries by using Chevallier
classification. Tumor measurements by PE and US were obtained before and after PST.
Different clinical measurements were compared with histology. Disease-free survival (DFS)
was assessed.
Results: Pathological complete remission (pCR=Chevallier I/II) was observed in 25 cases
(21.5%), amongst them triple negative histology dominated (44%), percent of high-grade
tumor was 76%. Of 116 patients, 24 received taxane-based PST, 48 combined
taxane+anthracycline treatment, 8 trastuzumab combinations, 21 antracycline-based
treatments, and 15 other were administered. In the taxane treated group, the pCR rate was
30%, in the taxane+anthracycline group 25%, in the anthracycline group 9.5% and in
trastuzumab group 37.5%. After PST, PE correlated better with pathology than US (p=0.00001
and p=0.0047, respectively). Concerning DFS, significant difference was observed between
the Chevallier III and IV group (p=0.0313). In the pCR group, fewer events were observed
during the follow-up period.
Discussion and conclusions: It seems that even limited, routinely used immunohistochemical
profiling of tumors is able to predict the likelihood of pCR to PST: patients with triple negative
and Her2-positive cancers are more likely to achieve pCR after PST. PE correlates with the
pathological findings better than US.
Funding/Research Support/Grants: None was involved.
Ethical Committee Approval: Ethical approval for the study was given by the Semmelweis
University Institutional Review Board. Date and number of the ethical approval: 76/2007.
Conflict of Interest: The authors declare that they have no conflict of interest.
Thionin-modified poly(glycidyl methacrylate) particles as labels of antibodies for
biosensing applications - C
Reviewed and accpeted but could not present
Zasońska B. A.1*, Cadkova M.2,3, Kovarova A2 , Korecka L.2, Bilkova Z.2, Horák D.1
1 Department of Polymer Particles, Institute of Macromolecular Chemistry, Academy of
Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic
(*zasonska@imc.cas.cz)
2 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology,
University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic

3 Department of Analytical Chemistry, Faculty of Chemical Technology, University of
Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic
Keywords: poly(glycidyl methacrylate), particles, horseradish peroxidase, biosensor
Introduction: Poly(glycidyl methacrylate) (PGMA) particles can be directly coupled with a

biological molecules or further modified into different functional groups. The purpose of this
research was to synthesized suitable particles to improve the sensitivity and selectivity of
electrochemical immunosensors.
Methods: Poly(glycidyl methacrylate) nanospheres were obtained by emulsifier-free emulsion

polymerization. In the next step, the carboxyl groups were introduced by hydrolysis and
oxidation with KMnO4 (PCMMA) and thionin was immobilized. The enzyme horseradish
peroxidase (HRP) and the electron mediator thionin (Th) were covalently attached to the
antibody indicator in the enzyme-based electrochemical immunosensor. The resulting particles
were characterized by FTIR spectroscopy and scanning electron microscopy combined with
energy dispersive X-ray spectroscopy.
Results and Discussion: The number-average diameter of the starting PGMA changed after
modifications from 350−420 nm. Particle size dependent on the monomer and initiator
concentrations. Covalently bound thionin electron mediator of the PCMMA-Th-HRP
nanospheres had a positive effect on the resulting electrochemical signal and in a higher
labelling rate.
Conclusions: Thionin-modified poly(glycidyl methacrylate) particles represent a highly

sensitive, potential tool for labeling of antibodies in enzyme-based sandwich-type
immunosensors.
Abstracts Young Scientist Forum – Infection and Inflammation
Induction of cytokines by CVB2-Ohio and its mutants
Benkoova B1, Borsanyiova M1, Sarmirova S1, Lindberg M2, Bopegamage S1

1EnterovirusLaboratory, Faculty of Medicine, Slovak Medical University in Bratislava,
Limbová 12, 833 03, Bratislava, Slovakia
2Linnaeus University, Kalmar, Sweden SE-391 82 Kalmar, Sweden
Corresponding author: E mail: brigita.benkoova@szu.sk
Keywords: coxsackieviruses, immunity, cytokines response, mice
Introduction: Coxsackieviruses (CV) show diverse clinical manifestations which are closely
linked to their genetics, complex tissue tropism and host immune response. They induce innate
and adaptive immune response which consists of monotypic and heterotypic antibodies. Our
aim was to study the cytokines of the innate immune response, induced by prototype CVB2
(Ohio) and its mutants in two different strain of mice.
Materials and Methods: Two strains of mice A/J (inbred) and CD1 (outbred) were infected
with prototype coxsackievirus B2 prototype strain Ohio-1 (CVB2O) and its four mutants was
CVB2-ORD, CVB2-118, CVB2-164K, CVB2-185R were used for infection. Cytokines
granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemo attractant
protein 1 (MCP-1), interleukin-1β (IL-1β), IL-2, IL-6, IL-9, IL-10, interferon gamma (IFNγ),
and tumor necrosis factor alpha (TNFα) were studied
Conclusion: difference in the cytokine induction by coxsackievirus (Ohio strain) and its four
mutants differed in the two groups of mice. The diversity of the response depended on the day
post infection, type of mouse strain and the mutants. We suggest that these differences may be
reflected in pathogenesis and outcome of infection by these viruses in the two mice strains.
Acknowledgement:
This work was supported by the Norwegian financial mechanism, Mechanism EEA and Slovak
Government and the State Budget of the Slovak Republic (SK 0082) and by the realization of
the project "Center of excellence of environmental health", ITMS No.24240120033 and Cedars
Sinai Medical Center’s International Research and Innovation in Medicine Program, the
Association for Regional Cooperation in the Fields of Health, Science and Technology
(RECOOP HST Association) for their support of our organization as participating Cedars –
The study was approved by the Ethical Committee of the Slovak Medical University Date: 5th
November 2006 and the State Veterinary and Food Control Authority of the Slovak Republic.
Date: 22nd March 2007. Number: C.k Ro 3035/07-221/3.
Possible mechanisms involved in the development of drug resistance in Ruk/CIN85-
overexpressing MCF-7 cells
Pasichnyk G.V.1, Horak I.R.1, Povorozniuk O.O.1, Samoylenko A.A.1, Kamińska-Kaczmarek

B.2, Drobot L.B.1
1 Laboratory of Cell Signaling, Palladin Institute of Biochemistry, National Academy of
Sciences of Ukraine, Kiev, Ukraine

2Laboratory of Molecular Neurobiology, Nencki Institute of Experimental
Biology,Warszawa, Poland
Corresponding author: GannaPasichnyk, e-mail: anya_p@meta.ua
Key words: adaptor proteinRuk/CIN85,NF-κB transcription factor, ABC transporters,
chemoresistance.
Introduction:Resistance to chemotherapeutic agents is a common clinical problem in the
treatment of cancer. Multidrug resistance formation is known to be a multifactor processthat
includes blockage of apoptosis, activation of detoxifying systems andABC transporters as well
as alterations in the cell cycle progression.This biological phenomenon can be realized through
aberrant activation of a set of signaling pathways such as MAPK, Akt/PI3K, NF-κB. The aim
of this study was to investigate the impact of NF-κB transcription factor in the development of
drug resistance inRuk/CIN85-overexpressing MCF-7 cells.
Methods:All experiments were carried out on wild type human breast adenocarcinoma MCF-
7 cells, cells with stable overexpression of Rukl/CIN85 (G4 cells) and G4 cells with down
regulation of Rukl/CIN85. Such approaches as MTT assay, flow cytometry, inhibitory
analysis,luciferase reporter assayand Western blotting were used.
Results and Discussion:Cells with overexpression of Rukl/CIN85 were revealed to bemore
resistant to doxorubicin and etoposide than control cells. To investigate the possible
mechanisms involved in increased chemoresistance of Rukl/CIN85-overexpressing cells, the
activity of ATP-binding cassette membrane transporters were evaluated by study the efflux of
Rhodamine 123 from treated cells. It was revealed that efficiency of Rhodamine 123
elimination was positively correlated with Rukl/CIN85 expression level.In addition, it was
demonstrated that Ruk/CIN85-overexpressing MCF-7 cells are characterized by constitutive
activity of NF-κBtranscription factor that was sensitive to NF-κB inhibitor BAY11-
7085.BAY11-7085 did not influenceRhodamine 123 efflux from MCF-7 cells with the highest
expression level of Ruk/CIN85 while it decreased the efficiency of Rhodamine 123 elimination
from wild type MCF-7 cells. Interestingly, knocking down of Rukl/CIN85 reversed the
observed effects of adaptor protein overexpression on MCF-7 drug resistance.
Conclusion: Overexpression of Ruk/CIN85 in MCF-7 cells results in the development of drug
resistance through non-NF-κB-dependent ABC transporters’ activation.
This work was partially supported by the collaboration program between the NAS of Ukraine
and Polish Academy of Science, by the target complex interdisciplinary program of the NAS
of Ukraine studies “Fundamental basis of molecular and cellular biotechnologies” and the grant
of STCU, #5974.
QSAR Studies of 4-thiazolidinone derivatives showing antitrypanosomal activity
Kryshchyshyn A.1, Deviniak O.2, Grellier P.3, Lesyk R.1*

1Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv
National Medical University, Pekarska 69, Lviv, 79010, Ukraine;
2Department of pharmaceutical disciplines, Uzhhorod National University, Narodna 1,
Uzhhorod, 88000, Ukraine

3National Museum of Natural History, CP 52, 57 Rue Cuvier, 75005 Paris, France;
*e-mail: dr_r_lesyk@org.lviv.net, roman.lesyk@gmail.com
Keywords: antitrypanosomal activity, 4-thiazolidinones, QSAR.
Introduction. Trypanosomiasis belongs to the world’s neglected tropical disease. Inadequate

funding of research and development programs, national health programs for new
antitrypanosomal agents search resulted in that no new drug has been approved during the past
3 decades. Also there are a number of problems associated with the existing drugs, consisting
in toxicity, availability and insufficient drugs’ effectiveness. Given the fact of the
antiproliferative activity of 2-hydrazonothiazolidones, aminoacyl-4-thiazolidinone, 5-
ylidenerhodanine-3-carboxylic acids, thiopyranothiazoles established in in vitro assays on
T.b.b. and T. cruzi, mentioned classes are the promising direction for antiparasitic agents
search. Presented work is the continuation of our study on antitrypanosomal activity of 4-
thiazolidonone’ diverse derivatives.
Methods. Structure-based design, synthesis, antitrypanosomal activity assay, Quantitative
structure–activity relationship (QSAR) analysis.
Results and discussion. QSAR Models development was carried out using obtained
antitrypanosomal activity data. Evaluation of the activity involved two subspecies: compounds
were tested on T.b.b. and T.b.g. The data from both has been combined together, but the
corresponding binary variable was included into predictor matrix aiming to detect possible
differences in the activity range. The geometry of compounds has been optimized within
MMFF 94x force field, and above 1600 molecular descriptors were calculated with E-Dragon
and MOPAC6; 774 descriptors were retained in the descriptor matrix. Random Forest (RF)
regression, Stochastic gradient boosting, Multivariate adaptive regression splines and Gaussian
processes regression were the algorithms used for QSAR modelling. The plots of actual versus
predicted values for both RF and Gaussian processes models are quite similar and the results
show that both models are valid for screening purposes due to their satisfactory predictive
abilities established in the test.
Conclusion. QSAR-models developed with RF and Guassian processes regression algorithms
describes antitrypanosomal activity and have good predictive ability. The obtained models are
using for current in silico screening of new 4-thiazolidinones in search for antitrypanosomal
hit-compounds.
organization as participating Cedars-Sinai Medical Center-RECOOP Research Centers
(CRRS).
Ethical Committeeof Danylo Halytsky Lviv National Medical University Animal Care and
Use Committee Approval: 18.03.2013, #3
Plasmid-mediated fluoroquinolone resistance in Enterobacteriaceae
O. Szabó
Institute of Microbiology, Semmelweis University, Budapest, Hungary
Key words: antibiotic resistance, plasmid, Enterobacteriaceae
Introduction. Fluoroquinolone resistance developes by chromosomal mutations and by

plasmid-mediated mechanisms in Enterobacteriaceae.
Methods. In our study we investigated plasmid-mediated fluoroquinolone resistance (PMQR)

genes in 214 Enterobacteriaceae strains from urinary tract infections at Semmelweis
University. The antibiotic susceptibility was determined by Mueller-Hinton broth
microdilution for ciprofloxacin, cefotaxime, ceftriaxone, amikacin and tobramycin. QnrA,
qnrB, qnrC, qnrD and qnrS resistance genes were investigated by PCR. In case of qnrD
positive strains gyrA, gyrB, parC and parE genes were investigated by PCR and DNA
sequencing; the results were analyzed by NCBI GenBank database.
Results. Ciprofloxacin MIC values of tested strains ranged between 0.06 and 128 μg/ml.
Altogether 15 qnr positive strains were detected: 6 Escherichia coli with qnrS, 2 E. coli with
qnrA and qnrS, an E. coli with qnrB, 4 Proteus mirabilis with qnrA and 2 Morganella morganii
with qnrD. The qnrD positive strains were susceptible to cefotaxime, ceftazidime and
ceftriaxone, but were resistant to ciprofloxacin and one was also resistant to tobramycin and
amikacin. A qnrD-harbouring plasmid was investigated by inverse PCR and primer walking,
with set of primers designed in this study. Positive PCR products were sequenced and a 2662
bp plasmid was identified. It coded qnrD1 gene and it was flanked by mobile insertion
cassettes. In parC gene an S80I aminoacid change was detected.
Discussion. In our study we found 26,6% prevalence for PMQR genes. We identified a new
plasmid carrying qnrD1 resistance gene, which is submitted to GenBank under KU160530
accession number.
Conclusion. Our novel plasmid showes 95-98% similarity to other qnrD-plasmids, that
suggests a stabile genetic structure, however mobile insertion cassettes provide ability for
recombination.
Acknowledgement
Our research team is funded by Hungarian Scientific Fund, Grant number: OTKA 108481.
Selected concepts and investigations among thiazolo[4,5-b]pyridines as perspective
anticancer and anti-inflammatory agents
A. Lozynskyi, B. Zimenkovsky, R. Lesyk

National Medical University, Lviv 79010, Ukraine
Corresponding author: Andrii Lozynskyi, lozisnskij@i.ua
Key words: thiazolo[4,5-b]pyridines, anticancer agents, anti-inflammatory agents.
Introduction. Thiazolo[4,5-b]pyridines are have received considerable attention from the

medicinal chemists owing to their capacity to mimic a biologically important 4-thiazolidinone
fragment in rigidfused system. Therapeutic applications of this template are very broad, and
range from cardiotonicagents (including cAMP PDE III inhibitors) to antimicrobial and anti-
inflammatory compounds. High affinity ligands have been obtained also for H3-histamine,
metabotropic glutamate 5 (mGluR5)and epidermal growth factor receptors. Owing to the high
number of positive hits encountered with this heterocycle and its congeners, thiazolo[4,5-
b]pyridines template certainly deserves the title of “hit-compounds” in medicinal chemistry.
Bioactive thiazolo[4,5-b]pyridines Optimization of core heterocyclic template
CH3 variety substituents

H 3C N H H 3C
N N H
N N N
O N
O N
S H 2N
S S O
HOOC
N H 3C S
O
Vasodilatatory activity Antimicrobial activity Anti-inflammatory activity
Methods. It has been analysed the corresponding research in synthesis and adopted synthetic
procedure of obtaining of appropriate biologically active thiazolo[4,5-b]pyridinesin the
reaction [3+3]-cyclocondensation .
Resultsand discussion. All the new thiazolo[4,5-b]pyridine derivatives were synthesized from
4-amino-5Н-thiazol-2-one and various arylidene pyruvic acids or chalcones in the reaction
[3+3]-cyclocondensation. The raw of thiazolo[4,5-b]pyridine-5-carboxylic acid amides were
synthesized by the interaction of corresponding acid chlorides with various amines in
anhydrous dioxane. As a result, the library of new thiazolo[4,5-b]pyridine-based heterocycles
for search of new anticancer and anti-inflammatory agents have been designed and synthesized.
The synthesized compounds were selected by National Cancer Institute (NCI) Developmental
Therapeutic Program (www.dtp.nci.nih.gov) for the in vitro cell line screening to investigate
their anticancer activity and evaluated for their and anti-inflammatory activity on the
carrageenan oedema model in rats.
Conclusion.The preliminary results allowed to identify the active compounds with promising
anticancer and anti-inflammatory activity.
(CRRS).
Ethical Committee or Institutional Animal Care and Use Committee
Approval:18/03/2013 № 3
Abstracts for Annual Review of the Research Activities in RECOOP
Session II
ORAL PRESENTATIONS
Relationship of soluble receptors for advanced glycation end products and metabolic
syndrome in adolescents from Bratislava region
Gurecká R1,2, Koborová I1, Csongová M1, Šebeková K1

1Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava,
Slovakia
2Institute of Medical Physics, Biophysics, Informatics and Telemedicine, Comenius
University, Bratislava, Slovakia
Corresponding author: radana.kollarova@gmail.com
Keywords: metabolic syndrome, adolecents, sRAGE, esRAGE
Introduction: Metabolic syndrome (MS) is defined as a constellation of several clinical

symptoms including central obesity, insulin resistance, atherogenic dyslipidemia and arterial
hypertension. Individuals with MS are at an increased risk for type 2 diabetes mellitus, and
cardiovascular diseases, and the risk increases with the number of its components. The aim of
this study was to estimate the prevalence of MS and its components in population of high-
school students in Bratislava region, and to evaluate the relationship between MS and two
forms of receptor for advanced glycation end products (RAGE). In adults, low sRAGE levels
are independent predictors of later cardiometabolic diseases and overall mortality.
Methods: Data from a cross-sectional study Respect for Health conducted on 2 991 students
aged 14-to-22 years were analyzed. To classify MS, we used modified IDF criteria. Circulating
concentrations of sRAGE (soluble RAGE) and esRAGE (endogenous secretory RAGE) were
assessed using commercial ELISA kits.
Results: Prevalence of MS in general population of Bratislava high-school students was 4.98%,

2,5-times higher in boys than in girls. Concentration of sRAGE and esRAGE decreased with
number of MS components in both genders. In boys, isolated central obesity and high blood
pressure were associated with lower circulating levels of both receptors.
Discussion: The prevalence of MS in our study was similar to those reported from other
European countries. We revealed an inverse relationship between concentrations of both
sRAGE and esRAGE with number of MS components. These relations were mostly, but not
exclusively, determined by presence of central obesity.
Conclusion: We showed that already in adolescence, MS and some of its components are
accompanied with changes in circualting concentrations of sRAGE and esRAGE.
Study was approved by the Ethics Committee of Bratislava Self-Governing Region on

