PSN 2018 POSTER SESSION

Ulatowski Filip
Dynamic and Supramolecular Chemistry Group, Institute of Organic Chemistry, PAS (Kasprzaka 44/52, 01-224 Warsaw, Poland)
Presenting author: Filip Ulatowski, Ph.D. (filip.ulatowski@icho.edu.pl)
NTRODUCTION: The activity of common drugs relies on the capability of given molecules to form specific supramolecular complexes. These properties depend on the adopted spatial arrangement of functional groups, which define the molecular geometry. Experimental determination or theoretical prediction of molecular geometry is a highly challenging task. Our aim is to employ a dynamic combinatorial libriary to act as a sensor array to determine certain aspects of molecular structure and evaluate geometrical and functional similarity between different chemical compounds.
METHODS: In the initial studies a set of molecules (templates) – carboxylates that vary in number of functional groups, size, shape and flexibility – was used to interact with dynamic combinatorial libraries of macrocyclic disulphides. The composition of the library, influenced by introduction of a template was evaluated by HPLC. Concentrations of the library members were in the next step analysed by principal component analysis (PCA) and artificial neural networks (ANN).
RESULTS: The dynamic combinatorial library is able to distinguish all of the used templates. After PCA certain clusters are easily recognised, which correspond to templates of similar length of bearing the same structural feature. After training, the ANN can be used to determine the number of functional groups in an unknown template with approx. 70% efficiency.
CONCLUSIONS: These results indicate that dynamic combinatorial library in combination with PCA or ANN can recognise specific structural features and assess structural similarity between chemical compounds. This property may be particularly useful in analysis of potential action and side effects of novel drugs.

Sulkowska Joanna I.
Centre of New Technologies, University of Warsaw (S. Banacha 2c, 02-097 Warsaw, Poland)
Presenting author: Joanna I Sulkowska, Ph.D. (jsulkows@gmail.com)
INTRODUCTION: KnotGenom server enables to analyze entanglement of a single chromosome and links between chromosomes in the entire cell. All types of entanglement can be determined by different deterministic and probabilistic methods. The knotting complexity of the chromosome is presented in the form of a matrix diagram that shows users the knot type of the entire polypeptide chain and of each of its subchains. Entanglement of links is also computed by Gaussian linking integral. Entangled chromosomes are presented graphically in an intuitive way. The stability of entanglement can be studied based on the relaxation method.
METHODS: Knot theory
RESULTS: The topological analysis of chromosomes has shown that single chromosome can be knotted and this knowledge is used to improve current data, 3D chromosome structure. However, according to our knowledge, a pair of chromosomes has never been analyzed from the point of view of links. A link is formed from at least two chains – e.g. a pair of chromosomes. Our review of available data shows that a significant number of stable links between chromosome pairs exist. Some links are even conserved between cells. In the paper we also present methods used to determine types of links and their locations along chromosomes.
CONCLUSIONS: Identified links might suggest that a small fraction of chromosomes are entangled not only locally. Presented methods should be used also as a quantitative assessment – descriptor – to distinguish the quality of modeled data.

Boniecki Michał J.1, Łach Grzegorz1, Tomala Konrad1, Dawson Wayne1, Sołtysiński Tomasz1, Łukasz Paweł1, Rother Kristian1, Bujnicki Janusz M.2,1
1 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw (ul. Trojdena 4, 02-109 Warszawa, Poland)
2 Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań (ul. Umultowska 89, 61-614 Poznań, Poland)
Presenting author: Michał J Boniecki, Ph.D. (mboni@genesilico.pl)
INTRODUCTION: The molecules of the ribonucleic acid (RNA) perform a variety of vital roles in all living cells. Their biological function depends on their structure and dynamics, both of which are difficult to experimentally determine, but can be theoretically inferred based on the RNA sequence. We have developed a computational method for molecular simulations of RNA, named SimRNA.
METHODS: SimRNA is based on a coarse-grained representation of a nucleotide chain, a statistically derived energy function, and Monte Carlo methods for sampling of the conformational space. The backbone of RNA chain is represented by two atomsper nucleotide, whereas nucleotide bases are represented by three atoms. All terms of the energy function were derived from a database of crystal RNA structures, as a statistical potential. Sampling of the conformational space was accomplished by the use of the asymmetric Metropolis algorithm coupled with a dedicated set of moves.
RESULTS: Recent tests demonstrated that SimRNA is able to predict basic topologies of RNA molecules with sizes up to about 50 nucleotides, based on their sequences only, and larger molecules if supplied with appropriate distance restraints. The user can specify various types of restraints, including restraints on secondary structure, distance and position.
CONCLUSIONS: SimRNA can be used for RNA folding and RNA 3D structure prediction. It is also able to fold/refine structures with irregular (non-helical) geometry of the backbone (RNA pseudoknots, ulges etc.). SimRNA is a folding simulations method, thus it allows for examining folding pathways, getting approximate view of the energy landscapes.

Janik-Superson Katarzyna1, Sobalska-Kwapis Marta1,2, Słomka Marcin1,2, Marciniak Błażej1, Strapagiel Dominik1,2
1 Biobank Lab, Department of Molecular Biophysics, University of Lodz (ul. Pilarskiego 14/16, 90-231 Lodz, Poland)
2 BBMRI.pl Consortium, Wroclaw Research Centre EIT+ (ul. Stabłowicka 147, 54-006 Wroclaw, Poland)
Presenting author: Katarzyna Janik-Superson, Ph.D. (katarzyna.superson@biol. uni.lodz.pl)
We work on a huge amount of data. We still collect and process thou-sands of genotypic and phenotypic information about one patient for the use in extensive genetic and medical research. We are helped by the biobanking system, which we are currently improving in Poland as a part of the European Consortium European Research Infrastructure Consortium BBMRI-ERIC. All procedures for collecting material, recruitment of volunteers and patients, data anonymization, entering declarative data and medical history into the system, storage procedures for a given biological and DNA samples must be subject to inspection and registration. For this we need platforms edited only by authorized administrators. Since 2010 such a system has been created in Biobank Lodz of University of Lodz. As part of the TESTOPLEK project, we have created the Sample Management System (SMS) to collect and manage biological samples and phenotype data. We collected samples from over 10 000 Polish adults including children, people suffering from cancers, endometriosis etc.
We created unique questionnaires, in which personal data were irreversibly anonymized, contained very detailed questions about appearance phenotype, ethnicity, addictions, diseases and treatment. As a member of BBMRI. pl consortium, we comprehensively tested freeware software BBMS for management of Biobanks, available online on the manufacturer’s website (LabMind). The purpose of BBMS is to manage sample storage and track all the experimental procedures. This homogeneous system makes possibilities to unify data collection and management procedures for broad sharing and learning others, cooperation and information exchange between other scientific units and medical centers.

Polak Maja1, Koj Natalia1, Shrubkovskyi Illia1, Janik-Superson Katarzyna1,
Sobalska-Kwapis Marta1,2, Słomka Marcin1,2, Borówka Paulina1,2, Marciniak Błażej1,2, Strapagiel Dominik1,2
1 Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection University of Lodz (Pilarskiego 14/16, 90-237 Lodz, Poland)
2 BBMRI.pl Consortium, Wroclaw Research Centre EIT+ (Stablowicka 147, 54-066 Wroclaw, Poland)
Presenting author: Maja Polak, M.Sc. (maja.polak@biol.uni.lodz.pl)
Within project “Digital sharing of biomolecular and descriptive resources of Biobank and Department of Anthropology, University of Lodz – characteristics of populations living in present-day Poland through the ages. Information platform e-Czlowiek.pl (e-Human.pl)” we obtain data of Polish individuals and after analysis we will publish it on platform e-Czlowiek.pl. This is long and difficult way but results are invaluable.
The platform will contain anthropological and genetic information of human populations living in the central Poland in the period from the Piasts to the beginning of the 19th century and also genetic data of contemporary Poles from the whole country. Analysis of modern and ancient DNA differ from each other.
First step of modern DNA analysis is acquisition of the data about over 0.5 million SNPs digitized on microarrays. After this stage we have to compare our data to databases such as tools delivered by NCBI, GWAS Catalog, PredictSNP and others. We check SNP location, description of occurrence SNP in world populations and functional consequences of SNP. We get results by PLINK 1.07 using casecontrol association analysis, covariative with environmental factors and we also build logistic and linear models. Important stage of analysis is to visualise our results by HaploView software where we obtain Manhattan plots and linkage disequilibrium blocks. For visualisation we also use LocusZoom to depict the location of genetic markers and its correlation with the reference SNP.
The objectives of present study is genetic characterization of Polish population living in present-day Poland through the ages.