19.04.2011
Supported by Bratislava Self-governing region, grants APVV 0447-12 and VEGA 1/0637/13.
The effects of anthocyanin-rich wheat diet on the oxidative status and behavior of rats
Janšáková K.1, Bábíčková J.1,2, Havrlentová M.3,4, Hodosy J.1,2,5, Kraic J.2,3, Celec P.1,2,6,7,
Tóthová Ľ.1,2
1
Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia
2
Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia
3
Research Institute of Plant Production, Piešťany, Slovakia
4
Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius,
Trnava, Slovakia
5
Institute of Physiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia
6
Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia
7
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava,
Slovakia
Corresponding author Katarína Janšáková jansakova.katarina@gmail.com
Key words: anthocyanins, functional food, advanced glycation end products,

malondialdehyde, behavioral phenotyping
Introduction: Flavonoid-rich food containing anthocyanins decreases oxidative stress in

systemic diseases and has positive behavioral effects. Anthocyanins in food have antioxidant
and anti-inflammatory effects, but wheat enriched with anthocyanins has not been tested yet.
The aim of this study was to evaluate the effect of food based on anthocyanin-enriched wheat
on oxidative status and behavior of healthy rats.
Methods: Twenty male Wistar rats were randomly divided into a control group receiving
common wheat food and an anthocyanin group receiving anthocyanin-enriched wheat for 2
months. Oral glucose tolerance test and proteinuria were analyzed monthly creatinine clearance
was analyzed at the end of the experiment. Behavioral analysis using Phenotyper cages was
performed to assess general locomotor activity and anxiety. Serum and tissue samples were
collected to measure markers of oxidative stress and antioxidant status.
Results: Anthocyanins increased total antioxidant status in serum, decreased advanced
oxidation protein products, but increased thiobarbituric acid reactive substances in the kidney.
In the liver and colon, markers of oxidative stress were comparable between the groups. No
significant differences were found in renal functions and glucose metabolism. Rats in the
anthocyanin group showed significantly higher anxiety-like behavior.
Discussion and conclusion: Food containing wheat enriched with anthocyanins had some
positive effects on antioxidant status and protein oxidation, but increased lipid peroxidation in
the kidneys and anxiety-like behavior. The molecular mechanisms explaining the negative
effects should be further elucidated. Follow-up studies will focus on the potentially beneficial
effects in disease models associated with increased oxidative stress such as inflammation.
Anthocyanin-rich whole grains did not affect the vital functions of the organism. Potential
incorporation of anthocyanins into common daily food may be safe. Anthocyanins have great
beneficial properties which could serve as prevention for systemic diseases of organisms.
Source of research support: This study was supported by the Center of Excellence for
Information Biomacromolecules in Prevention of Diseases and Quality of Life Improvement
ITMS 26240120003.
Ethical approval number 05/2013/SKP1012 received from the Ethics Committee of the
Institute of Molecular biomedicine, Faculty of Medicine, Comenius University on 29.1.2013
Interleukin 6/Wnt interactions in rheumatoid arthritis: Interleukin 6 inhibits Wnt
signaling in synovial fibroblasts and osteoblasts
Malysheva Kh.1,2, de Rooij K.3, Löwik C.3, Baeten D.4, Rose-John S.5, Stoika R.1,
Korchynskyi O.1,3,4
1Institute of Cell Biology, NAS of Ukraine, 14/16, Drahomanov St., Lviv 79005, Ukraine
2Insitute of Animal Biology, NAAS of Ukraine, 38, V. Stus St., Lviv 79034, Ukraine
3Leiden University Medical Center, 2, Albinusdreef, 2333 ZA Leiden, The Netherlands
4Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
5Institute of Biochemistry, Christian-Albrechts-University, Kiel, D-24098, Germany
Corresponding author: khrystyna.malysheva@gmail.com.
Keywords: rheumatoid arthritis, inteleukin-6, bone morphogenetic protein, Wnt signaling

pathway, mesenchymal precursor cells.
Introduction. Rheumatoid arthritis (RA) is a severe autoimmune inflammatory disorder,

which etiology remains unknown. It has been demonstrated that interleukin (IL) 6 and tumor
necrosis factor-α (TNFα) play a crucial role in RA pathophysiology. The bone morphogenetic
protein (BMP) and Wnt proteins are key players that induce and support cartilage and bone
formation and maintenance.
Aim. To evaluate an impact of previously unrecognized negative interaction between the Wnt
and IL-6 signaling pathways in skeletal tissues as a possible major mechanism leading to age-
and inflammation-related destruction of bone and joints.
Methods. Luciferase reporter assays were performed to monitor Wnt pathway activation upon
IL-6 and TNFα treatment in primary human synovial fibroblasts and mouse NIH-3T3
fibroblasts. In vitro evaluation of functional contribution of IL-6 and TNFα interaction to
inhibition of bone formation was performed by using small hairpin RNAs (shRNA) in mouse
mesenchymal precursor cells (MPC) of C2C12 and KS483 lines induced to differentiate into
osteoblasts by BMPs.
Results and Discussion. We found that IL-6 inhibits activation of Wnt signaling in primary
human synoviocytes, and IL-6 cooperatively with TNFα and Dickkopf-1 inhibits the activation
of Wnt response. ShRNA-mediated knockdown of IL-6 mRNA significantly increased early
BMP2/7-induced osteogenesis and rescued it from the negative effect of TNFα in C2C12 cells
as well as intensified bone matrix mineralization in KS483 cells.
Conclusion. IL-6 is an important mediator in inhibition of the osteoblast differentiation by
TNFα, and knockdown of IL-6 partially rescues osteogenesis from negative control of
inflammation. The anti-osteoblastic effects of IL-6 and DKK-1 cooperation are most likely
mediated by their negative action in Wnt signaling pathway.
BioEthics Committee Approval. All human subject samples were collected after approval by
the Institutional Review Board of the Academic Medical Center/University of Amsterdam,
Amsterdam, The Netherlands (Protocol MEC 07/079 #10.17.0708) and provision of informed
consent by the patients.
Acknowledgement. The study was supported by Cedars Sinai Medical Center’s International
Exploiting Hydrogen Sulfide of Novel 4-Thiazolidinone Derivatives in Cytoprotection of
Small Intestine Under Indometacin-Induced Injury
I. Ilkiv, R. Lesyk, O. Sklyarov

Department of Biochemistry, Danylo Halytsky Lviv National Medical University, Lviv
79010, Ukraine
Corresponding author: Iryna Ilkiv, ira9ilkiv@gmail.com
Key words: hydrogen sulfide, small intestine, indomethacin-induced injury, 4-thiazolidinones.
Introduction. Various endogenous and exogenous (NSAIDs, stress) agents can lead to
gastrointestinal mucosa injury. Considerable interest belongs to mucosal defense factors such
as prostaglandins, nitric oxide and H2S. For this reason, the purpose of our study was to
investigate the influence of novel 4-thiazolidinone, which release H2S, in protective
mechanisms of small intestine.
Methods. The experiment was performed on 40 white rats. Indometacin-induced injury (IMII)
caused in the ulcerogenic dose (35 mg/kg, subcutaneously). A series of 4-thiazolidinones (Les-
5054 and Les-5055) were administered three times intragastrically at a single dose 10 mg/kg.
In homogenates of the mucous of small intestine (MSI) were determined the activity of NO-
synthases, myeloperoxidation, superoxide dismutase (SOD), the content of nitrite anion (NO2-
) and MDA.
Results. The activity of iNOSin MSI increased in 2 times, as well as the content of MDA and
NO2-(p<0,05; 43% and 138 % respectively) under condition of IMII as compared to the indiсes
of the control group. Les-5054 on the background of IM effect decrease activity of iNOS for
38 % as well as content of MDA and NO2- (p<0,05; 29 % and 57 % respectively) and increase
the activity of cNOS for 37 % (p<0,05) compared with indeces of IMII group. Parameters of
NO-synthase system in Les-5055-treated group showed the same tendency as under the effect
of Les-5054.
Conclusion. The preliminary results allowed us to identify the tested compounds as perspective
H2S-donors in the searching of cytoprotective agents among biologically active 4-
thiazolidinones.
(CRRS).
Ethical Committee or Institutional Animal Care and Use Committee Approval:

DHLNMU 16/03/2015 № 3
Application of novel polymeric nanocarrier for temozolomide delivery to glioblastoma
cells
Finiuk N.1, Senkiv J.1, Molochiy N.2, Zaichenko O.3, Avdieiev S.4, Stoika R.1
1 Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, NAS
of Ukraine, Lviv, Ukraine
2 Department of Biochemistry, Ivan Franko National University of Lviv, Lviv, Ukraine
3 Department of Organic Chemistry, Lviv National Polytechnic University, Lviv, Ukraine
4 Department of Functional Genomics, Institute of Molecular Biology and Genetics, NAS of
Ukraine, Kyiv, Ukraine
Corresponding author: nataliyafiniuk@gmail.com
Key words: glioblastoma, temozolomide, apoptosis, polymeric nanocarrier
Introduction. Human glioblastoma is the most common primary brain tumor with poor
prognosis (3% survival). Currently, anti-neoplastic treatment combines chemotherapy
(temozolomide, TMZ), radiotherapy, and surgery.
Aim. Study of cytotoxity of temozolomide immobilized on novel PEGylated polymeric

nanocarrier (TMZ-PC) and of mechanisms of TMZ-induced glioma cell death.
Methods. MTT and trypan blue exclusion assays, fluorescent microscopy, Western-blot
analysis, cell culture.
Results and discussion. TMZ dose-dependently inhibited the viability of human glioblastoma
T98G and rat brain glioma C6 cell lines. TMZ-PC action was more pronounced than that of
free TMZ. TMZ’s IC50 was 243 uM for T98G cells, while its immobilization by novel
polymeric nanocarrier reduced IC50 to 166 uM. TMZ-PC induced apoptosis in malignant
glioma cells (Annexin V-FITC/PI staining). TMZ-PC-induced apoptosis was accompanied by
phosphorylation of JNK, STAT3 and Rb proteins. Immobilization of TMZ on the PC also
increased drug intercalation into DNA. Synergistic effect of Doxorubicine and TMZ was
detected when human glioblastoma T98G cells were treated.
Conclusion. Immobilization of TMZ with novel polymeric nanocarrier leads to an increase in

cytotoxity of TMZ and does not change the mechanism of its action. This makes studied PC-
TMZ complex perspective for future preclinical investigations.
Acknowledgement. The study was supported by Cedars Sinai Medical Center’s International
Mapping of Residues of Fibrinogen Cleaved by Protease II of Bacillus thuringiensis var.
israelensis IMV B-7465
Stohniy E.M.1, Chernyshenko V.O.*, 1, Nidialkova N.A. 2, Rebriev A.V. 1, Varbanets L.D. 2,
Hadzhynova V.E. 1, Chernyshenko T.M. 1, Kolesnikova I.M. 1, Lugovskoy E.V. 1
Palladin Institute of Biochemistry, NAS of Ukraine

Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
Corresponding author: Volodymyr Chernyshenko, e-mail: bio.cherv@gmail.com
Keywords: protease, Bacillus thuringiensis, fibrinogen, αC-domain
Introduction: Proteases are known to have strong specificity to distinct peptide bonds of
proteins. The limited proteolysis of macromolecules allows obtaining of the fragments that
possess the features or preserve the structure of the whole molecule and could be used in the
study of proteins structure and functions. Proteases targeted to fibrinogen and fibrin are of
interest as the source for obtaining of physiologically active fragments of fibrin(ogen) and for
direct defibrination in vivo. That is why the aim of our work was to study the proteolytic action
of Protease II (PII) purified from Bacillus thuringiensis var. israelensis IMV B-7465.
Methods: Hydrolysis products of fibrinogen by PII were analysed by SDS-PAGE under

reducing conditions with further immunoprobing using the mouse monoclonal 1-6B (anti-
Aα509-610) and ІІ-5С (anti-Aα20-78) antibody. MALDI-TOF analysis of fibrinogen
hydrolysis products was performed using a Voyager-DE. Results were analyzed by Data
Explorer 4.0.0.0.
Results & Discussion: SDS-PAGE showed that PII cleaved preferentially the Aα-chain of
fibrinogen. Western-blot analysis carried out using monoclonal antibodies allowed us to detect
the product with apparent molecular weight of 10 kDa that corresponded the C-terminal part
of Aα-chain of fibrinogen molecule. MALDI-TOF analysis of products of hydrolysis of
fibrinogen by PII allowed to detect the main peak occurs at mass/charge (M/Z) ratio of 11 441
Da. According to «Peptide Mass Calculator» this peptide corresponded to fragment Аα505-
610 of fibrinogen molecule. It showed that PII cleaves the peptide bond AαAsp-Thr-Ala504-
Ser505.
Conclusions: Western-blotting using monoclonal antibody and MALDI-TOF analysis showed

that PII derived from cultural medium of B. thuringiensis var. israelensis IMV B-7465 cleaves-off
the C-terminal peptide of Аα-chain of human fibrinogen. Thus, PII can be used for the
obtaining of unique fragments of fibrinogen molecule. As far as αC-domain contains numerous
sites of intermolecular interactions we can consider PII as a prospective agent for the
defibrination.
Synthesis of potential antiviral agents among chromeno[4’,3’:4,5]thiopyrano[2,3-
d][1,3]thiazole-5-carboxylates
Nataliya Zelisko, Roman Lesyk

National Medical University, Pekarska 69, Lviv, 79010, Ukraine
Corresponding author : Nataliya Zelisko, NataljaZelisko@gmail.com
Key words: chromeno[4’,3’:4,5]thiopyrano[2,3-d][1,3]thiazoles, hetero-Diels-Alder reaction,

antiviral activity.
Introduction. The hetero-Diels–Alder cycloaddition is the one of the most powerful methods
for the synthesis of polycyclic compounds. It has been used as an excellent synthetic instrument
for obtaining thiopyrano[2,3-d][1,3]thiazole derivatives. The mentioned compounds are
promising area of research because of their diverse biological activities. Maleic and fumaric
acids and their derivatives are good dienophiles and react with a variety of dienes to afford the
corresponding cyclohexene system. We decided to evaluate the reaction between 5-(ortho-
hydroxybenzylidene)-substituted isorhodanines and esters of fumaric or maleic acids as
dienophiles.
Method. It has been analysed the corresponding research in synthesis and adopted synthetic
procedure for obtaining appropriate biologically active chromeno[4’,3’:4,5]thiopyrano[2,3-
d][1,3]thiazoles in hetero-Diels-Alder reaction.
Results and discussions. Compounds 1a-h were studied with above-mentioned esters and we
have got interesting acylation-hetero-Diels-Alder process forming only tetracyclic fused
derivatives 2a-h. Completely unexpected results were obtained for the esters of fumaric acid
with the same stereochemistry, as starting with maleic acid. In our opinion, the formation of
tetracyclic products 2 with cis-configuration of the protons at positions 5 and 5a, 5a and 11b in
case of esters of fumaric acid could be explained by the mechanism that includes retro Michael
addition reaction followed by stereoselective Michael addition cyclization.
1
COOR
Method A 2a R = H, R1 = n-C7H15,
1 72% (A)
COOR
2b R = 10-NO2, R1 = n-C7H15,
H
N 71% (A)
S OH AcOH, reflux, 4h O S H
61-81% COOR
1 2c R = 8,10-Br2, R1 = n-C7H15,
S 80% (A)
HN H
R COOR
1 2d R = 9-OH, R1 = n-C7H15,
S H O
73% (A)
O 1 Method B O 2e R = H, R1 = Et,
10 81% (A), 79% (B)
1a R = H COOR
1 3
R 8 2f R = 8,10-Br2, R1 = Et
1d R = 5-Br 9
1f R = 5-NO2 72% (A), 70% (B)
AcOH, reflux, 4h 2g R = H, R1 = Me,
1h R = 3,5-Br2 63-79% 61% (A), 63% (B)
1i R = 4-OH
2h R = 10-NO2, R1 = Me,
65% (A), 70% (B)
We also investigated antiviral activity for synthesized compounds in vitro against Influenza
Virus Type A. Compounds 2a and 2f showed significant activities with a 50% effective
concentration (EC50) = 0.6 mg/ml, selective index (SI) of >170 and EC50=0.31, SI>320
respectively, which are higher than commercial Ribavirin.
Conclusion. 5-(2-Hydroxybenzylidene)-4-thioxothiazolidin-2-ones and esters of maleic or
fumaric acids undergone tandem acylation-hetero-Diels-Alder reaction diastereoselectively
providing rel-(5R,5aR,11bS)-2,6-dioxo-3,5a,6,11b-tetrahydro-2Н,5H-chromeno[4’,3’:4,5]
thiopyrano[2,3-d][1,3]thiazole formation regardless of dienophile isomerism. The bioassay
results allowed to identify the active compounds with promising antiviral activity.
(CRRS).
Ethical Committee or Institutional Animal Care and Use Committee Approval:

19/03/2014 № 2
Experimental Works
of RECOOP Members
(Ukraine)
78
UDC 577.151. 6
Mapping of Residues of Fibrinogen