Chojnowska Katarzyna, Niewiadomski Paweł
Laboratory of Molecular and Cellular Signaling, Centre of New Technologies, University of Warsaw (Banacha 2c, 02-097 Warszawa, Poland)
Presenting author: Paweł Niewiadomski, Ph.D. (p.niewiadomski@cent.uw. edu.pl)
Despite recent revolutionary progress in the understanding of the mechanisms of oncogenic transformation, personalized cancer therapy remains largely elusive. Instead, to eradicate the tumor we still rely on surgery, broad-spectrum chemotherapeutics, and radiation therapy, all of which severely reduce the patient’s quality of life. And yet, cells in each tumor rely on just a handful of deregulated signaling pathways to maintain their growth. In principle, if we were able to precisely identify these pathways, we could target them therapeutically and eliminate the tumor with minimal side effects. Finding such pathways has become feasible in recent years due to progress in automation, DNA sequencing, and data analysis methods, which enabled screening of potential targets on an unprecedented scale. Recently, two largescale loss-of-function screens yielded a trove of data regarding the sensitivity of cancer cells to the loss of different genes. We searched the data generated in these screens to identify previously unknown cancer type-specific factors that determine the growth capacity of cancer cells. We hope that these factors will serve as targets for personalized therapy or as biomarkers to stratify cancers for different therapeutic interventions.
This work was supported by the SONATA BIS grant 2014/14/E/NZ3/00033 from the National Science Centre.

Grochowalski Łukasz1, Jarczak Justyna2,1, Marciniak Błażej2,1, Słomka Marcin2,1, Sobalska-Kwapis Marta2,1, Strapagiel Dominik2,1
1 Biobank Lab, Department of Molecular Biophisics, University of Lodz (Pilarskiego 14/16, 90-231 Lodz, Poland) 2 Wroclawskie Centrum Badan EIT+ Sp. z o o, BBMRI.pl Consortium (Stablowicka 147, 54-066 Wroclaw, Poland)
Presenting author: Łukasz Grochowalski, B.Sc. (lukasz.grochowalski@gmail. com)
In population studies, getting the right population division can be difficult. Administrative regions are useful however they do not reflect the structure of specific population. When sample size increases and samples start to come in from different places this problem occurs. The solution could be clustering, the machine learning method of sample grouping, based on their spatial coordinates and creating new intermediate division that are best suited to data. The aim of the present study was to find the method to visualize trait distribution in Polish population. We had at our disposal two types of data: genetic, based on single nucleotide polymorphism in DNA and survey, based on information from questionnaire. The experimental group included samples taken from 5852 individuals representing administrative units of both levels of local administration in Poland. Many different methods for clustering were tested with scikit-learn package in Python including Ward, Birch but K-Means turned out to be the simplest way to obtain reliable results. K-means can give cluster centres or cluster’s borders which can be further used in analysis for example to interpolate trait distribution in population on maps using GIS software.
Funding: ”Biobank network in Poland, within the BBMRI-ERIC Research Infrastructure of Biobanks and Biomolecular Resources” no. DIR/WK/2017/01 and POPC.02.03.01-00-0012/17: „Digital sharing of biomolecular and descriptive resources of Biobank and Department of Anthropology, University of Lodz – characteristics of populations living in present-day Poland through the ages. Information platform e-Czlowiek.pl” (Operational Programme Digital Poland for 2014–2020).