Cleaved by Protease II of Bacillus thuringiensis
var. israelensis IMV B-7465
E. M. Stohniy1, V. O. Chernyshenko1*, N. A. Nidialkova2, A. V. Rebriev1,
L. D. Varbanets2, V. E. Hadzhynova1, T. M. Chernyshenko1,
I. M. Kolesnikova1, E. V. Lugovskoy1
1
Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv;
2
Zabolotny Institute of Microbiology and Virology, National Academy
of Sciences of Ukraine, Kyiv;
*e-mail: bio.cherv@gmail.com
The limited proteolysis of macromolecules allows obtaining the fragments that preserve the structure
and functional properties of the whole molecule and could be used in the study of proteins structure and
function. Proteases targeted to fibrinogen and fibrin are of interest as the tool for obtaining of functionally
active fragments of fibrin(ogen) and for the direct defibrination in vivo. That is why the aim of the present work
was to study the proteolytic action of Protease II (PII) purified from Bacillus thuringiensis var. israelensis
IMV B-7465 on fibrinogen.
Hydrolysis products of fibrinogen by PII were analysed by SDS-PAGE under reducing conditions
with further immunoprobing using the mouse monoclonal 1-5A (anti-Aα509-610) and ІІ-5С (anti-Aα20-78)
antibodies. It was shown that PII cleaved preferentially the Aα-chain of fibrinogen splitting off the peptide
with apparent molecular weight of 10 kDa that corresponded the C-terminal part of Aα-chain of fibrinogen
molecule.
MALDI-TOF analysis of hydrolysis of fibrinogen was performed using a Voyager-DE. Results analyzed
by Data Explorer 4.0.0.0 allowed to detect the main peak occurring at mass/charge (M/Z) ratio of 11 441 Da.
According to «Peptide Mass Calculator» this peptide corresponded to fragment Аα505-610 of fibrinogen
molecule. The result showed that PII cleaves the peptide bond AαAsp-Thr-Ala504-Ser505.
Thus, PII can be used for the obtaining of unique fragments of fibrinogen molecule. As far as αC-
domain contains numerous sites of fibrin intermolecular interactions we can consider PII as a prospective
agent for their study and for the defibrination.
K e y w o r d s: protease, Bacillus thuringiensis, fibrinogen, αC-domain, limited proteolysis.
all three chains of fibrinogen molecule [9]. Most of
P
roteases could be found in pathogenic and
nonpathogenic species of microorganisms bacterial fibrinogenases cleave both fibrinogen and
could be targeted to proteins of human and polymeric fibrin [10, 11]. These properties allowed
other mammals [1-4]. Fibrinogen as big and labile authors to conclude the potential anti-thrombotic use
molecule could be a cleaved by proteases more or of the enzymes in peptide-based cardiovascular drug
less specifically. Fibrinogen-specific metallopro- development [7-9, 12]. Some of them were already
teases were purified from Serratia sp. [5] and Pseu- tested in animal models [8] or were cloned for these
domonas aeruginosa [6], serine proteases from purposes [13].
Brevibacillus brevis [7], Bacillus sp. [8], Bacillus Proteases targeted to fibrinogen and fibrin also
cereus [9], Vibrio metschnikovii [10], Aeromonas are of interest as the source for obtaining of physio
sobria [11]. Their molecular weight ranges from 30 logically active fragments of fibrin(ogen) and for
to 60 kDa. Some of them preferentially degrade the direct defibrination in vivo. That is why the aim of
Aα-chain of fibrinogen [9, 11], others are targeted to our work was to study the proteolytic action of PII
both Aα- and Bβ-chains of fibrinogen [6, 8] or even purified from Bacillus thuringiensis var. israelensis
79
IMV B-7465 on human fibrinogen and detection the Solubilised samples were separated by SDS-PAGE
site(s) of proteolysis on fibrinogen molecule. and immunoprobed in Western blot analysis.
Chromogenic substrate assay. Cleavage of
Materials and Methods chromogenic substrates was studied in microtiter
Materials. PII was purified from B. thuring- plates by mixing of 0.05 М Tris-НСl buffer of рН 7.4
iensis var. israelensis IMV B-7465 [14]. Fibrinogen containing 0.13 М NaCl with chromogenic substrates
used in this study was purified from human citrated in the concentration range from 25 to 160 µМ, and
blood plasma according to the method described by PII (0.0005 mg/ml), at 25 °С. Amidase activity of PII
Varetskaya [15] and was further plasminogen deplet- was continuously monitored at 405 nm. The amount
ed on a Lysine-Sepharose affinity column. Chromo- of hydrolyzed substrate was calculated using a mo-
genic substrates used in the study were S2238 (H- lar extinction coefficient of 10.500 M−1·cm−1 for free
D-Phe-Pip-Arg-pNA), S2251 (D-Val-Leu-Lys-pNA), pNA on reader Multiskan EX [18].
S2765 (Z-D-Arg-Gly-Arg-pNA), S2236 (pyro-Glu-
Results and Discussion
L-Pro-L-Arg-pNA), S2302 (H-D-Pro-Phe-Arg-pNa)
and S1040 (Glp-Ala-Ala-Leu-pNа) (Chromogenics, Previously was shown that PII purified from the
Sweden). Mouse monoclonal 1-6B (anti-Aα509-610) B. thuringiensis var. israelensis IMV B-7465 could
and ІІ-5С (anti-Aα20-78) antibodies were designed effectively degrade fibrinogen [19]. To identify the
and purified in Protein Structure and functions de- region of fibrinogen molecule attacked by PII, the di-
partment of Palladin Institute of biochemistry, NAS gestion mixture was analyzed by SDS-PAGE under
of Ukraine. reduced conditions. Plasminogen-free human fibrin-
SDS-PAGE/Western blot. The molecular ogen was incubated with PII at ambient temperature.
weights and purity of proteins were determined by Upon incubation of fibrinogen with PII, the Bβ- or
SDS-PAGE using 10 or 12% gels accordingly to γ-chains of the molecule appeared to be the same
Laemli [16]. Hydrolysis products of fibrinogen and as those of control fibrinogen whereas the Aα-chain
fibrin obtained by PII action were also analyzed by gradually disappeared, resulting in the formation of
SDS-PAGE under reducing conditions. The sepa- a truncated form of about 58 kDa (Fig. 1). The low-
rated proteins were further transferred to a nitrocel- molecular weight fragment of Aα-chain cleaved-off
lulose membrane in order to specify the bands by by PII was detected using SDS-PAGE in 15% po-
immunoprobing. The membrane was blocked with lyacrilamide gel without β-mercaptoethanol as a
5% milk in PBS for an hour, incubated with a mouse polypeptide with molecular weight of about 12 kDa
monoclonal antibody to Bβ26-42 or to Aα20-78 for (Fig. 2).
another hour and then developed with a secondary To localize the PII-sensitive area within the
HRP-labeled goat anti-mouse antibody. The bands fibrinogen Aα-chain, we used specific monoclonal
were visualized using 0,001 M 4-chloro-1-naphtol antibody to the C-terminal Aα509-610 (1-5A) [20]
solution in 0.5 M Tris-НСl) pH 7.5 and 0.03% H2O2. and to the N-terminal Aα20-78 (ІІ-5С) [21] portions
Mass-spectrometry. MALDI-TOF analysis of of fibrinogen Aα-chain for immunoprobing of SDS-
purified fibrinogenase was performed using a Voya PAGE separated proteins of the digestion mixture
ger-DE (Applied Biosystems, USA). Н+-matrix ioni- (Fig. 3, 4). In the case of I-5A antibody use the traces
zation of polypeptides under sinapic acid (Sigma- of the native fibrinogen Aα-chain which was slightly
Aldrich) was used. Results were analyzed by Data visible after 30 minutes of incubation with PII and
Explorer 4.0.0.0 (Applied Biosystems) [17]. almost completely disappeared after 60 minutes
Fibrinogenolytic activity of protease. Fibrino- while the low-molecular weight fragment of Aα-
gen (1.5 mg/ml) was mixed with PII (0.005 mg/ml) chain appeared (Fig. 3). On the other hand, when ІІ-
in TBS-buffer pH 7.4. The molar enzyme/substrate 5С antibody was used we observed high-molecular
ratio was 1:30. The mixture was incubated during weight fragment of Aα-chain that contain N-termi-
5, 10, 15, 20, 25, 30, 40, and 60 minutes at 25 °C for nal portions of the Aα-chain (Fig. 4).
SDS-PAGE or for 30 and 60 minutes for the Western Thus we can conclude that PII selectively de-
blot analysis. The hydrolysis was terminated by the grades Aα-chain of fibrinogen releasing a polypep-
addition of electrophoresis sample buffer containing tide with average molecular weight of about 12 kDa.
2% SDS, 5% glycerine and 2% β-mercaptoethanol. This molecular weight was checked by MALDI-TOF
80
M C 5 10 15 20 25 30 40 60
170–
130–
95–
72–
55–
43–
34–
26–
17–
11–
Fig. 1. SDS-PAGE of fibrinogen (1.5 mg/ml) digested by PII (0.005 mg/ml). Incubation time 5-60 min. C – na-
tive fibrinogen; M – molecular weight markers. Samples were analyzed under reducing conditions (0.2%
β-mercaptoethanol)
1 2 M 3 2 1
72 Aα
55
55
43
40
34
33
26
24
17
αC′′
17
Fig. 3. Western blot analysis of fibrinogen digested

by PII (0.01 mg/ml). M – molecular weight markers;
11 1 – native fibrinogen; 2 – fibrinogen after 30 min of
hydrolysis; 3 – fibrinogen after 60 min of hydrolysis.
Samples were analyzed under reducing conditions
Fig. 2. SDS-PAGE of fibrinogen digested by PII
and immunoprobed by anti-fibrinogen antibody
(0.005 mg/ml) during 60 min. 1 – molecular weight
1-5A (anti-Aα509-610) targeted to the C-terminal
markers; 2 – hydrolyzed fibrinogen
parts of αC-domains
81
M 1′ 2′ 3′ 4′ drophobic Leucine (as in S1040 substrate) and was
less specific to the bond formed by C-group of posi-
tively charged Lysine proceeded by the hydrophobic
72 Aα Leucine (as in S2251 substrate) (Fig. 6). Proteases
55 α′′ targeted to peptide bonds formed by C-groups of
43 hydrophobic amino acids are not numerous but Leu-
34 cine-specific proteases were reported [22] and one of
26 such proteases was purified from Bacillus sp. [23].
It is also known that substrates containing Alanine
or Valine in the P1 position are specific for elastase
[24]. So the data on specificity of PII confirmed the
data of MALDI-TOF analysis of fibrinogen-derived
peptide formed by this enzyme.
Fibrinogen αС-regions are distant C-terminal
parts of Аα-chains (Аα392-610) that binds tothe cen-
Fig. 4. Western blot analysis of fibrinogen digested tral portion of the molecule. After the conversion of
by PII (0.01 mg/ml). M – molecular weight markers; fibrinogen to fibrin αС-regions dissociate from the
1 – native fibrinogen; 2 – fibrinogen after 30 min of central region and are available for intermolecular
hydrolysis; 3 – fibrinogen after 60 min of hydrolysis. interaction [25-27]. These parts of molecule take part
Samples were analyzed under reducing conditions in polymerisation of fibrin [28], they contain Arg-
and immunoprobed by anti-fibrinogen antibody ІІ- Gly-Asp residues (Аα572-574) that interacts with
5С (anti-Aα20-78) targeted to the N-terminal part platelet receptors [29] and support endothelial cells
of α-chain migration and proliferation [30]. Using the forms of
fibrinogen with removed αС-regions is known as an
analysis. We compared the spectra of the mixture approach for the study of their role in biological pro-
of fibrinogen with PII before the incubation (A) and cesses [31, 32]. That is why characterisation of new
after 60 min of incubation (B). The main peak that proteases targeted to the residues of αС-regions is
appeared after hydrolysis occurred at mass/charge of interest for the study of fibrinogen structure and
(M/Z) ratio of approximately 11441 (Fig. 5) gener- functions.
ated by a polypeptide of 11.441 kDa bearing one On Fig. 7 are shown C-terminal residues of
charge. Other peaks were minor and did not repeat fibrinogen Aα-chain with points of proteolysis by
themselves across multiple spectra. several enzymes. Plasmin that is serine protease in-
Analysis of C-terminal portions of the fin- volved in fibrinolysis has numerous proteolytic sites
brinogen Aα-chain in "Peptide Mass Calculator" at the αС-regions [33]. Among them Lys509-Thr510
(http://www.peptidesynthetics.co.uk/tools/) showed and Lys584-Met585 are located in distant C-termi-
that peptide with molecular weight of 11445.317 Da nal parts of the Aα-chains [34].
corresponded to the peptide "stgktfpgffspmlgefvs- There are the list of reports on proteases that
etesrgsesgiftntkessshhpgiaefpsrgksssyskqftsstsyn- attack αС-regions but the specificity is not revealed
rgdstfesksykmadeagseadhegthstkrghaksrpv", that for most of these enzymes [35-37]. Recently de-
could be formed after cleavage of the peptide bond scribed serine protease from the venom of Echis
AαAsp-Thr-Ala504-Ser505. multisquamatis cleaves Arg491-His492 peptide
Surprisingly this observation showed that PII bond releasingpeptide Aα492-610 [38]. Macrophage
predominantly cleaves fibrinogen at the carboxyl elastase characterised in the work [39] attacks
side of the non-polar hydrophobic amino acid Ala- Glu520-Phe521. Hementin purified from the poste-
nine. To approve this conclusion we performed the rior salivary glands of the leech Haementeria ghilia-
chromogenic substrate assay and compared the ac- nii is able to cleave the peptide bond Ala498-Ala499
tivity of PII towards chromogenic substrates S2238 [40]. Remarkably hementin is specific to the bond
(H-D-Phe-Pip-Arg-pNA), S2251 (D-Val-Leu-Lys- formed by C-terminal group of the hydrophobic Ala-
pNA), S2302 (H-D-Pro-Phe-Arg-pNa), S1040 (Glp- nine. According to our results PII of B. thuringien-
Ala-Ala-Leu-pNа). We showed that the PII was more sis var. israelensis IMV B-7465 cleaved-off peptide
specific to peptide bonds formed by C-group of hy- АαAla505-Ser610 from fibrinogen molecule.
82
A
60
50
40
% Intensity
30
20 10528.19
12427.83
10
0 0
10000 11000 12000 13000
Mass (m/z)
B 11441.15
60
50
40
% Intensity
10528.19
30
20
12427.83
10
0 0
10000 11000 12000 13000
Mass (m/z)
Fig. 5. MALDI-TOF spectrum of fibrinogen hydrolyzed by PII. A – MALDI-TOF spectrum before hydrolysis;
B – MALDI-TOF spectrum after hydrolysis
A new fibrinogen-specific protease from the Addendum

B. thuringiensis var. israelensis IMV B-7465 was All authors contribute to the work equally. Eu-
described. It was established that the target of its gene Stohniy performed SDS-PAGE and Western
proteolytic action on fibrinogen is AαAla494- Blot experiments, Volodymyr Chernyshenko con-
Ser495 peptide bond. As far as proteases with such tributed to the study design and acquisition of data.
specificity have not been described previously we Natalya Nidialkova purified and characterized the
could suggest its potential use for obtaining of the enzyme. Andriy Rebriev performed MALDI-TOF
unique digested forms of fibrinogen. Fibrinogen analysis. Tamara Chernyshenko purified and charac-
desAα505-610 could be used in the study of the role terized human fibrinogen used in the study. Veronika
of distant C-terminal portions of fibrinogen αС- Hadzhynova and Iryna Kolesnikova constructed the
regions in the protein and cellular interactions. Fur- monoclonal antibody that was used in study. Eduard
ther studies of coagulability of digested fibrinogen Lugovskoy and Liudmyla Varbanets contributed to
as well as the study of PII action in vivo will allow the analysis and interpretation of data, as well as the
to assume the possible use of PII as anticoagulant drafting of the article. All authors approved the final
agent. version of the article to be published.
83
100
90
80
Activity, μM/(min·mg) 70
60
50
40
30
20
10
0
S1040 S2251 S2238 S2302
Chromogenic substrates
Fig. 6. Amidase activity of PII towards some chromogenic substrates: S2238 (H-D-Phe-Pip-Arg-pNA), S2251
(D-Val-Leu-Lys-pNA), S2302 (H-D-Pro-Phe-Arg-pNa), S1040 (Glp-Ala-Ala-Leu-pNа)
Fig. 7. Amino acid residue of C-terminal points of Aα-chain of fibrinogen. Points of hydrolysis by different
proteases are marked with the spaces. Terminal amino acids of resulting peptides are marked
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86
UDC 57.08+576.5+54.057+547.311
Modulation of temozolomide action

towards rat and human glioblastoma cells
in vitro by its combination with doxorubicin
and immobilization with nanoscale
polymeric carrier
N. S. Finiuk1, J. V. Senkiv1, A. O. Riabtseva2,
N. Y. Mitina2, N. I. Molochii1, M. O. Kitsera1,
S. S. Avdieiev3, O. S. Zaichenko2, R. S. Stoika1
1
Institute of Cell Biology, National Academy of Science of Ukraine, Lviv;
2
Lviv National Polytechnic University, Ukraine;
3
Institute of Molecular Biology and Genetics, National Academy
of Science of Ukraine, Ukraine;
е-mail: stoika@cellbiol.lviv.ua
Malignant gliomas (glioblastoma multiforme and anaplastic astrocytoma) occur more frequently than
other types of primary central nervous system tumors, having a combined incidence of 5–8/100,000 popula-
tion. Even with aggressive treatment using surgery, radiation, and chemotherapy, median reported survival
is less than one year. Alkylating agents, such as temozolomide (TMZ), are among the most effective cyto-
toxic agents used for malignant gliomas, however, the responses still remain poor. Here, we present data
about an enhancement of TMZ treatment effect towards rat and human glioma cells in vitro by immobilizing
this drug with a new nanoscale polymeric-phospholipidic delivery system. It is a water-soluble comb-like
poly(PM-co-GMA)-graft-PEG polymer consisting of a backbone that is a copolymer of 5-tert-butyl-peroxy-
5-methyl-l-hexene-3-yne (PM) and glycidyl methacrylate (GMA) and polyethylene glycol (PEG) side chains.
The molecular weight of the carrier was 94,000 g/mol. Conjugation of TMZ with a novel polymeric carrier
functionalized with phosphatidylcholine resulted in approximately 2 times enhancement of anticancer activity
of TMZ. Combining of TMZ with doxorubicin (50 nM) resulted in further enhancement by 23% of the anti-
proliferative effect of TMZ. TMZ caused apoptosis in glioma cells via activation of MAPK signaling pathway,
inhibition of STAT3, and affected a transition through G2/M phase of cell cycle. These features make the novel
nano-formulation of TMZ a perspective strategy for further development of this drug.
K e y w o r d s: glioblastoma, apoptosis, temozolomide, doxorubicin, polymeric carrier.
G
liomas (glioblastoma multiforme (GBM) studies is not possible. As a result, there is reliance
and anaplastic astrocytoma (AA)) are the on nonrandomized studies as the principal design
most common types of primary tumors of for identification of potentially active therapies that
central nervous system (CNS) and have a combined should be studied in more definitive, randomized tri-
incidence of 5-8/100,000 population. The five-year als [1].
survival rates for brain tumors are the third lowest The methylating drugs, such as temozolomide
among all types of cancer where pancreas and lung (TMZ), are widely used in treatment of brain tu-
cancers are the first and second, respectively [1]. The mors, particularly the malignant gliomas [2]. TMZ
prognosis for patients with the advanced glioblasto- is a small (194 Da) lipophilic molecule that can be
ma multiforme is very poor, and the mean survival administered orally, and it crosses effectively the
period is 8-10 months. One of the challenges for test- blood–brain barrier (BBB) [3]. TMZ is an imidazo-
ing new agents in this disease is the fact that brain tetrazinone with activity attributed to the formation
tumors are uncommon, one-tenth as frequent as of a reactive methyldiazonium cation and methyla-
breast or lung cancer. Therefore, an unlimited num- tion of O6-guanine in the DNA molecule. Clinical
ber of large, prospective, randomized, controlled responses to TMZ are closely linked to the activity
87
of O6-alkyl-guanine-DNA alkyl-transferase (AGT), and human malignant glioma cells grown in vitro.
a DNA repair protein that removes O6-alkyl-guanine The mechanisms of the proapoptotic action of TMZ
adducts in the DNA [4]. TMZ advantages attractive in free and polymer-immobilized forms have been
for use in treatment of CNS tumors include excel- evaluated.
lent oral bioavailability and good circumvention of
the BBB [5]. Although a survival for glioblastoma Materials and Methods
multiforme has not changed significantly over the Cell lines and culture conditions. The C6 rat
past three decades, the emergence of novel treatment glioma and T98G human glioblastoma multiform
strategies for these tumors has led to heightened in- cell lines were obtained from a collection at the In-
terest and optimism among oncologists [1]. New stitute of Molecular Biology and Genetics, National
treatment strategies are emerging that target steps in Academy of Sciences of Ukraine (Kyiv, Ukraine).
the molecular pathogenesis of these tumors. Cell lines were cultured in Dulbecco’s modified Ea-
A perspective strategy to improve the activity gle’s medium (DMEM, Sigma-Aldrich, USA) sup-
of anticancer drugs is the combining of two or more plemented with 10% fetal bovine serum (APP, Aus-
drugs possessing different mechanisms of action that tria). Cells were cultivated in the CO2-thermostate at
could produce the additive or synergistic effects. 37 °C in atmosphere of 95% air and 5% CO2 at 100%
Generation of combinatorial approaches that act on humidity.
the compensatory pathways in tumors may notably Reagents. Temozolomide (TMZ, 3,4-dihy-
improve the effectiveness of the targeted therapy dro-3-methyl-4-oxoimidazo [5,1-d]-as-tetrazine-
used alone [6]. 8-carboxamide, Fig. 1) was kindly supplied by the
New directions for the use of TMZ will likely Department of Functional genomics (Institute of
focus on drug delivery to the targets in CNS. Since Molecular Biology and Genetics, National Academy
90% of malignant gliomas recur within 1-2 cm of of Sciences of Ukraine). Doxorubicin (Dox) was
the original site, local therapy may be an effective purchased from Arterium (Ukraine) via a standard
strategy [1]. One of the methods here is the use of pharmacy distribution.
polymeric carriers to deliver drugs via diffusion The polymeric carrier (Fig. 2) was synthesized
from the micropores in the polymer matrix or by a at the Department of Organic Chemistry at Lviv
release of drug within the interstices of a degradable Polytechnic National University (Lviv, Ukraine). It
matrix. Strategies to circumvent the BBB (polymers, was a water-soluble comb-like polymer poly(PM-co-
bradykinin analogues, gene therapy) are important GMA)-graft-PEG consisting of backbone that was
advances that have also shown an efficacy in early copolymer of 5-tert-butyl-peroxy-5-methyl-l-hexene-
clinical trials. Future treatment strategies for malig- 3-yne (PM) and glycidyl methacrylate (GMA) and
nant gliomas will likely involve synergistic combi- polyethylene glycol (PEG) side chains. The molecu-
nations of agents aimed at different pathways in the lar weight of the carrier was 94.000 g/mol [11, 12]. To
molecular pathogenesis of this type of cancer. prepare TMZ delivery system, solution of 100 mg/ml
A central approach is the development of the
nanoparticle formulations to reduce the acute toxici
ty of free drug and improve its therapeutic efficacy.
To reach that goal, various dendrimer-, polymer-, li-
posome-, and micelle-based systems were designed
as delivery vehicles [7, 8]. Besides, a decrease of
adverse effects, several nanoparticle-based drug
delivery systems [9, 10] were developed in order to
bypass specific efflux proteins and to selectively in-
crease drug accumulation in drug-resistance tumor
cells.
In this article, we developed the conjugate of
TMZ with novel polymeric carrier (A24-p-TMZ)
functionalized with the phosphatidylcholine. We
aimed to examine whether such modification of
TMZ can enhance its treatment effect towards rat Fig. 1. Temozolomide
88
Fig. 2. Structure of the of the polymeric carrier functionalized with the phosphatidylcholine
poly(PM-co-GMA)-graft-PEG was prepared in the conjugated with polymeric carrier TMZ, and poly-
dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA). meric carrier. FITC-conjugated Annexin V (Sigma-
For preparation of phosphatidylcholine, containing Aldrich, USA) and PI (Sigma-Aldrich, USA) were
system 50 mg of phosphatidylcholine were dissolved added directly to the culture medium of drug-treated
in 1 ml of chloroform, then chloroform was evapo- C6 rat glioma cells to reach a final concentration of
rated to obtain thin film of the phosphatidylcholine. 250 μM of free and polymeric carrier functionalized
100 mg/ml solution of poly(PM-co-GMA)-graft- with phosphatidylcholine, polymer-encapsulated
PEG in DMSO was added to the phosphatidylcho- TMZ, and A24-p-TMZ. In 72 h after the addition of
line film and kept at room temperature for 1 h. Then, the tested compound, the C6 cells were pelleted by
3.0 mg of TMZ were dissolved in 0.5 ml DMSO and centrifugation at 2,000 rpm, washed twice with 1x
the resulting solutions of TMZ and polymer were Phosphate Buffered Saline (PBS), and incubated for
mixed and added drop by drop to 8.5 ml water con- 15 min in the Annexin V binding buffer containing
taining 0.9% NaCl. Finally, the solution was stirred 1/20 volume of FITC-conjugated Annexin V solu-
for 1 h and then sonicated for 20 sec. tion and PI (20 μg/ml). 10 μl of cell suspension were
Anti-proliferative assays. Screening of antican-
added on slides and cover glasses were placed over
cer activity in vitro of free TMZ and its encapsulated
them. The cells were examined under Zeiss Axio-
form with the polymeric carrier functionalized with
Imager A1 fluorescent microscope (Carl Zeiss, Ger-
the phosphatidylcholine and Dox (the last was used
many).
here as a reference drug control) was measured using
Western blot analysis. After 48 h exposure of
MTT assay [13]. Tumor cells were grown for 24 h in
96-well plates (0.1 ml) at 5,000 cells/well. After that, the tested compound, cell proteins were isolated, re-
cells were incubated for 72 h with various additions solved by SDS/PAGE, and transferred onto a polyvi-
of the synthesized compounds (final concentration nylidene difluoride (PVDF) membrane for Western
of 0; 50; 100; 250 μM). MTT reagent which is con- blotting, as described [14]. The following antibodies
verted to dark blue, water insoluble MTT formazan were used at a 1:1,000 dilution: anti-ERK ½, anti-
by the mitochondrial dehydrogenases, was used to STAT3, anti-phospho-cdc2 (Tyr 15) (Cdk1), anti-
determine viable cells according to the manufac- phospho-Rb (Ser 807/811), anti-cleaved Caspase 3
turer’s protocol (Sigma-Aldrich, USA). The IC50 of (Cell Signaling Technology, USA), anti-JNK (sc-
the tested compounds was calculated as a lethal con- 571), anti-Cdk2 (sc-6248) (Santa Cruz Biotec., USA).
centration of drug killing 50% cells in comparison Equal protein loading of each lane was evaluated by
with an untreated cell culture. The viability of the the immunoblotting of the same membrane with an-
untreated cells was regarded as 100%. ti-beta-actin monoclonal mouse AC-15 (Sigma-Al-
Fluorescent microscopy. FITC-conjugated An- drich, USA). All secondary peroxidase-labelled an-
nexin V and Propidium Iodide (PI) double staining tibodies (Cell Signaling, USA) were used at working
were performed in order to detect early apoptotic dilution of 1:5,000.
events in C6 rat glioma cells treated with free and
89
Statistical analysis. All data are presented as Cytotoxic effect of free and polymeric carrier en-
mean ± SD. Results were analyzed and illustrated capsulated temozolomide
with GraphPad Prism (version 6; GraphPad Soft-
ware, San Diego, CA). Statistical analyses were TMZ A24-p-TMZ
performed using two-way ANOVA with Bonferroni Cell line
ІС50 ± SD, μМ ІС50 ± SD, μМ
post-tests (apoptosis induction, tumor growth). A P-
value of < 0.05 was considered statistically signifi- С6 rat glioma 74.6 ± 5.1 72.4 ± 4.1
cant. T98G human
Results and Discussion glioblastoma
multiform 243.1 ± 3.2 165.9 ± 3.1
Anticancer activity of temozolomide. To exam-
ine the antitumor effect in vitro of free TMZ and its
complex with the polymeric carrier functionalized ner. The cytotoxity of free TMZ and its form encap-
with the phosphatidylcholine (A24-p-TMZ) towards sulated by the polymeric carrier functionalized with
glioma cells, we treated glioma cell lines (rat C6 and the phosphatidylcholine towards glioma cells was
human T98G) with 0-250 μM of studied compounds carried out in 3, 24, 48 and 72 h. It was found that
for 72 h. Then, cell viability was determined by the IC50 value of A24-p-TMZ complex in case of treating
MTT assay. C6 cell line was reached as soon as in 3 h of incuba-
When chemo-sensitivity of C6 rat glioma cell tion (Fig. 4). While the IC50 value at the action of free
line towards studied compounds was studied, simi- TMZ was reached only in 48 h of incubation. In 6 h
lar dose-dependent changes in cellular viability was of incubation, the cytotoxic effects of A24-p-TMZ
found. The IC50 (concentration resulting in cell via- complex and free TMZ were almost the same. We
bility of 50% of control) of TMZ was 74.6 ± 5.1 μM, have observed difference in the cytotoxicity of drugs
while the IC50 of A24-p-TMZ was 72.4 ± 4.1 μM after 24 h and 48 h incubation, where A24-p-TMZ
(Table). However, T98G human glioblastoma mul- complex possessed higher cytotoxic effect at lower
tiform cells showed to be more resistant to TMZ concentration of used substances. However, similar
(IC50 = 243.1 ± 3.2 μM) than to the A24-p-TMZ IC50 of free and conjugated form of TMZ towards C6
(IC50 = 165.9 ± 3.1 μM) (Table). cell line were found (Fig. 4).
It should be noted that the polymeric carrier Next, we studied the cytotoxic effect of free
functionalized with phosphatidylcholine itself did and polymer-conjugated form of TMZ, and the pol-
not affect proliferation of C6 and T98G glioma cells ymeric carrier towards T98G human glioblastoma
considerably (Fig. 3). multiform cell line (Fig. 5).
As shown in Fig. 4, 5, TMZ inhibited viability We have found that free TMZ achieved its IC50
of glioma cells in a dose- and time-dependent man- at 48 h treatment of human T98G glioblastoma mul-
1.2 C6 1.2 T98G
1.0 1.0
-fold of growth
-fold of growth
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
0 50 100 150 200 250 0 50 100 150 200 250
Concentration, μM Concentration, μM
Fig. 3. Comparison of cytotoxicity of the polymeric carrier functionalized with the phosphatidylcholine
(A24-p) towards different glioma cell lines. After a total experimental time of 72 h, cell vitality was measured
by the MTT assay
90
1.2 3h 1.2 24 h
1.0 1.0
-fold of growth
-fold of growth
0.8 0.8
0.6 0.6
0.4 A24-p 0.4 A24-p

TMZ TMZ
0.2 0.2
A24-p-TMZ A24-p-TMZ
0.0 0.0
0 50 100 150 200 250 0 50 100 150 200 250
1.2 48 h 1.2 72 h
1.0 1.0
-fold of growth
-fold of growth
0.8 A24-p
0.8
0.6 TMZ 0.6

A24-p-TMZ
0.4 0.4 A24-p
TMZ
0.2 0.2
A24-p-TMZ
0.0 0.0
0 50 100 150 200 250 0 50 100 150 200 250
Fig. 4. Exposure time-dependency of the action of free temozolomide and its form encapsulated by the poly-
meric carrier functionalized with the phosphatidylcholine towards C6 rat glioma cells. Measurements were
performed after 3, 24, 48 and 72 h exposition to compounds. Viability of cells was determined using MTT
assay
tiform cells (IC50 = 245.7 ± 2.4 μM). After 72 h of to improve the antitumor effect in vitro of free and
incubation, the IC50 was 243.14 ± 3.21 μM that is al- conjugated form of TMZ, we have additionally used
most 3 times higher value than the IC50 for rat C6 traditional anticancer drug – the Dox. To investigate
glioma cells (IC50 = 74.55 ± 5.06 μM). TMZ conju- possible synergistic effect of Dox in combination
gated with the polymeric carrier functionalized with with various forms (free and encapsulated) of TMZ,
the phosphatidylcholine reached its IC50 at 48 h of we used MTT assay after 72 h of incubation of cells
incubation. The IC50 of the conjugated form of TMZ with the studied drugs.
was 165.9 ± 3.1 μM that is 1.5 times lower than that The obtained results demonstrated that the
of the free form of TMZ. presence of Dox in cultural medium did not affect
Thus, conjugation of TMZ with novel the antitumor action of free form of TMZ (Fig. 6).
polymericcarrier functionalized with the phos- However, Dox increased the cytotoxic action of
phatidylcholine leads to enhancement of the anti- TMZ conjugated with the polymeric carrier func-
proliferative activity of this drug. tionalized with the phosphatidylcholine towards rat
The synergistic effect of doxorubicin combined C6 glioma cells. The addition of Dox (50 nM) to the
with free and conjugated forms of temozolomide. culture medium led to a decrease in the value of ІС50
Combination of two or more drugs possessing dif- from 67.9 μM to 52.46 μM. So, the effectiveness of
ferent mechanisms cytotoxic action or demonstra A24-p-TMZ complex was improved by 23% with its
ting different mechanisms of circumventing drug combination with the Dox.
resistance can produce additive or synergistic effects Mode of temozolomide action. Previously, it
in the action of anticancer drugs [6]. That is why, was reported that TMZ-induced apoptosis in the
91
1.2 3h 1.2 24 h
1.0 1.0
-fold of growth
-fold of growth
0.8 0.8
0.6 0.6
0.4 A24-p 0.4 A24-p

TMZ TMZ
0.2 0.2
A24-p-TMZ A24-p-TMZ
0.0 0.0
0 50 100 150 200 250 0 50 100 150 200 250
1.2 48 h 1.2 72 h
1.0 1.0
-fold of growth
-fold of growth
0.8 0.8
0.6 0.6
0.4 A24-p 0.4 A24-p
TMZ TMZ
0.2 0.2
A24-p-TMZ A24-p-TMZ
0.0 0.0
0 50 100 150 200 250 0 50 100 150 200 250
Fig. 5. Exposure time-dependency of free temozolomide and its form encapsulated by the polymeric carrier
functionalized with the phosphatidylcholine towards T98G human glioblastoma multiform cell line. Meas-
urements were performed after 3, 24, 48 and 72 h of exposition to studied compounds. Viability of cells was
determined using MTT assay
1.2 1.2
TMZ
TMZ + Dox, 50 nM
1.0 1.0 A24-p-TMZ
A24-p-TMZ + Dox, 50 nM
-fold of growth
-fold of growth
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
0 50 100 150 200 250 300 0 50 100 150 200 250
Fig. 6. Patterns of cytotoxic action of free temozolomide and its form conjugated with the polymeric carrier
functionalized with the phosphatidylcholine towards rat C6 glioma cells under addition of 50 nM doxorubicin.
After a total experimental time of 72 h, cell vitality was measured by the MTT assay
glioblastomas, however precise mechanisms of dergo apoptosis, we performed a supra-vital double