Seweryn Michał, Ludwig-Słomczyńska Agnieszka, Kapusta Przemysław, Pitera Ewelina, Wołkow Paweł
OMICRON, Jagiellonian University Medical College (Kopernika 7c, 31-008 Kraków, Poland)
Presenting author: Michał Seweryn, Ph.D. (michal.t.seweryn@uj.edu.pl)
INTRODUCTION: Mitochondrial RNA synthesis is a proces which originates from the D loop – a regulatory region of the mitogenome. This study is aimed to detect significant interactions between the level of heteroplasmy in the D-loop region of the mitochondrial genome and the expression of mitochondrial transcription factors encoded in the nuclear genome in the context of the body mass index (BMI).
METHODS: The analysis of genetic variants was performed using Whole Genome Sequencing and RNA-seq data from the Genotype-Tissue Expression (GTEx) project. Two tissues were selected for analysis – subcutaneous adipose tissue (89 individuals) and skeletal muscle tissue (107 individuals).
RESULTS: The analysis revealed a statistically significant association between the expression level of the transcription factor TFAM in the skeletal muscle tissue and the levels of heteroplasmy at position 16293 of the mitochondrial DNA in the context of BMI. This site is a variable locus exhibiting an A to G or T nucleotide change (rs878890610) and an AC to GT or TT (rs386828867). The increased expression of the TFAM gene and elevated levels of heteroplasmy correlate with BMI values [p(nuc) = 0.01 i p(mito)0.003′]. Their interaction leads to a compensatory effect and correlates with lower BMI (p = 0.001). In the subcutaneous adipose tissue an association was identified between: (1) TFB2M expression and a variant at position 493 of the mitochondrial genome in the context of BMI (p = 0.03); (2) expression of TFB1M andavariant at position 302 in the context of BMI (p = 0.02) and (3) TFAM expression and a variant at position 16189 in the context of BMI (p = 0.05).
CONCLUSIONS: The presented results indicate the existence of statistically significant interactions between expression of mitochondrial transcription factors and polymorphic variants in the D loop of the mitogenome in the context of BMI.

Walczak Tomasz1, Michałowska Martyna1, Grabski Jakub K.1, Stopa Marcin2, Rakowicz Piotr2, Wojciechowska Joanna2, Schwarc Kamila2
1 Institute of Applied Mechanics, Poznan University of Technology (Jana Pawła II 24, 60-965 Poznan, Poland)
2 Department of Ophthalmology. Chair of Ophthalmology and Optometry, Poznan University of Medical Sciences (Grunwaldzka, 16/18, 60-780 Poznan, Poland)
Presenting author: Kamila Schwarc, M.D. (kamilaschwarc@gmail.com)
INTRODUCTION: Our purpose was to evaluate the effectiveness of artificial neural networks (ANN) in predicting cataract development after pars plana vitrectomy in phakic patients.
METHODS: Thirty-seven patients were included in the study group. The examinations were performed before vitrectomy, one month and three months after. Visual acuity and the grade of opacity of the lens based on the LOCS III scale (N, C, P) were analyzed. The results were used in the ANN learning process. Input parameters included the patient’s age and stage of cataract before and one month after the surgery. The stage of cataract 3 months after the operation was the output parameter. For each patient, 2 000 MLP networks of the different structure with distinct activation functions were used. The network with the highest learning, validation, and testing rates was selected for analysis.
RESULTS: The accuracy of prediction the stage of cataract advancement 3 months after the pars plana vitrectomy was checked both for the combined grade of cataract (N + C + P) and for each variable separately. At the learning stage, the ANN has achieved high rates of learning, validation, and testing for combined N + C + P (over 90%). For the N – ANN produced poor learning, validation, and testing parameters, while the P and C were good and very good respectively.
CONCLUSIONS: Our results indicate the possibility of prediction with ANN the combined grade of cataract and the grade of posterior subcapsular cataract and cortical cataract. The patient’s age and the stage of cataract before and one month after vitrectomy were sufficient input parameters.