such action are still not clearly understood [15]. To stainingwith FITC-conjugated Annexin V and PI of
determine whether TMZ-treated glioma cells un- rat glioma cells treated with free TMZ and its form
92
60
Annexin V PI
50
40
% of cells
30
20
10
0
Control A24-p TMZ A24-p-TMZ
Fig. 7. Ratio of dead cells treated for 72 h with free temozolomide and its form conjugated with the polymeric
carrier functionalized with the phosphatidylcholine. Ratio of dead cells was determined by the Annexin V
(indicator of apoptosis) and PI (indicator of necrosis) co-staining of rat C6 glioma cells. Percentage of the
apoptotic cells was analyzed using the fluorescent microscopy
conjugated with the polymeric carrier functionalized Treatment of C6 glioma cells with TMZ conju-
with the phosphatidylcholine. As shown in Fig. 7, gated with the polymeric carrier caused a reduction
48.9% of apoptotic cells were detected in rat C6 glio in the amount of STAT3 protein. A combination of
ma cells treated for 72 h with TMZ (75 μM). While TMZ conjugated with polymeric carrier with Dox
the polymer-conjugated form of TMZ induced ap- also caused a decrease in STAT3 protein amount.
pearance of 44.8% apoptotic C6 glioma cells. Thus, Free form of TMZ and combination of Dox with
the cytotoxic action of TMZ is due to induction of TMZ did not affect the amount of STAT3 protein
apoptosis in these cells. (Fig. 8). According to literature data, the phospho-
At the next stage of study, we explored the rylated STAT3 is a transcription activator acting as
signaling pathways for apoptosis during action of a signaling molecule for many cytokines and growth
free and conjugated forms of TMZ in combination factors. This protein was activated at different types
of Dox. The reason to conduct such study was that of cancer and possessed an oncogenic effect and an-
we have shown that low dose of Dox (50 nM) en- ti-apoptotic properties [16].
hanced the cytotoxic action of polymer-conjugated We have also found an increased amount of
form of TMZ (see: Fig. 6), while other investigators CDK2 protein under the action of TMZ conjugated
found that Dexamethasone decreased temozolomide- with the polymeric carrier and under the action of
induced apoptosis in human gliobastoma T98G cells combination of Dox with free TMZ and its conju-
[15]. gated form with the polymeric carrier (Fig. 8).
It is known that cellular stresses and some other Next, we studied the mechanism of the pro-
stimuli can induce apoptosis via modulating a sign- apoptotic action of free TMZ and its polymer-
aling pathway of the mitogen-activated protein ki- conjugated form in combination with the Dox in
nases (MAPK) [16]. Here we found that conjugated human T98G glioblastoma cells. It was found that
TMZ enhanced phosphorylation of JNK kinases. the amount of cleaved Caspase 3 increased more
A combination with Dox (50 nM) of free TMZ and definitely under the effect of free form of TMZ than
its conjugated form with the polymeric carrier also under the effect of polymer-conjugated TMZ. Howe
caused an activation of JNK kinases (Fig. 8). These ver, the combination of TMZ with Dox decreased the
proteins are activated as a result of negative impact level of cleavage of Caspase 3 (Fig. 9).
of some external factors. In turn, pJNK induces acti- The obtained results also showed an increase
vation of a large number of specific proteins involved of JNK kinase in T98G cells under the effect of all
in apoptosis (p53, ATF-2, c-Jun and others). Thus, an forms of TMZ and its combination with the Dox
increase in the amount of pJNK could be a charac- (50 nM). Besides, we have found that free TMZ and
teristic feature of cells that die by apoptosis. its form conjugated with the polymeric carrier inde-
93
1 2 3 4 5 6 7
JNK
STAT3
CDK2
b-actin
Fig. 8. Pattern of expression of the pro-apoptotic and cell cycle regulation proteins in T98G cells after their
treatment for 24 h with free temozolomide and its form conjugated with the polymeric carrier functionalized
with the phosphatidylcholine. 1 – Control; 2 – polymer A24-p (300 μM); 3 – Dox (50 nM); 4 – A24-p-TMZ
(300 μM); 5 – TMZ (300 μM); 6 –A24-p-TMZ (300 μM) combined with the Doxorubicin (50 nM); 7 – TMZ
(300 μM) combined with the Doxorubicin (50 nM)
1 2 3 4 5 6 7
Cleaved
Caspase 3
JNK
ERK1/2
STAT3
b-actin
Fig. 9. Pattern of expression of the pro-apoptotic proteins in human T98G glioblastoma cells treated for
24 h with free temozolomide and its form conjugated with the polymeric carrier functionalized with the
phosphatidylcholine. 1 – control; 2 – polymer A24-p (250 μM); 3 – Dox (50 nM); 4 – TMZ (250 μM); 5 – A24-
p-TMZ (250 μM); 6 – TMZ (250 μM) combined with Doxorubicin (50 nM); 7 – A24-p-TMZ (250 μM) combined
with Doxorubicin (50 nM)
pendent on a combination with the Dox caused an Combination with Dox of TMZ conjugated
increase in the amount of ERK 1/2 protein (Fig. 9). with the polymeric carrier decreased an amount of
Treatment of the T98G glioma cells with free cyclin-dependent kinase 1 (Cdk1). It is known that
TMZ and its form conjugated with the polymeric an elevation of the Cdk1 plays a key role in control-
carrier independent on a combination with the Dox ling the eukaryotic cell cycle through modulating
caused a reduction in the amount of STAT3 protein. centrosome cycle, as well as a mitotic onset. Cdk1
Combination of Dox with TMZ conjugated with the promotes the G2/M transition and regulates G1 pro-
polymeric carrier caused more pronounced decrease gress and the G1-S transition via association with the
in the amount of STAT3 protein (Fig. 9). multiple interphase cyclins [17].
94
While the amount of phosphorylated form of liferative activity. It could be due to the selectively
Rb protein under the effect of TMZ alone was rather increase of the drug accumulation in tumor cells and
weak, TMZ combined with the Dox caused more slow drug release into the interstitial space of the
pronounced increase in the amount of phosphoryla tumor [18, 19]. Application of such kind of complex
ted pRb. Although, combination of Dox with TMZ stimulates the cells to accumulate the drug by endo-
conjugated with the polymeric carrier did not affect cytosis pathway and by macropinocytosis pathway.
the level of phosphorylated pRb (Fig. 10). Endocytotic drug uptake could be the promising
Thus, the obtained results demonstrated that strategy to diminish the effects of chemotherapy re-
cytotoxicity of TMZ towards glioma cells is rela sistance [20]. In addition, many nanoparticles that
ted to induction of their apoptosis via activation of are PEGylated are characterized by reduced binding
MAPK signaling pathway, inhibition of STAT3, and to serum proteins or (immune) cells and, such, evade
affecting a transition of G2/M phase in cell cycle. their degradation via incorporation by macrophages
Patients suffering from malignant glioma, in [18].
particular glioblastoma multiforme, have a very poor The mechanism of apoptosis induction by the
prognosis. Standard therapy, in addition to surgery O6-methylating agents has been elucidated in detail
and radiotherapy, includes treatment with alkylating in various experimental models such as rodent cell
agents, specifically TMZ, and the chloroethylating lines [21], lymphoblastoid cells [22] and peripheral
drugs, such as carmustine and nimustine. In future, human lymphocytes [2]. However, this mechanism
the use of TMZ as well as other methylating drugs remains enigmatic in the malignant gliomas and
is expected to become more dominant than a use of some other tumor types. It was reported that in hu-
chloroethylating agents because of less severe nega- man glioma cells TMZ might induce either a p53-as-
tive side effects [2]. sociated G2/M arrest followed by cell senescence or
Application of the nanocarriers for delivery of p53-independent mitotic catastrophe [23]. In another
anthracyclines including the Dox can be a successful study, TMZ was proposed to induce an autophagy,
strategy aimed at improvement of the pharmacologi- while it failed to induce apoptosis in the malignant
cal characteristics and, thus, at reduction of adverse glioma cells [3], although glioma cells growing as
effects. Most of available nanoparticles were de- spheriods were shown to be sensitive to apoptosis
signed for addressed tumor targeting. In this study, induced by the alkylating agent [24].
we present a novel polymeric/phospholipid conjugate These contradictory data prompted us a neces-
of TMZ which resulted in an enhanced anticancer sity of studying in more detail the mechanism of
activity in vitro. It was found that nanocomplexation induction of glioma cell death upon treatment with
of TMZ increased approximately twice its antipro- TMZ. Here, we demonstrated that glioma cells un-
1 2 3 4 5 6 7
CDK1
pRb
b-actin
Fig. 10. Pattern of expression of the pro-apoptotic and cell cycle regulation proteins in human T98G glioblas-
toma cells treated for 24 h with free temozolomide and its form conjugated with the polymeric carrier func-
tionalized with the phosphatidylcholine. 1 – control; 2 – polymer A24-p (250 μM); 3 – Dox (50 nM); 4 – TMZ
(250 μM); 5 – A24-p-TMZ (250 μM); 6 – TMZ (250 μM) combined with Doxorubicin (50 nM); 7 – A24-p-TMZ
(250 μM) combined with Doxorubicin (50 nM)
95
dergo apoptosis following their treatment with free [26]. Also, ERK can regulate cell growth at the
TMZ and its form conjugated with the polymeric transcription level affecting an early response gene,
carrier functionalized with the phosphatidylcholine. such as c-fos, that is mediated by STAT3 (Ser727)
Other strategy aimed at enhancement of the activation. STAT3 mediates the expression of a va-
activity of anticancer drugs is the combining the ac- riety of genes in response to cell stimuli, and, thus,
tion of two or more drugs. Favorable safety profile plays a key role in many cellular processes such as
of TMZ allows it to be co-administered with various growth and apoptosis. It was reported that STAT3
additional agents. The antitumor activity of TMZ acts as a general positive regulator of cell transfor-
is dependent on the level of alkyl-guanine alkyl- mation and oncogenesis in various tissues including
transferase (AGT) in tumor cells. The results of the brain [28, 29]. So, STAT3 inhibition under the ac-
preclinical studies indicate that inhibition of AGT tion of free TMZ and its form conjugated with the
potentiates the activity of TMZ in several human tu- polymericcarrier could provide the tumor suppres-
mor cell lines [6]. sor action.
Current chemotherapy approaches in glioblas- Cdk1 is a catalytic subunit of highly conserved
toma treatment are based on using high doses of protein kinase complex known as M-phase promo
TMZ (75-200 mg per square meter of body surface ting factor (MPF) which is essential for G1/S and
per day). Unfortunately, such regiment induces neu- G2/M phase transitions of the eukaryotic cell cycle.
rotoxicity and cognitive dysfunction expressed as a pRb remains phosphorylated throughout S, G2 and
treatment-related negative side effects [25]. There is M phases. When pRb is hyper-phosphorylated, it is
a preclinical evidence indicating that cisplatin can unable to form a complex with transcription factor
enhance the antitumor activity of TMZ [6]. E2F and, therefore, to restrict a progression from the
In our study, combining of TMZ and Dox G1 to the S phase of cell cycle [30].
(50 nM) actions resulted in an enhancement of the Thus, we found that TMZ induces apoptosis in
anti-proliferative effect of TMZ. Besides, the an- glioma cells via activation of MAPK signaling path-
ticancer activity of A24-p-TMZ complex was en- way and inhibition of STAT3 and affects a transi-
hanced by 23% at its combination with the Dox. tion through G2/M phase. A novel polymeric carrier
The precise mechanism of action of TMZ in functionalized with the phosphatidylcholine is capa-
glioblastoma cells remains to be elucidated [26]. It ble of enhancing approximately twice an anticancer
was shown that treatment of human tumor cells with therapeutic efficacy of TMZ in vitro. Combination
TMZ induced an increase in the activity of PARP - of TMZ with the Dox (50 nM) resulted in further
DNA reparation enzyme involved in nucleotide improvement by 23% of the anti-proliferative effect
excision repair. The inhibition of PARP has been of TMZ. These features make the novel nano-for-
reportedto be a mechanism of enhancing a cytotoxi mulation of TMZ a perspective candidate for further
city of the methylating drug agents [6]. In this study, development of this drug.
we showed an increased caspase-3 cleavage under Acknowledgements
the action of TMZ towards glioma cells. The authors thank RECOOP HST Association
A number of intracellular signaling pathways and CSMC International Research and Innovation
was described in the regulation of growth and sur- Management Program for support. Personal thanks
vival of GBM. They include the activation of the epi- are also expressed to Dr. Sandor Vari (Director, In-
dermal growth factor receptor (EGFR) in these cells, ternational Research and Innovation in Medicine
and EGFR dysfunction correlates with poor progno- Program, Cedars - Sinai Medical Center, Los An-
sis in cancer patients [26]. Activation of EGFR leads geles, USA, President of the RECOOP HST Asso-
to induction of downstream signaling pathways such ciation) for inspiring collaboration between research
as phosphorylation of AKT, MAPK and JNK [27]. teams involved in this work. The authors thank Pro-
Our data showed an increase in the level of fessor Vadym Kavsan (Department of Biosynthesis
phospho-ERK1/2 and phospho-JNK in T98G glio- of Nucleic Acids, Institute of Molecular Biology and
ma cells. These kinases function downstream of the Genetics, National Academy of Science of Ukraine)
EGFR signaling which is commonly associated with who was a founder of studies in the field of glioma
GBM pathology. Thus, auto-trans-phosphorylation treatment in Ukraine.
of EGFR could activate AP-1 via ERK1/2 and JNK
96
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98
UDC 612.33:[615.012].014.44
The influence of novel 4-thiazolidinone

derivaties in cytoprotective mechanisms of small
intestine under nsaid-induced damage
I. I. Ilkiv, R. B. Lesyk, O. Ya. Sklyarov
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine;

e-mail: ira9ilkiv@gmail.com
The aim of investigation was to compare the action of novel 4-thiazolidinone derivaties (compounds
Les-5054 and Les-5055) toward parameters of nitroso-oxidative processes in mucous membrane of small
intestine (MMSI) in rats on the background of indomethacin induced injury. The activity of nitric oxide syn-
thases, myelopeoxidase, content of NO, and parameters of lipoperoxidation processes were measured in
MMSI and the level of H2S and L-arginine in blood serum. Administration of indomethacin caused significant
destructive damages in distal part of small intestine and increase in activity of inducible nitric oxide synthase
(iNOS) and intensity of lipoperoxidation processes in comparison to control were observed. Also indometha-
cin injection was accompanied by decrease of H2S and L-arginine level in blood serum. Administration of
4-thiazolidinone derivaties on the background of indomethacin induced injury reduce the activity of iNOS,
myeloperoxidase, intensity of lipid peroxidation and increase generation of H2S, that may be linked with the
structure of this compounds. However compound Les-5054 showed more significant cytoprotective effect and
antioxidant properties than compound Les-5055. Thus, the novel 4-thiazolidinone derivaties led to reduce of
nitroso-oxidative processes caused by administration of NSAIDs.
K e y w o r d s: hydrogen sulfide, 4-thiazolidinones, small intestine, indomethacin-induced injury.
H
ydrogen sulfide (H 2S) is now recognized mucus secretion by the epithelium, decrease blood
as an important gasotransmitter together flow, that play key role in the process of gastrointes-
with nitric oxide (NO) and carbon monox- tinal injury caused by NSAIDs [4]. Also these drugs
ide (CO). H2S has been implicated in the induction reduce H2S generation by modulating the expression
of such processes as inhibition of leukocytes adher- of activity of cystathionine-γ-lyase, which is mainly
ence to blood vessels, increase of endogeneous pros- expressed in smooth muscle cells in the cardiovascu-
taglandins production in small intestine, induction lar system and in the gastrointestinal tract [5].
of vasodilatation, increases cyclic AMP (cAMP) Thus, 4-thiazolidinones are one of important
production in neural retina, modulates epithelial source of organic sulfurcontaining compounds and
secretion and promotes resolution of colitis[1]. The have been widely investigated regarding their thera-
deficiency of hydrogen sulfide could lead to various
peutic applications. Thiazolidinone-based molecules
pathological changes in digestive tract, such as gas-
are attaractive targets in rational design of “drug-
tric mucosal injury, liver cirrhosis etc [2].
like” compounds which possess anti-inflammatory,
Nowadays nonsteroidal anti-inf lammatory
drugs (NSAIDs) are the most widely used class of antioxidant, antitumor, choleretic, diuretic and other
drugs for treating inflammatory conditions such as: activities [6-8].
osteochondrosis, polyarthritis, headaches. However, In the present study the effects of a novel 4-thi-
they cause significant adverse reaction in the mucous azolidinones (compounds Les-5054 and Les-5055),
membranes of the digestive system in the form of as potential H2S donors or mediators of its signal-
erosions, ulcers, impaired motor skills [3]. Owing to ing pathways were investigated. The effects of these
the inhibition of cyclooxygenase, NSAID suppress compounds in terms of nitroso-oxidative and cyto-
synthesis of prostaglandins which leads to leukocyte protective effects, ability to decrease small intesti-
adherence to the vascular endothelium in the gastro- nal injury, and acute anti-inflammatory effects were
intestinal microcirculation, reduce bicarbonate and compared.
99
Materials and Methods
Animals. The experimental protocols were ap-
proved by the Ethical Committee of Lviv National
Medical University (Ukraine). Male albino rats
weighing 200-250 g were used. The rats were fed
standart chow and water ad libitum, and were housed
in room with controlled temperature (22 ± 1 °C), hu-
midity (65-70%) and light cycle (12 h light/dark).
The study comprised of the following series of Les-5054 Les-5055
experiments: 1 – intact animals were used as con-
trols (n = 10); 2 – small intestine lesions in rats were Fig. 1. Structures of 4-thiazolidinone derivatives
induced by indomethacin (Sigma, USA) in dose of
35 mg/kg subcutaneously (n = 10) as previously dehyde) concentration in homogenates of MMSI. It
described (n = 10) [9]; 3,4 – experimental groups, was measured according to the procedure of Timir-
animals received compounds Les-5054, Les-5055 bulatow et al. [15]. MDA levels were expressed as
(10 mg/kg) per os once daily per 72 h on the back- mmol/l.
ground of indomethacin-induced injury (n = 10). The Intracellular myeloperoxidase activity. My-
compounds Les-5054, Les-5055 were synthesized by eloperoxidase (MPO) content in homogenates of
prof. Lesyk R. in the Department of Pharmaсeutical, MMSI measured at 460 nm according to the pro-
Organic and Bioorganic Chemistry Danylo Halytsky cedure of Bradley et al. [16]. MPO level were ex-
Lviv National Medical University (Fig. 1) [8, 10]. pressed as U/mg.
Under general anesthesia, rats were sacrificed Antioxidant enzymes defence determination.
by decapitation and 10 sм of distal part of small in- Activity of superoxide dismutase (SOD) was de-
testine was then blindly evaluated for hemorrhagic termined by the reaction of reduction of nitrotetra-
damage. This involved measuring the lengths (mm) zoliume blue to nitroformazan [17]. SOD activity
of all hemorrhagic lesions. The intestinal damage was expressed in mmol/min×mg of protein. Cata-
scores were then calculated by summing the lengths lase (CAT) activity was determined by measuring
of all lesions for each rats. The mucous membrane of of the decrease in hydrogen peroxide concentration
small intestine (MMSI) samples were homogenized at 410 nm by the Korolyuk method [18]. Colon mu-
in phosphate buffer pH 6.0 1:4 and centrifuged at cosal catalase activity was expressed in mmol H2O2/
3000 rpm, supernatant was used to determine values min×mg of protein.
of biochemical parameters. Statistics. The statistical processing of the data
Determination of NO-system in mucous mem- was done by conventional methods for analysis of
brane of small intestine. The content of NO in ho- variance using MS Excel software for Student’s t-
mogenate was determined as nitrites by the method test. The difference was considered to be significant
of Green et al. [11]. The absorbance was read in a at P < 0.05.
Stat fax at 550 nm. NO concentration was expressed
as mmol/g. NO-synthases (general NOS, iNOS, and Results and Discussion
eNOS) activity was measured by the method de- In our study, injection of indomethacin (35 mg/
scribed in detail [12]. NOS activity was expressed in kg) manifested by erosions and hemorrhages, with
nmol L-citrylline/min×mg of protein. a total area of 74 ± 5,94 mm2 (Fig. 2, 3). Indometa-
Measurement of L-arginine and H2S in blood cin induced injury in the MMSI was associated
serum. The level of L-arginine in plasma samples with change of the activity of NO-synthases: activi
was measured by Sakaguchi reaction [13]. Plasma ty of general NOS decreased (from 815.5 ± 49.8 to
L-arginine level was expressed as mmol/l. H2S con- 595.54 ± 73.7 nmol/min×mg) (P < 0.05), activity of
centration was determined by reaction with N,N- eNOS was decreased by 55% (P < 0.01), and activity
dimethyl-para-phenylenediamine in the presence of of iNOS increased more than threefold (P < 0.01) as
FeCl3 and expressed as mmol/g [14]. compared with indeces of control group. In MMSI
Lipid peroxidation determination. Lipid peroxi- concentration of NO was markedly elevated in two
dation level was expressed as MDA (malonic dial- times and, concomitantly (P < 0.01), content of L-ar-
100
ginine in serum blood decreased for 33% (P < 0.05). 90
It was found that H2S formation from L-cysteine was 80
dependently inhibited by addition of indomethacin 70
by 10% (P < 0.05) (Table 1).
60
MMSI, affected with indometacin induced
mm2
injury, was subjected to the following changes: en- 50
hanced activity of lipoperoxidation processes mani- 40
fested by a steep rise of MDA concentration – for 30