Marciniak Błażej1, Borówka Paulina2,1, Janik-Superson Katarzyna1, Shrubkovskyi Illia1, Polak Maja1, Koj Natalia1, Karkus Justyna2, Mietlińska Joanna2, Lorkiewicz Wiesław2, Strapagiel Dominik3,1
1 Department of Molecular Biophysics/Biobank Lab, University of Lodz (Pilarskiego street 14, 90-237 Lodz, Poland)
2 Department of Anthropology, University of Lodz (Pilarskiego street 14, 90-237 Lodz, Poland)
3 BBMRI.pl Consortium, Wroclaw Research Centre EIT+ (Stabłowicka street 147, 54-066 Wroclaw, Poland)
Presenting author: Błażej Marciniak, M.Sc. (blazej.marciniak@biol.uni.lodz.pl)
Obtaining of biomedical data is essential for science and constitutes the most expensive and time consuming part of scientific projects. Publishing data as a scientific paper attachments does not resolve the problem of the lack of available data in public resources, which could be used for additional analyses. Main question is: is it the right way? The answer is not so simple and explicit. We think, that one of the reasons of this situation can be Legal Property issue. Therefore, we would proudly announce new approach in data sharing. Project “Digital sharing of biomolecular and descriptive resources of Biobank and Department of Anthropology, University of Lodz – characteristics of populations living in present-day Poland through the ages. Information platform e-Czlowiek.pl (e-Human.pl)” gained European Union founding in “Digital Poland Operational Program”. The idea is to share as many data as we can, but what makes us different from other already existing repositories is that usage of data is under licensing embargo. Our web platform will provide advanced search capabilities, we are planning to enable searching even at the level of individual sample details. The designed platform will be open for all scientists to upload and share theirs data on conditions that they define (MTA/DTA licensing, co-authorship, citation of resources, etc.)
Launch of platform is planned on 3rd quarter of 2020. Initial data set: 10000 individuals with phenotype data collected by a questionnaire and DNA Microarray results (over 550K SNPs/Donor); 3D scans of 200 skeletons dated from XI to XIX century, isolated DNA, genomes.

Latoch Przemyslaw1, Starosta Agata L.2
1 Wydział Informatyki, Polsko-Japońska Akademia Technik Komputerowych (Koszykowa 86, 02-008 Warszawa, Poland)
2 Ecotech-COMPLEX/ Laboratorium Ekspresji Genów, Uniwersytet Marii Curie-Skłodowskiej (Głęboka 39, 20-612 Lublin, Poland)
Presenting author: Przemyslaw Latoch, M.Sc. (przemyslaw.latoch@poczta. umcs.lublin.pl)
INTRODUCTION: The Next Generation Sequencing (NGS) technology has revolutionized the biological sciences. With its ultra-high throughput, scalability, and speed, NGS enables researchers to perform a wide variety of applications and study biological systems at a level never before possible. Due to the increasing popularity and lower price we get more and more data to analyze.
METHODS: The most important analysis we are interested in is identification of differentially expressed genes (DEGs) between specific conditions. Highthroughput transcriptome sequencing (RNA-Seq) has become the main technique for these studies. Thus, the number of methods and softwares for differential expression analysis from RNA-Seq data also increased rapidly.
RESULTS AND CONCLUSIONS: There are several different approaches to analyzing such data, each consisting of several steps. Each step of the analysis can be carried out by many different tools. Most of them give slightly different results for the same input. The question is which approach and tools we should use, since there is no consensus about the most appropriate pipeline or protocol. In our research we use data from bacteria and yeasts to find the optimal pipeline for this type of analysis, by comparing the results obtained from different tools.