56% (P < 0.01) at that, MPO activity enhanced more 20
than 4-fold (P < 0.01), and catalase activity – for 32% 10
(P < 0.01). The activity of SOD was not statistically 0
significant (Table 2). Control Indomethacin Les-5054 Les-5055
+ IM + IM
In the presence study the development of
NSAID-induced small intestinal damage via indo- Fig. 3. The area of the structure-hemorrhagic dama
methacin injection was acompanied by enhanced ge of the MMSI. *P < 0.05 versus the indexes in in-
processes of lipid peroxidation, increase activity of domethacin (IM)
iNOS and myeloperoxidase that led to development
of hemorrhagic lesions in distal part of small intes-
Compound Les-5054 displayed significant cyto-
tine. This destructive changes caused by inflamma-
protective effect that manifested by separate hemor-
tion processes and acomplished throught decrease
rhages with the absence of considerable destructive
contents of H2S which enhance gastrointestinal mu-
changes of the MMSI. The total area of hemorrhagic
cosal resistance to injury [19].
lessins decreased for 63% (P < 0.05) (Fig. 2, 3). The
On another hand, the injection of NSAIDs such
administration of Les-5054 on the background of
as diclofenac, naproxen, indomethacin caused the
indomethacin-induced injury decrease the activity
non selective inhibition of both COX-1 and COX-2
of iNOS for 35% (P < 0.01), and activity of eNOS
and, as result, a significant reduction in production
of prostaglandins that is the key factor in develop- increased for 52% (P < 0.01) as compared with inde-
ment of enteropathies. pendent action of indomethacin. Intensity of lipop-
eroxidation processes were determined much lower
than under the effect of indomethacine, MDA con-
centration declined for 32% (P < 0.01). Administra-
tion of compound Les-5054 reversed the inhibition
of H2S caused by indomethacin and increase it for
24% (P < 0.05) as compared with indices of the sec-
ond group.
1
Compound Les-5054 led to 3 time decreased
MPO activity as compared with indices of 2 group
(P < 0.05). It was found the administration of com-
pounds Les-5054 and Les-5055 as H 2S releasing
compounds can inhibit peroxidase activity of MPO,
key role is production of reactive oxidants (HOCl
2 etc) which influence to a healthy tissue is the main
indication of MPO’s pathophysiological functions
[20].
In our investigations it was shown that admin-
istration of Les-5055 on the background of indo-
methacin-induced injury also shows cytoprotective
3 effect but not as much as compound Les-5054. The
total area of hemorrhagic lessins decreased for 37%
Fig. 2. Representative photographs of the small in- (P < 0.05) as compared with independent action of
testinal injury: 1 – indomethacin; 2 – Les-5054 + indomethacin (Fig 2, 3). Activity of eNO-synthases
indomethacin; 3 – Les-5055 + indomethacin had a tendency to increase, whereas iNOS activity
101
T a b l e 1. Effect of novel 4-thiazolidinones at the background of indomethacin-induced injury on concen-
tration of malonic dialdehyde, nitrite anion, activity of nitric oxide synthases and arginase in MMSI and
concentration of H2S and L-arginine in serum blood
Experimental Groups
Variable Indomethacin Les-5054 + Les-5055 +
Control group
35 mg/kg Indomethacin Indomethacin
Malonic dialdehyde,
(μmol/g) 186.6 ± 8.1 291.4 ± 26.7** 199.1 ± 11.1## 242.6 ± 16.0##
Nitrite anion, (μmol/g) 1.2 ± 0.1 2.8 ± 0.2** 1.8 ± 0.2 1.9 ± 0.1
Total nitric oxide synthase –
NOS, (nmol/min·g) 815.5 ± 49.8 595.5 ± 73.1 637.9 ± 42.8 578.2 ± 64.7
Inducible nitric oxide
synthase – iNOS,
(nmol/min·g) 66.1 ± 24.9 203.6 ± 26.8** 132.9 ± 27.5## 162.3 ± 27.7#
Constitutive nitric
oxide synthase – cNOS,
(nmol/min·g) 728.6 ± 66.1 331.9 ± 62.5** 506.3 ± 45.3## 415.9 ± 44.0#
Arginase, (μmol/ min·g) 0.20 ± 0.03 0.05 ± 0.02** 0.15 ± 0.04# 0.09 ± 0.006
L-Arginine, (μg/ml) 46.7 ± 3.6 31.2 ± 2.8 42.6 ± 4.1## 40.7 ± 4.2
H2S (μmol/g×min) 88.4 ± 2.7 79.5 ± 1.0* 98.5 ± 2.6# 93.0 ± 2.5#
Here and for table 2 results are expressed as mean ± SD for 10 rats per group; *p < 0.05, **p < 0.01 in comparison of
control group; #p < 0.05, ##p < 0.01 versus the indices of indomethacin action.
T a b l e 2. The activity of myeloperoxidase and antioxidant enzymes in MMSI of rats injected indomethacin
with investigated compounds
Experimental Groups
Variable Indomethacin Les-5054 + Les-5055 +
Control group
35 mg/kg Indomethacin Indomethacin
SOD, (mmol/min×mg) 23.9 ± 1.0 27.8 ± 1.3 24.9 ± 0.9# 27.9 ± 1.5
CAT, (mmol H2O2/min×mg) 16.9 ± 1.6 22.4 ± 2.6** 20.9 ± 1.2 20.0 ± 2.4
МРО, (U/mg) 1.2 ± 0.3 5.0 ± 0.5* 1.6 ± 0.5# 4.3 ± 1.2
was reduced for 20% (P < 0.05), compared to their decrease of NO, IL-6 and TNF-a secretion in intes-
activity in indomethacin induced damages. Contents tine mucosa [1].
of NO and MDA also showed a tendency to decrease In experimental model of indomethacin-in-
(P < 0.05). duced injury characteristic damages in distal part of
Many reports showed that several H 2S-based small intestine was observed. In our investigation we
therapeutics corresponded to target disorders and demonstrated that novel 4-thiazolidinones displayed
were characterized by oxidative stress and associ- significant cytoprotective effect, manifested by the
ated tissue injury. It was shown that their cytopro- decreased area of the MMSI lesions. Normalization
tection was accompanied by the decrease of mRNA of NO-synthases activities and the intensity of li-
expression for pro inflammatory cytokines, such as poperoxidation processes were found. Also admini
IL-10 or TGF-b. They also prevent inflammation by stration of investigated compounds was associated
decrease of MDA content, increase GSH level and with increase of H 2S level in serum blood. Thus,
102
novel 4-thiazolidinones showed cytoprotective, anti- 7. Lozynskyi AV, Kaminskyy DV, Roman
inflammatory effects and antioxidant properties, and chyshyn KB, Semenciv NG, Ogurtsov VV,
may be promising substances for new pharmacologi- Nektegayev IO, Lesyk RB. Screening of
cal preparations. antioxidant and anti-inflammatory activities
among thiopyrano[2,3-d]thiazoles. Biopolym
Acknowledgement
Cell. 2015; 31(2): 131-137.
Thank you for Cedars Sinai Medical Center’s
8. Lesyk RB, Zimenkovsky BS. 4-Thiazolidones:
International Research and Innovation in Medicine
centenarian history, current status and
Program, the Association for Regional Cooperation
perspectives for modern organic and medicinal
in the Fields of Health, Science and Technology (RE-
chemistry. Curr Org Chem. 2004; 8(16): 1547-
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104
UDC 547.818:547.489.4:542.91:615.359
New thiopyrano[2,3-d][1,3]thiazole derivatives

as potential antiviral agents
N. I. Zelisko, I. L. Demchuk, R. B. Lesyk*
Danylo Halytsky Lviv National Medical University, Ukraine;

*e-mail: dr_r_lesyk@org.lviv.net
A series of novel thiopyrano[2,3-d][1,3]thiazole derivatives were synthesized and evaluated for their
antiviral activity in vitro within AACF NIAID programme. Compounds were studied towards Dengue Virus
Type 2, Venezuelan Equine Encephalitis Virus, Respiratory Syncytial Virus, SARS Coronavirus, Rift Valley
Fever Virus, Tacaribe Virus, Influenza Virus Types A and B. Among the tested thiopyrano[2,3-d][1,3]thia-
zoles, alkyl rel-(5R,5aR,11bS)-2,6-dioxo-3,5a,6,11b-tetrahydro-2Н,5H-chromeno[4’,3’:4,5]thiopyrano[2,3-d]
[1,3]thiazole-5-carboxylates 8 and 11 were found to be the most active and showed significant antiviral activ-
ity against Influenza Virus Types A H3N2 and H5N1.
K e y w o r d s: thiopyrano[2,3-d][1,3]thiazoles, screening, antiviral activity.
V
iruses are known as common pathogens thiopyrano[2,3-d][1,3]thiazole-6-carboxylic acids
that cause a variety of diseases. Search for (14-17) 3, 2,6-dioxo-3,5a,6,11b-tetrahydro-2H,5H-
new efficient antiviral agents is an impor- cromeno[4′,3′:4,5]thiopyrano[2,3-d]thiazol-5a-yl]
tant worldwide problem among scientists and clini- acetic acids (18,19)3,14, 7′-(R-phenyl)-1-(R1-phenyl)-
cians, because of rapid emergence of drug resistant 3′,7′-dihydro-2H,2′H,5H-spiro[pyrolidin-3,6′-
strains. Even influenza virus may cause life-threate thiopyrano[2,3-d]thiazol]-2,2′,5-triones (20-24)3 and
ning events in high-risk patients. To overcome the their characteristics were described in our previous
drawbacks of the current antiviral drugs and to reports. The structure of compounds involved in the
obtain more efficacious drugs, new antiviral drugs study is presented in Fig. 2.
with a novel mode of action should be developed. The antiviral activity screening of the com-
Thiopyrano[2,3-d][1,3]thiazoles have become a pounds was performed at the National Institute of
promising area of research because of their diverse Allergic and Infectious Diseases of the National In-
biological activities, such as anticancer, antitrypa- stitute of Health (Bethesda, MD, USA) within AACF
nosomal, antimycobacterial, anti-inflammatory and (Antimicrobial Acquisition and Coordinating Facili-
antioxidant 1-7 (Fig. 1). ty (http://www.niaid-aacf.org) programme. Antiviral
In the last two decades, there has been an activity was determined against Dengue Virus Type
increase in the number of studies on 4-thiazoli- 2 (New Guinea C strain, Vero 76 cell line), Venezue-
dinone derivatives as potential antiviral agents 8-13. lan Equine Encephalitis Virus (TC-83 strain, Vero
Thiopyrano[2,3-d][1,3]thiazoles could be of special cell line), Respiratory Syncytial Virus (A2 strain,
interest as cyclic mimetics of biologically active (in- MA 104 cell line), SARS Coronavirus (Urbani
cluding antiviral) 4-thiazolidinones. The aim of pre- strain, Vero 76 cell line), Influenza Virus Type A
sent study was to estimate the antiviral activity of H5N1 (Vietnam/1203/2004H strain, MDCK cell line),
new thiopyrano[2,3-d][1,3]thiazole derivatives. Rift Valley Fever Virus (MP-12 strain, Vero 76 cell
line), Tacaribe Virus (TRVL-11573 strain, Vero cell
Materials and Methods line), Influenza Virus Type A H3N2 (Perth/16/2009
The synthesis of 2-oxo-3,7-dihydro-2Н-thio strain, MDCK cell line), Influenza Virus Type A
pyrano[2,3-d]thiazole-6-carboxylic acids (1-5)15 , H1N1 (California/07/2009 strain, MDCK cell line),
rel-(5R,5aR,11bS)-2,6-dioxo-3,5a,6,11b-tetrahydro- Influenza Virus Type B (Florida/4/2006 strain,
2Н,5H-chromeno[4′,3′:4,5]thiopyrano[2,3-d][1,3] MDCK cell line) using standart AACF screening
thiazole-5-carboxylic acids derivatives (6-13) 16 , assay protocols 17-19.
6-carboxymethylene-2-oxo-3,5,6,7-tetrahydro-2H-
105
Fig. 1. Biologically active compounds among thiopyrano[2,3-d][1,3]thiazoles
Fig. 2. Compounds tested for antiviral activity
106
Antiviral assay. Antiviral assay was performed Results and discussions
at a virus’s panel with a protocol of the NIAID’s an- Antiviral activity assay of synthesized com-
timicrobial acquisition and coordinating 17. Results pounds allowed us to identify some highly active
for each tested compound were reported as virus- thiopyrano[2,3-d]thiazoles, which demonstrated
inhibitory concentration; 50% endpoint (EC50) and certain sensitivity profile towards, Influenza Virus
cell-inhibitory concentration, 50% endpoint (CC50) Types A H3N2 and H5N1, as well Dengue Virus. The
were determined. A general selectivity index (SI) obtained results are summarized in Table.
was calculated as a ration of (EC50)/(CC50). An SI A m o n g 2 - oxo -3,7- d i h yd r o -2 Н- t h i o
of 3 or greater indicates that confirmatory testing is pyrano[2,3-d]thiazole-6-carboxylic acids (1-5)
needed. compound 1 showed a weak activity against Den-
Inhibition of Viral Cytopathic Effect (CPE). gue Virus (EC50 = 71 µg/ml, SI > 1.4), and deriva-
This test, run in 96 well flat-bottomed microplates, tives 3-5 exhibited an efficiency against Venezue-
was used for the initial antiviral evaluation of com- lan Equine Encephalitis Virus (EC50 = 21÷45 µg/
pounds. In this CPE inhibition test, four log10 di- ml, SI = 1,1÷1,7), Dengue Virus (EC50 = 18÷32 µg/
lutions of each test compound (e.g. 1000, 100, 10, ml, SI = 1,0÷1,1), Respiratory Syncytial Virus
1 µg/ml) were added to 3 cups containing the cell (EC50 = 26 µg/ml, SI = 1,1÷> 3,8), SARS Coronavi-
monolayer; within 5 min. On the next step, the virus rus (SI = 1,2÷2,3). The substituents in the positions
was added and the plate was sealed and incubated 3, 4 and 5 of aryl frgment contributed to increase
at 37°C. CPE read microscopically when untreated of antiviral activity for a above-mentioned group of
infected controls develop a 3 to 4+ CPE (approxi- compounds. Moderate effect against influenza virus
mately 72 to 120 hr). A known positive control drug A (H5N1, Vietnam strain) is identified for derivative
Ribavirin was evaluated in parallel with test drugs 4 (EC50 = 3.6 ÷ 6.8 µg/ml and SI = 5.0÷8.9).
in each test. The data are expressed as 50% effective According to the results of study compounds
concentrations (EC50). 14-17 haven’t shown an antiviral effect, in the same
Increase in Neutral Red (NR) Dye Uptake. This time tetracyclic derivatives 18 and 19 pocessed a
test was run to validate the CPE inhibition seen in weak activity against influenza virus A (H5N1, Vi-
the initial test, and utilized the same 96-well micro etnam strain). It has been identified compound 20
plates after the CPE has been read. When neutral red among 7′,-(R-phenyl)-1-(R1-phenyl)-3′,7′,-dihydro-
was added to the medium cells that were not dama 2H,2′,H,5H-spiro[pyrolidin-3,6′,-thiopyrano[2,3-d]
ged by virus take up a greater amount of dye, which thiazol]-2,2′,5-triones (20-24), which had some
is desplayed on a computerized microplate autorea activity against Dengue virus (EC50 = 8÷18 µg/
der. An EC50 was determined from this dye uptake. ml, SI = 4,7÷> 13.0). Other viruses were resistant
Cytotoxicity. In the CPE inhibition tests, two to the action of compounds 20-24. The SAR study
wells of uninfected cells treated with each concen- revealed that combination of a chlorine atom in the
tration of tested compounds was run in parallel with para-position of the 1-aryl substituent and 2-hy-
the infected, treated wells. At the time CPE was de- droxy-5-nitrophenyl (20), 4-methoxyphenyl (23), and
termined microscopically. The toxicity control cells 4-dimethylaminophenyl (24) fragments in position 7
were also examined microscopically for any chang- of basic heterocycle are important for new antiviral
es in cell appearance compared to normal control thiopyrano[2,3-d]thiazoles design.
cells run in the same plate. These changes may be Derivatives of rel-(5R,5aR,11bS)-2,6-dioxo-
enlargement, granularity, cells with ragged edges, 3,5a,6,11b-tetrahydro-2Н,5H-chromeno[4′,3′:4,5]
filmy appearance, rounding, detachment from the thiopyrano[2,3-d][1,3]thiazole-5-carboxylic acids
surface of the well, or other changes. These changes (6-13) belong to the most promising group of com-
were given a designation of T (100% toxic), PVH pounds. The substituents in the positions 8 and 10
(partially toxic–very heavy – 80%), PH (partially of the basic tetracyclic heterosystem and the ester
toxic–heavy – 60%), P (partially toxic – 40%), Ps group in position 5 are desirable for antiviral ac-
(partially toxic–slight–20%), or 0 (no toxicity – 0%), tivity. It was found, the increase of the alkyl moie
conforming to the degree of cytotoxicity seen. A ty length for alkyl rel-(5R,5aR,11bS)-2,6-dioxo-
50% cell inhibitory (cytotoxic) concentration (CC50) 3,5a,6,11b-tetrahydro-2Н,5H-chromeno[4′,3′:4,5]
was determined by regression analysis of these data. thiopyrano[2,3-d][1,3]thiazole-5-carboxylates (8-13)
107
T a b l e . Antiviral activity of the synthesized compoundsa
Сom- EC50 c, СC50 d,

Virus / strain / cell line Methodb SI e
pound µg/ml µg/ml
1 Dengue Virus Type 2 / New Guinea C / Vero 76 NR 71 >100 >1.4
3 Venezuelan Equine Encephalitis
Virus / TC -83 / Vero NR 35 59 1.7
NR 32 34 1.1
Dengue Virus Type 2 / New Guinea C / Vero 76
V 18 32 1.8
Flu B / Florida /4/2006/ MDCK NR 32 39 1.2
Respiratory Syncytial Virus / A2 / MA 104 V 32 >100 >3.1
NR 26 33 1.3
SARS CoV / Urbani / Vero 76
V 56 >100 >1.8
4 Venezuelan Equine Encephalitis NR 24 35 1.5
Virus / TC -83 / Vero V 21 28 1.3
NR 6.8 34 5.0
Flu A (H5N1) / Vietnam /1203/2004H / MDCK
V 3.6 32 8.9
NR 30 41 1.4
Flu B / Florida /4/2006/ MDCK
V 32 >100 >3.1
Rift Valley Fever Virus / MP -12 / Vero 76 NR 28 41 1.5
SARS CoV / Urbani / Vero 76 NR 32 38 1.2
Virus / TC -83 / Vero NR 45 50 1.1
NR 32 36 1.1
V 32 32 1.0
Tacaribe Virus / TRVL -11573 / Vero NR 40 53 1.3
NR 26 >100 >3.8
Respiratory Syncytial Virus / A2 / MA 104
V 37 >100 2.7
Flu A (H3N2) / Perth /16/2009 / MDCK NR 32 43 1.3
NR 27 63 2.3
V 32 68 2.1
Virus / TC -83 / Vero NR 41 58 1.4
Flu B / Florida /4/2006/ MDCK NR 29 >100 >3.4
NR 54 >100 >1.9
Flu A (H1N1) / California /07/2009/ MDCK
V 100 >100 >1.0
108
T a b l e . Continuation
7 Venezuelan Equine Encephalitis NR 24 65 2.7

Virus / TC -83 / Vero V 27 42 1.6
Dengue Virus Type 2 / New Guinea C / Vero 76 V 32 42 1.3
NR 3.1 45 15
V 10 32 3.2
NR 8.8 18 2.0
V 13 24 1.8
Flu A (H1N1) / California /07/2009/ MDCK NR 31 37 1.2
NR 35 49 1,4
Flu A (H3N2) / Perth /16/2009 / MDCK
V 32 >100 3.1
SARS CoV / Urbani / Vero 76 V 42 >100 >2.4
8 NR 32 >100 3,1
V 32 >100 3,1
NR 32 >100 3.1
V 32 >100 3.1
NR 0.6 >100 >170
V 2,5 >100 >40
NR 56 >100 >1.8
V 23 28 1.2
Virus / TC -83 / Vero NR 77 >100 >1.3
Flu A (H5N1) / Vietnam /1203/2004H / MDCK NR 8.0 >100 >13
Tacaribe Virus / TRVL -11573 / Vero NR 50 >100 >2.0
Rift Valley Fever Virus / MP -12 / Vero 76 NR 34 >100 >2.9
SARS CoV / Urbani / Vero 76 NR 48 >100 >2.1
11 NR 0.31 >100 >320
V 0.32 >100 >310
NR 34 >100 >2.9
V 32 >100 >3.1
NR 32 >100 >3.1
V 32 >100 >3.1
Flu A (H3N2) / Perth /16/2009 / MDCK NR 45 >100 >2.2
13 NR 32 >100 >3.1
V 32 >100 >3.1
NR 32 >100 >3.1
V 32 >100 >3.1
NR 27 >100 >3.7
V 32 100 3.1
NR 30 >100 >3.3
V 42 >100 >2.4
109
T a b l e . Continuation
17 Tacaribe Virus / TRVL -11573 / Vero NR 28 38 1.4

18 Flu A (H5N1) / Vietnam /1203/2004H / MDCK NR 50 >100 >2.0
NR 32 >100 >3.1
V 32 >100 >3.1
Virus / TC -83 / Vero NR 67 >100 >1.5
Flu A (H5N1) / Vietnam /1203/2004H / MDCK NR 31 >100 >3.2
20 NR 18 84 4.7
V 8 >100 >13
Flu A (H5N1) / Vietnam /1203/2004H / MDCK NR 30 43 1.4
Respiratory Syncytial Virus / A2 / MA 104 NR 46 >100 >2.2
23 Flu A (H5N1) / Vietnam /1203/2004H / MDCK V 32 >100 >3.1
Flu B / Florida /4/2006/ MDCK NR 32 >100 >3.1
NR 27 >100 >3.7
V 32 100 3.1
24 Flu A (H5N1) / Vietnam /1203/2004H / MDCK V 32 >100 3.1
Respiratory Syncytial Virus / A2 / MA 104 NR 23 50 2.2
SARS CoV / Urbani / Vero 76 NR 43 >100 >2.3
Riba- Respiratory Syncytial Virus/ A2 / MA 104 NR 6.1 > 250 > 41
virin NR 8.9 > 1000 > 110
Rift Valley Fever Virus / MP -12 / Vero 76
V 9.1 > 1000 > 110
NR 5.4 > 1000 > 190
Tacaribe Virus / TRVL -11573 / Vero
V 5.6 > 1000 > 180
NR 4.9 > 100 > 20
V 3.2 > 100 > 31
NR >11.0 > 100 > 9,1
V >11.0 > 100 > 9.1
NR 2.3 > 100 >43.0
V 5.3 > 100 > 19
NR 2.4 > 100 > 42
V 1.7 > 100 > 59
a
Compounds 2, 9, 12, 14-16, 21 and 22 did not show any antiviral activity. bNR – neutral red, V – visual cEC50 – com-
pound concentration that reduces viral replication by 50% dCC50 – compound concentration that reduces cell viability
by 50% eSI – CC50/EC50.
contributes for antiviral activity increasing (Fig. 3). and derivative 11 – against Influenza Virus Type A
Moreover, two the most active hits 8 and 11 belong (H5N1, Vietnam strain) with EC50 = 0.31÷0.32 µg/ml
to the above-mentioned group of thiopyrano[2,3-d] and SI = >310.0÷>320.0.
[1,3]thiazoles. Compound 8 showed a higher activity In general, it should be noted that compounds
against Influenza Virus Type A (H3N2, Perth strain) 6, 7, 8, 10, 11, 13 have the specific antiviral activity
with EC50 = 0.6÷2.5 µg/ml and SI = 40.0÷>170.0, against influenza viruses.
110
Fig. 3. SAR study of the thiopyrano[2,3-d][1,3]thiazoles.
The screening of 24 thiopyrano[2,3-d][1,3] References

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112
April 10, 2016 (Sunday)
Departure
Goodbye
See you CSMC - RECOOP

RESEARCHER at 7th TriNet Meeting in
Budapest on October 7 - 9, 2016
RECOOP Visegrad Scholarship Program
Visegrad Scholarship http://visegradfund.org/scholarships/
The top ten young scientists selected during the Bridges in Life Sciences Annual Conferences
have the opportunity to apply for International Visegrad Fund (IVF) Scholarship and receive
the RECOOP Young Scientists Matching Fund. The Visegrad Scholarship is the Visegrad Four
European Macro-Region’s Fulbright Program. Therefore it could be important to link the
Visegrad Scholarship and the Fulbright Foreign Student Program.
RECOOP HST Association in 2014 won two Visegrad Scholarships:
Post-Master’s Scholarship:
Ivana Koborová
Institute of Molecular Biomedicine
Medical Faculty, Comenius University, Bratislava, Slovakia
Research project at the Department of Pharmacodynamics, Semmelweis University, Budapest,

Hungary from September 2014 to January 2015:
“Relationship of SSAO/VAP-1 and insulin resistance in adolescents”
In-Coming Scholarship:
Alexander Karmash
Intern at the Department of Regulation of Cell Proliferation and Apoptosis
Institute of Cell Biology, NASU, Lviv, Ukraine
(Department of Biochemistry, Ivan Franko Lviv National University, Ukraine)
Research project at the Horváth Laboratory of Bioseparation Sciences at the Research Centre
for Molecular Medicine, University of Debrecen, Hungary, September 2014 – January 2015:
“Role of disease-related changes in immunoglobulin IgG glycosylation”
Visegrad Scholarship Program (VSP)
The International Visegrad Fund offers Master’s and Post-Master’s scholarships awarded to
selected scholars for periods of 1 or 2 semesters (with the exception of Master’s scholarships
within the Visegrad Scholarships schemes where 1– to 4-semester scholarships can be
awarded).
The following scholarship schemes are available:
Intra-Visegrad Scholarships
In-Coming Scholarships
Out-Going Scholarships
Scholarship Program for Belarusian Students
Scholarship Program for Ukrainian Students
Visegrad Scholarships at OSA Archivum (separate program)
If selected each scholar receives the scholarship funding at the beginning of each five-month
period (semester) upon a written confirmation from the host university/institution.
Deadline for all scholarship applications is 31 January. Results are announced by mid-May.
CSMC – RECOOP Research Centers (CRRC) the Center of Excellences of the RECOOP HST
Association. They host young scientists, Ph.D. students with CSMC – RECOOP (IVF – CSMC
- RECOOP) Scholarship. The RECOOP HST Association Scientific Advisory Board selects
the young scientists who could compete for IVF – CSMC - RECOOP Scholarship.
The selected young scientists (preferably Ph.D. students) will spend maximum four semesters
at the host organization and receive: €2,300 / semester and the corresponding host
universities/institutes receive €1,500/semester/scholar. The host CRRC will get $1,500 for
laboratory expense and consumables from CSMC – RECOOP HST Association. Applicants
whose current (i.e. at the time of applying) university or employer is further than 1,500 km
from the selected host university/institute are eligible for a one-time travel grant.
RECOOP HST Association Members from the Visegrad Group Countries:

IKEM - Institute for Clinical and Experimental Medicine, Prague, Czech Republic
Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic
Slovak Medical University, Bratislava, Slovakia
RECOOP HST Association Member Organizations allegeable for the In-Coming scheme
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine
Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine
RECOOP HST Association’s Cedars – RECOOP Research Center (CRRC) could

participate
Semmelweis University, Budapest, Hungary
Comenius University in Bratislava, Slovakia
Institute of Physics, Wroclaw University of Technology, Wroclaw, Poland
University Hospital in Hradec Kralove, Czech Republic
Participant List
CROATIA
School of Medicine, Josip Juraj Strossmayer University of Osijek, Croatia
Professor Ines Drenjančević, MD, PhD.

Department of Physiology and Immunology
Vice Dean for Science, Faculty of Medicine Osijek (since 2005)
University J. J. Strossmayer Osijek
Honorary university professor, University of Pecs, Hungary (since 2012),
J. Huttlera 4, 31000 Osijek, Croatia
Tel: +38531399606 and +38531512882
Mobile:+385 91 224 1406
E-mail: ines.drenjancevic@mefos.hr; inesdp@yahoo.com
Anita Ćosić MS
PhD Student
School of Medicine University Josip Juraj Strossmayer Osijek
J. Huttlera 4 31000 Osijek, Croatia
Tel: +385 31 512 869
fax: +385 31 512 833
Mobile: +385958251645
Mail: anitaa3006@gmail.com
Zrinka Mihaljević
Assistant professor
School of Medicine University Josip Juraj Strossmayer Osijek
J. Huttlera 4 31000 Osijek, Croatia
Tel: +385 031 512 869; +385 031 512 893
Mob: +385 098 533 261
e-mail: zrinka.ivanovich@gmail.com; zivanovic@mefos.hr;
Department of Medical Biology
Professor Marija Heffer, MD, PhD

Department of Medical Biology
School of Medicine,
Josip Juraj Strossmayer University of Osijek, Croatia
Huttlerova 4, 31 000 Osijek, Croatia
Phone: +385 31 512 845
Mob. 385 91 504 36 77
E-mail: mheffer@mefos.hr; marija.heffer@gmail.com;
Marta Balog
PhD Student
Department of Medical Biology,
School of Medicine,
J. J. Strossmayer University of Osijek, Faculty of Medicine
Huttlerova 4, 31000 Osijek, Croatia
Mobile: +385 98 655 705
E-mail: marthab007@gmail.com
Vedrana Ivic, research assistant

Laboratory for Neurobiology
Faculty of Medicine,
Josip Juraj Strossmayer University of Osijek, Croatia
J. Huttlera 4, 31000 Osijek, Croatia
Tel. +385 31 512 825
Fax. +385 31 505 615
Mob. +385 95 805 1221
E-mail: vedrana.ivic@mefos.hr;
Andrijana Müller, MD. Ph.D

Department of Obstetrics and Gynecology
University Hospital Center Osijek
School of Medicine Osijek
J. Huttlera 4, 31 000 Osijek, Croatia
Tel: + 38531512302
Mob: +385915767478
E-mail: andrijana.muller@os.t-com.hr
Milorad Zjalic, MS
Graduate student
Department of Biology
University Josip Juraj Strossmayer of Osijek,
Cara Hadrijana 8/A, Osijek, Croatia
Mob:+385989146425
Email: milzjalic@gmail.com
Department of Biology, University J.J. Strossmayer
Professor Elizabeta Has-Schön, PhD

Department of Biology,
University J.J. Strossmayer
Osijek, 31000 Osijek, Cara Hadrijana 8A, Croatia
Land line: +385-31-399933;
Mobil: +385 91 224 1413; +385-91-530 3066
Fax:+385 31-399339
E-mails: hasschon@biologija.unios.hr; hasschon.elizabeta@gmail.com;
University of Zagreb School of Medicine
Srećko Gajović, MD, PhD

Professor of Histology and Embryology
Editor-in-Chief, Croatian Medical Journal
Zagreb University School of Medicine
Šalata 3b, 10 000 Zagreb
Mob: +385 98 962 4800
E-mail: srecko.gajovic@cmj.hr; srecko.gajovic@hiim.hr
www.cmj.hr
Ivan Alić, DVM, PhD

Department of Anatomy, Histology and Embryology
Faculty of Veterinary Medicine
University of Zagreb
Heinzelova 55, 10 000 Zagreb, Croatia
Tel: 00 385 1 2390 259
Fax: 00 385 1 2441 390
Mobile: 00 385 98 903 39 27
E-mail: ialic@vef.hr
Marta Skelin MS, MD

PhD student
Croatian Institute for Brain Research,
School of Medicine, University of Zagreb, Croatia
Šalata 3b, 10 000 Zagreb
Mob: +38598205181
E-mail: marta.skelin@gmail.com
School of Medicine, University of Split
Ivancica Pavlicevic, MD,GP,PhD

Assistant Professor
Department of Family Medicine
University of Split School of Medicine
Croatian Cochrane Branch Training Coordinator
Šoltanska 2, 21000 Split, Croatia
E-mail: ipavlicevic@gmail.com
Slavica Jurić Petričević

Department of Emergency Medicine in Split,
Department of Family Medicine,
University of Split School of Medicine,
Šoltanska 2, Split, Croatia 21 000
Mob: +385 91 790 1002
E-mail: slavica.juric01@gmail.com
CZECH REPUBLIC
Institute for Clinical and Experimental Medicine (IKEM), Prague
Jan Pitha M.D., Ph.D.

Head of Laboratory for Atherosclerosis Research,
Centre for Experimental Research
Institute for Clinical and Experimental Medicine (IKEM)
Videnska 1958/9 14021 Prague 4, Czech Republic
Tel.: +420 26 136 3069; Fax: + 420 24 1721 574
Mob: + 420 60 764 3119
E-mail: japi@medicon.cz; japi@ikem.cz; pithajan@volny.cz
Tereza Blahová, Mgr.

Laboratory for Atherosclerosis Research,
Centre for Experimental Research
Institute for Clinical and Experimental Medicine (IKEM)
Videnska 1958/9 14021 Prague 4, Czech Republic
Tel.: +420 26 136 3069; Fax: + 420 24 1721 574
Mob:
E-mail: tereza.blahova@ikem.cz
Institute of Macromolecular Chemistry AS CR
Beata Zasonska
Department of Polymer Particles
Heyrovského nám. 2, 162 06 Praha 6, Czech Republic
Tel. 420-296809260
Fax. 420-296809410
E-mail: beata.zasonska@gmail.com
Maksym Moskvin
PhD. students
Department of Polymer Particles
Heyrovského nám. 2, 162 06 Praha 6, Czech Republic
Tel. 420-296809260
Fax. 420-296809410
E-mail: moskvin@imc.cas.cz
Faculty of Military Health Sciences, University of Defence
Pavel Bostik, MD, PhD

Associate Dean for Research
Professor and Director
Center for Advanced Studies
Faculty of Military Health Sciences
University of Defence
Trebesska 1575, 50001 Hradec Kralove
Czech Republic
Tel: (420)973253245,
Mob: (420)72 469 2609
E-mail: bostik@pmfhk.cz
GERMANY
RWTH University Aachen, Germany
Peter Boor, MD, PhD

Scientist and pathologist
Institute of Pathology and Department of Nephrology,
RWTH University Aachen, Germany
Kullenhofstr. 54B, App.023, 52074 Aachen, Germany
Academic status: (MD, PhD)
E-mail: pboor@ukaachen.de; boor@email.cz
HUNGARY
Semmelweis
Dr. Gyongyver Szentmartoni, M.D.

1st Department of Internal Medicine
Oncology Division
Medical Faculty, University of Semmelweis, Budapest
1083 Budapest Tömő utca 25-29
E-mail: gyszentmartoni@gmail.com
Prof. Éva Szökő, PhD, DSc.,

Department of Pharmacodynamics
Faculty of Pharmacy, Semmelweis University
Nagyvárad tér 4, Budapest, Hungary H-1089
Tel: (36-1) 210 2930/ 56324
Fax: (36-1) 210 4411
M: +36 30 329 6621
E-mail: szoko.eva@pharma.semmelweis-univ.hu
Tamás Tábi, PhD

Associate Professor
Nagyvárad tér 4., Budapest, H-1089 Hungary
Tel.: +36-1-210-2930/56412
Fax: +36-1-210-4411
M: +36 20 920 6408
E-mail: tabi.tamas@pharma.semmelweis-univ.hu
Fruzsina Bagamery
PhD Student
Nagyvárad tér 4., Budapest, H-1089 Hungary
M: +36 30 3435219
E-mail; bfruzsina11@gmail.com
Korányi Frigyes Science Dormitory
Andras Makkos
Medical Student VI
Semmelweis University
Tel: +36 70 370-7325
E-mail: makkosandras@gmail.com
Tim Frederik Svanya

Medical Student V.
Department of Physiology
Semmelweis Universtiy
Faculty of Medicine
Phone number: 49/173-5929799
E-mail: timsvanya@gmail.com
Orsolya Szabo
Medical Student V.
Institute of Microbiology
Semmelweis University
Faculty of Medicine
Tel: + 36 70 418-4506
E-mail: orsolya910120@gmail.com
Klara Aliz Stark

3rd year Medical Student
Faculty of Medicine, Semmelweis University, Budapest, Hungary
HUNGARY 1074 Budapest, Hársfa utca 59/b
Mobile phone: +36306760894
Email: klara.stark@t-online.hu
Dezso Kristof Gyarfas

HUNGARY 3530 Miskolc, Arany János u. 33.
Email: gyarfas.dezso.kristof@gmail.com
Tamás Daniel Csury

Email: daniel.csury@gmail.com
Balint Egyed
2nd year Medical Student
Address: HUNGARY
Email: canis.lynx@gmail.com
Imre Gonda
3rd year Pharmacological Student
Faculty of Pharamacy, Semmelweis University, Budapest, Hungary
Address: HUNGARY
Email: gonda.imre@yahoo.com
Semmelweis Publishing
Julia Mallasz
Semmelweis Publishing and Multimedia Studio
Semmelweis University, Budapest
Nagyvarad ter 4, Budapest, Hungary, H-1089
E-mail: skadi_@hotmail.com
Metropolitan College of Budapest, Institute of Applied Arts
Julia Gacs
Metropolitan College of Budapest, Institute of Applied Arts
Budapest Rozsa ut 4-6, Hungary, H-1077,
Tel: +36 1 273 3099
E-mail: gacsjulia@hotmail.com
Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, University

of Szeged
Robert Gaspar Pharm. Dr., Ph.D.

Associate Professor
Department of Pharmacodynamics and Biopharmacy
Faculty of Pharmacy
University of Szeged
Szeged Eotvos u 6, Hungary H-6720
Tel: + +36 62 341 971
Mob: + 36 30 975 5401
Fax: 36-62-545-567
E-mail: gaspar@pharm.u-szeged.hu
Judit Hajagos-Tóth PharmD, PhD

Assistant Professor
Faculty of Pharmacy
Szeged, Eotvos u 6, Hungary H-6720
Tel: 36-62-341-330
Tel/fax: 36-62-545-567
Mob: 36 30 314 8680
E-mail: judittoth@pharm.u-szeged.hu
Anita Sztojkov-Ivanov, PharmD, PhD
Assistant professor
Faculty of Pharmacy
Tel: 36-62-341 - 974
Tel/fax: 36-62-545-567
E-mail: Ivanov.Anita@pharm.u-szeged.hu
Kálmán Szűcs, PharmD, PhD Student

Dept. of Pharmacodynamics and Biopharmacy
Faculty of Pharmacy University of
Szeged, Hungary
Tel: 36-62-545-569
Tel/fax: 36-62-545-567
Mob:+36 30 530 6545
E-mail: szucs.kalman@pharm.u-szeged.hu
University of Debrecen
Eva Nagy, MSc

Bioengineer, PhD Student
Doctorate School of Animal Husbandry
Faculty of Agriculture and Nutritional Sciences
University of Debrecen
Egyetem ter 1.
Elettudomanyi epulet 2.027-28.
H 4032, Debrecen
Hungary
Mob: +36 30 401 8241
E-mail: nagyevacska@gmail.com; nagy.eva@agr.unideb.hu
Boglarka Donczo
Medical and Health Sciences Center
Research Centre for Molecular Medicine
Horváth Laboratory of Bioseparation Sciences
H-4032, Nagyerdei krt. 98. Theoretical Building 1/111, Debrecen, Hungary
M: +36 30 483 0825
E-mail: boglarka1112@gmail.com
Máté Szarka Biotechnology MSc.

Horváth Csaba Laboratory of Bioseparation Sciences
Medical and Health Sciences Center
Research Centre for Molecular Medicine
Horváth Laboratory of Bioseparation Sciences
University of Debrecen, Debrecen, Hungary
H-4032, Nagyerdei krt. 98. Theoretical Building 1/111, Debrecen, Hungary
Mobil: +3630 5841954
E mail: szarka.mate@med.unideb.hu szarkamatthew@gmail.com
University of Pecs
Professor Tibor Ertl, MD PhD DSc

Professor of Neonatology
Department of Obstetrics and Gynecology
Medical School, University of Pécs
Édesanyák u. 17, Pécs, Hungary, H-7624
Telephone # 36 72 536 381, 36 30 22 60 947
Fax # 36 72 536 381
Mob: +36 30 226 0947
E mail: tibor.ertl@aok.pte.hu
Professor Judit Horvath, MD, PhD

Medical School, University of Pécs, Hungary
E-mail: judit.e.horvath@aok.pte.hu
Tímea Kvárik, MD
PhD student
Department of Obstetrics and Gynaecology,
Medical School, University of Pécs
Édesanyák útja 17., Pécs, 7624, Hungary
Mob: +36 30 408 8076
E-mail: kvarik.timi@gmail.com
László Adorján
Business informatics engineer
email:adorjanl@gmail.com
POLAND
Department of Experimental Physics, Wroclaw University of Technology, Poland

Wroclaw
Artur Podhorodecki, PhD, D.Sc.

Associate Professor
Department of Experimental Physics, Wroclaw University of Technology
Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland
Tel: +48 71 320 2358
Mob: +48 607 244 799
E-mail: artur.p.podhorodecki@pwr.edu.pl
Maciej Chrzanowski
Department of Experimental Physics,
Wroclaw University of Technology
Tel: +48713202358
E-mail: 193919@student.pwr.edu.pl
Anna Lesiak
Department of Experimental Physics,
Wroclaw University of Technology
Tel: +48713202358
E-mail: annaa.lesiak@gmail.com
ROMANIA
“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
Iuliana Ceausu, M.D., Ph.D

The Department of Obstetrics and Gynecology
of “Dr. I. Cantacuzino” Hospital,
“Carol Davila” University of Medicine and Pharmacy,
Bucharest, Romania
Ion Movila Street, no 5-7, sector 2, Bucharest 70266 Romania
Tel: +40 1-210 2806; Fax: +40-1-310 1213; +40-1-210 6435,
Mobile: +40-72 279 0093
E-mail: iulianaceausu2004@yahoo.com; iceausu@hotmail.com
SLOVAKIA
Comenius University
Katarina Jansákova
Institute of Molecular Biomedicine
Comenius University
Sasinkova 4, 811 08 Bratislava, Slovakia
E-mail: jansakova.katarina@gmail.com
MSc. Radana Gurecká

Institute of Molecular BioMedicine
Faculty of Medicine, Comenius University
Sasinkova 4, 811 08 Bratislava, Slovak Republic
Phone: +421-2-59357-371
Fax: +421-2-59357-631
Mobile: +421 907 539 253
Email: radana.kollarova@gmail.com
Slovak Medical University
Assoc. Prof. Shubhada Bopegamage, MSc., PhD.

Head, Entervirus Laboratory
Medical Faculty
Slovak Medical University
Limbova 12, 83303 Bratislava, Slovak Republic
Phone: +421 25 937 0777 +421 25 937 0777
Mobile: +421 90 724 6365
E-mail: shubhada.bopegamage@szu.sk; s.bopegamage@gmail.com
Brigita Benkoova MSc

PhD student
Enterovirus Laboratory,
Faculty of Medicine,
Slovak Medical University in Bratislava,
Limbová 12, 833 03, Bratislava, Slovakia
Mob:00 421 905 884 811
E mail: brigita.benkoova@szu.sk
UKRAINE
Department of Pharmaceutical, Organic and Bioorganic Chemistry

Danylo Kaminskyy
Associate professor
Department of Pharmaceutical & Organic and Bioorganic Chemistry
Danylo Halytsky Lviv National Medical University
Pekarska 69, Lviv-10, Ukraine
Tel: +380322755966
Mob: +380677380471
E-mail: dankaminskyy@gmail.com
Anna Kryshchyshyn
Assistant professor
Pekarska 69, Lviv-10, Ukraine
Tel: +380322755966
Mob: +380678885827
E-mail: kryshchyshyn.a@gmail.com
Andrii Lozynskyi
assistant professor
Address: Pekarska 69, Lviv 79010
Tel: +38 032 2757734
Mob: +38 063 3517637
E-mail: lozisnskij@i.ua
Department of Biochemistry
Dr. Lesya Kobylinska, PhD

Associate Professor
Department Biochemistry
Pekarska 69, Lviv, Ukraine 79010
Tel: +380(322)757602
Mob: +380677223896
E-mail: lesya8@gmail.com
Iryna Ilkiv
assistant professor
Pekarska 69, Lviv 79010
Tel: +38 032 2757734
Mob: +38 093 618 77 39
E-mail: ira9ilkiv@gmail.com
Nataliya Denysenko
Assistant professor
69 Pekarska str., Lviv, Ukraine
Tel:+38 (032) 275 76 02
Mob: +38(063)307 56 65
E-mail: denysenko.natalka@gmail.com
Department of Pharmacology
Professor Oleh Pinyazhko, MD,PhD

Head of Pharmacology Department
Danylo Halytsky National Medical University
69, Pekarska Str, Lviv 79010, Ukraine
Tel: +380677993999
E-mail: olehpinyazhko@gmail.com
Internal Medicine
Mykhailo Pliatsko, M.D, Ph.D

Associate professor
Department of Internal Medicine №1,
Lviv National Medical University
79010, st., Lychakivska 32/12, Lviv, Ukraine
Tel +380 275 58 21
Mob +380 67 66 38 303
E-mail drplzk@gmail.com
Physiology Department Lviv National Medical University
Nazar Bula
PhD student
Physiology Department
Pekarska, 69, Lviv 79010, Ukraine
Tel : +38 032 2603007
Mob: +38 067 7770987
E-mail: bula_lnmu@ukr.net
Maryana Zvir
PhD student
Pekarska 69, Lviv 79010, Ukraine
Tel: +38 032 2603007
Mob: +38 098 5891689
E-mail: maryana.zv@gmail.com
Jaroslav Pavlovskyi
PhD student
Address: Pekarska 69, Lviv 79010, Ukraine
Tel: +38 032 2603007
Mob: +38 097 9535176
E-mail: pavl_jarik@ymail.com
Irena Pshyk-Titko
Teach Assistant, PhD
Tel: +38 032 2603007
Mob.: +38 050 5026346
E-mail: irena.pshyk@gmail.com
Professor Oksana Zayachkivska, MD, PhD, DSc

Tel: +380 32 260 3007
Mob.: +380 67 910 5686
E-mail: ozayachkivska@gmail.com
Institute of Cell Biology, National Academy of Sciences of Ukraine
Professor Rostyslav Stoika, PhD, D.Sc.

Head and Professor
Department of Regulation of Cell Proliferation and Apoptosis
Institute of Cell Biology
Drahomanov Street 14/16
79005, Lviv, UKRAINE
phone/fax: +38 032 261 22 87
Mobil: +380 66 303 2152
E-mail: stoika@cellbiol.lviv.ua; stoika.rostyslav@gmail.com
Aryana Stoyka
Institute of Cell Biology
Oleksandr Korchynskyy
Senior Scientist
Institute of Cell Biology, Natl. Acad.Sci. of Ukraine
14/16 Drahomanov Str., 79005 Lviv, UKRAINE
Tel: +38 (032) 261-2108
Mob +38 (095) 939-9926
E-mail: olexkor@hotmail.com
Nataliia Korchynska
Institute of Animal Biology
NAAS of Ukraine, Institute of Cell Biology NAS of Ukraine
Address: 14/16 Drahomanov Str., 79005 Lviv, UKRAINE
Tel +38 (032) 261-2108
Mob +38 (095) 939-9926
E-mail: olexkor@hotmail.com
Rostyslav Panchuk, PhD

Scientific Fellow,
Department of Regulation of Cell Proliferation and Apoptosis,
Dragomanov Str 14/16, 79005, Lviv, Ukraine
tel/fax +38-032-261-22-87
mobile +38-097-798-26-20
rpanchuk@ukr.net
Nataliya Finiuk
Junior Scientist
Drahomanov Street 14/16, 79005, Lviv, Ukraine
Tel: +38 032 261 22 87
Mob: +38 050 239 2957
E-mail: nataliyafiniuk@gmail.com
Khrystyna Malysheva
PhD student
Institute of Animal Biology NAAS of Ukraine,
Institute of Cell Biology NAS of Ukraine
14/16 Drahomanov Str. 79005 Lviv, UKRAINE
Tel +380 32 261-2108
Mob +380967659128
E-mail: khrystyna.malysheva@gmail.com
Julia Senkiv, Ph.D.,

Junior Scientist at the Institute of Cell Biology
Drahomanov Str. 14/16, 79005, Lviv, Ukraine,
Tel/fax +38-032-261-22-87,
Mobile +38-093-11-527-59,
E-mail: yu.senkiv@gmail.com
Palladin Institute of Biochemistry National Academy of Sciences Ukraine
Tatiana Borisova, PhD, DSc

Group Head, Leading scientist,
Department of Neurochemistry,
Palladin Institute of Biochemistry NAS of Ukraine
9 Leontovicha str., 01601 Kiev, Ukraine
Tel.: 380 44 234-32-54
Fax 380 44 279-63-65
E-mail: tborisov@biochem.kiev.ua
Maryna Dudarenko
a junior scientist of Department of Neurochemistry,
Palladin Institute of Biochemistry, NAS of Ukraine;
9 Leontovicha Str., 01601, Kiev, Ukraine.
Lead engineer; 26 y.o.;
mob. +38(097)2559572
E-mail: marina.dudarenko@gmail.com
Arsenii Borysov
Lab.Assistant
Department Neurochemistry
National Academy of Sciences of Ukraine 9 Leontovicha str., 01601 Kiev, Ukraine
Tel.: 380 44 234-32-54
Fax 380 44 279-63-65
E-mail: arsenjkeee@ukr.net
Mazur Iuliia
Lab Assistant
Department of Muscle Biochemistry
National Academy of Sciences of Ukraine 9 Leontovicha str., 01601 Kiev, Ukraine
Tel. 380 44 234 10 53
Fax 380 44 279-63-65
E-mail: yuliya.vorona@gmail.com
Mariia Dekaliuk
a junior scientist of Department of Molecular Immunology,
Ph.D. student; 24y.o.;
E-mail: dekalyuk_m@mail.ru
Ganna Pasichnyk
a researcher of Laboratory of Cell Signaling,
researcher; 29y.o.;
mob (097)670-66-79
E-mail: anya_p@meta.ua
Volodymyr Chernyshenko
Position: fellow scientist
Affiliation: Palladin Institute of biochemistry NAS of Ukraine
Address: 9, Leontovych Str., Kyiv, 01030.
Tel: +380442355172
Mob: +380675906710
E-mail: bio.cherv@gmail.com
USA
Cedars – Sinai Medical Center
Edward Prunchunas
Senior Vice President for Finance and Chief Financial Officer
Cedars-Sinai Medical Center and
Chairman of the Supervisory Board of the RECOOP HST Association
8700 Beverly Boulevard
Los Angeles, California 90048-1860
Telephone (310) 423 2312
Fax (310) 423 0120
E-mail: Edward.Prunchunas@cshs.org
URL: www.csmc.edu
Charles F. Simmons, Jr., MD

Professor and Chairman, Department of Pediatrics
Ruth and Harry Roman Chair in Neonatology
Director, Division of Neonatology
Cedars – Sinai Medical Center
8700 Beverly Blvd., Room 4228
Los Angeles, CA 90048
Tel: (310) 423-4416
Fax: (310) 423 - 0460
E-mail: charles.simmons@cshs.org
Sandor G. Vari, MD
Director, International Research and Innovation in Medicine Program
Cedars-Sinai Medical Center, Los Angeles, CA, USA &
President of the RECOOP HST Association
6500 Wilshire Blvd., 23rd floor Room 2311, Los Angeles, CA 90048-4903
Tel: 1 323-866-8122
Mob: 1 818 398 2642
E-mail: vari@cshs.org
URL: https://www.cedars-sinai.edu/Research/Research-Administration/index.aspx
James D. Laur, Esq.
Vice – President, Department of Legal Affairs
Cedars-Sinai Medical Center, Los Angeles, CA, USA
Member of the Technology Transfer Working Group of the of the RECOOP HST
Association
8701 West Third Street, Suite 290
Los Angeles, CA 90048
Telephone: (310) 423 5284
Fax : (310) 423 0101
E-mail : James.Laur@cshs.org
URL: www.csmc.edu
Linn Defensor RN, CCRP

Office of Research Compliance and Quality Improvement
Cedars-Sinai Medical Center
&
RECOOP HST Association Clinical Research Site
Management Network (CRSMN) Leader
Phone (310) 423-3783
Fax (310) 423-4195
M: 1 818 516 6017
E-mail: defensor@cshs.org
Aranya Seals
Executive Assistant
Cedars-Sinai Medical Center and
WEB Master of the
RECOOP HST Consortium
8700 Beverly Boulevard
Los Angeles, California 90048-1860
Telephone (310) 423 2312
Fax (310) 423 0120
E-mail: Aranya.Seals@cshs.org
Veronika Puska
Grant and Project Manager
Budapest, Szalanci str 5, H-1124, Hungary
6500Wilshire Blvd., 23rd floorRoom 2311
Los Angeles, CA 90048-4903
E- mail: Recoop.RA@gmail.com
Mob: +36 – 30 – 350 - 3292
Skype: recoop.ra
TM
Cycle of Knowledge
Bridges in Life Sciences 11th Annual Scientific Conference in Prague,
Czech Republic
April 7 – 10, 2016
Happier Scientist

Spec Iss 2016

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National Academy of Sciences of Ukraine

Palladin Institute of Biochemistry

Bridges in Life Sciences 11th Annual

April 7 – 10, 2016

Agenda of the Bridges in Life Sciences 10th Conference 4

RECOOP HST Association 17

Croatian Medical Journal 20

Common Mechanism of Diseases 22

Cardiovascular Research in Progress 31

RECOOP Research in Progress 56

Infections and Inflammation 64

RECOOP Research in Progress 70

Experimental Works of RECOOP Members (Ukraine) 78

RECOOP Visegrad Scholarship Program 114

Conference Participants 116

USA - Cedars-Sinai Medical Center, Los Angeles, California

Shlomo Melmed, M.D.

RECOOP HST Association

Veronika Puska, Grant and Project Manager, RECOOP HST Association

Editor: Sandor G. Vari, M.D.

April 8, 2016 (Friday)

8:30 – 9:00 Message from the President of RECOOP

Message from the President of RECOOP HST Association on April 8, 2016

9:00 – Keynote lecture:

9:00 – 9:15 RECOOP Project Common Mechanism of Diseases

9:15 – 9:25 Young Scietists Contribution – Electronic Interactive Poster Presentation

Plenary Session: Common Mechanism of Disease

9:25 – 9:40 Alteration of inflammatory markers and SSAO activity in diet

11:00 – 11:30 Coffee Break Conference Room 3

11:30 – 11:45 Panel discussion Common Mechanism of Disease

Electronic Interactive Poster Presentation

12:15– 12:25 Influence of acute and intermittent hyperbaric oxygenation on the

12:25 – 12:35 Comprehensive analysis of ischemic conditioning techniques in a porcine

12:35 – 12:45 The Role of Oxidative Stress in Development of Impaired Vascular

12:45 – 13:00 Evaluation of putative cannabinoid ligands as modulators of

13:00 – 14:00 Lunch Break

14:30 – 14:45 Biocompatibility and stem cell labelling properties of maghemite

14:45 – 15:00 S, N - containing carbon dots: fluorescent properties and application in

EIPP – Electronic Interactive Poster Presentations

15:00 – 15:10 Neurotoxic properties of carbon dots synthesized from sulphur-containing

15:00 – 15:20 CdSe/CdS quantum dots functionalization by d-penicillamine

15:20 – 15:30 Synthesis of Cd-based core shell nanocrystals for bio-imaging

16:00 – 17:00 Coffee Break

17:00 – 19:30 RECOOP Research in Progress – Young Scientists Forum

17:00 – 17:15 Sex-specific oxidative-antioxidative status in the liver of the chronically

17:15 – 17:30 Search for New Antifibrotic Agents among (amino)iminothiazolidinones

17:30 – 17:40 Birth outcome yearly dustrubution at Department of Gynecology and

Young Scientists Forum

EIPP – Electronic Interactive Poster Presentations

17:40 – 17:50 Metformin may influence neurodegeneration and neuroinflammation in

17:50 – 18:00 The importance of autophagy in the antiapoptotic effect of resveratrol

18:10 – 18:20 ARHGAP25 Rac-GAP has an important role in autoantibody-induced

18:20 – 18:30 The use of formalin-fixed paraffin-embedded samples in early diagnosis of

18:40 – 18:50 Analyses of impact circadian dysfunctions on the occurrence of

18:50 – 19:00 Plasma membrane Ca2+-pump selective regulators – calix[4]arene C-90

20:00 – 22:00 Dinner

8:30 – 9:00 Summary of the Conference’s Progress

9:00 – 11:00 Annual Review of the Research Activities in RECOOP

9:30 – 9:45 Diurnal variation in cholesterol 7α-hydroxylase activity is determined by

9:45- 10:00 Putative anticancer potential of novel 4-thiazolidinone derivatives: toxicity

11:00 – 11:30 Coffee Break

12:00 – 13:00 Young Scientist Forum - Infection and Inflammation

12:00 – 12:10 Induction of cytokines by CVB2-Ohio and its mutants