Napieralska Aleksandra, Majewski Wojciech, Latusek Tomasz, Miszczyk Leszek
Radiotherapy Department, Centrum Onkologii Instytut im. M. Skłodowskiej-Curie, Oddział w Gliwicach (Wybrzeże Armii Krajowej street 15, 44-100 Gliwice, Poland)
Presenting author: Aleksandra Napieralska, Ph.D. (olanapieralska@gmail.com)
INTRODUCTION The aim of the study is to evaluate stereotactic body radiotherapy (SBRT) results of cancer patients with small number of metastases (so called oligometastases). Evaluation of prognostic and predictive factors.
METHODS: Inclusion criteria were: histological confirmation of cancer, 1 to 3 metastases, except brain metastases, SBRT as local treatment of metastatic lesion.
RESULTS: Group consisted of 542 consecutive cancer patients (186 female, 356 male; age 21–85) treated in with SBRT due to 698 metastases, (241 lung, 227 lymph node, 106 bone, 105 liver and 19 adrenal/soft tissue metastases). Among them 120 metastases were synchronous and 422 metachronous. In 91% of them primary radical treatment of tumor was employed. SBRT total dose was 6–60 Gy (median 36). Median follow-up was 5.6 years and 5-year overall survival was 54%. Patients with synchronous metastases had worse OS compared to patients with metachronous metastases (37% vs 58%, p = 0.0003). Factors that statistically significantly affected OS in multivariate analysis were: the type of primary tumor treatment (radical vs. palliative, p = 0.0075), age (p = 0.0047), SBRT treatment year (p = 0.000) and location of metastatic lesions (p = 0.0009). The percentage of patients who had local response to SBRT was 91%, and 1-, 2-, and 5-year local control rates were: 88%, 74% and 65%. Metastases, outside irradiated area, occurred in 51% of patients after completion of SBRT, and 1-, 2-, and 5-year progression-free survival was 62%, 37% and 27%.
CONCLUSIONS: Radical treatment of the primary tumor in patients with oligometastases is associated with better overall survival. Patients with metastases diagnosed synchronously with the primary tumor have worse prognosis compared to patients with metachronous metastases.

Palittiya Sintusek , , Anna Rybak1,4, Mohamed Mutalib , Nikhil Thapar1, Osvaldo Borrelli1, Keith Lindley1
PS and AR contributed equally to this manuscript
OB and KJL acted equally as senior authors
which is characterized by IAS relaxation upon rectal distension. The RAIR is mediated by the myenteric plexus and therefore absent in Hirschsprung disease. In the present study, we retrospectively assessed the presence and the characteristics of the colo-anal reflex in children in whom large bowel continuity had been surgically disrupted.
METHODS: High-resolution (HR) colonic manometry and HR-anorectal anometry were used to evaluate both colonic and anal motor activity in ten children with treatment-unresponsive slow transit constipation (STC), who had previously undergone left sided colostomy formation with consequent disruption of the bowel continuity, and in two children with Hirschsprung’s disease (HSCR), who had previously undergone distal colon resection followed by Duhamel pull-through. Eight children with STC, normal colonic motor activity and preserved large bowel continuity served as a control group. The presence and characteristics of colo-anal reflex were analysed.
KEY RESULTS: In the study group, all patients showed the presence of both the normal HAPCs and the colo-anal reflex. In two cases of HSCR, RAIR was absent; however both patients demonstrated a colo-anal reflex.
CONCLUSIONS: In children with disrupted continuity of the colon and/ or abnormal anal myenteric reflex, the colo-anal reflex is still preserved suggesting that it is mediated by a different pathway from the RAIR, possibly an extrinsic neural pathway.

Prełowska Monika1,2, Ćwiek Aleksandra2, Mehlich Dawid2, Kamińska Klaudia2, Marusiak Anna2, Nowis Dominika3,2
1 Postgraduate School Of Molecular Medicine, Medical University of Warsaw (Żwirki i Wigury 61, 02-091 Warsaw, Poland)
2 Centre of New Technologies, University of Warsaw (Banacha 2c, 02-097 Warsaw, Poland)
3 Genomic Medicine, Medical University of Warsaw (1A Banacha st. F Building, 02-097 Warsaw, Poland)
Presenting author: Monika Prełowska, M.Sc. (m.prelowska@cent.uw.edu.pl)
Plasma cell myeloma (PCM), with the 5-year survival rate of 50%, remains still an incurable disease. Although the therapeutics such as proteasome inhibitors, including bortezomib, show initial strong antimyeloma effects, the disease frequently relapse due to acquired resistance.
Recently, the role of glutamine metabolism in response to chemotherapy has been identified as an emerging topic in tumor biology. It has been shown by Jeon et al., that in breast cancer, degradation of glutamine transporter ASCT2 by ubiquitin ligase RNF5 in response to endoplasmic reticulum (ER) stress is crucial for paclitaxel anticancer effects. It suggests that the treatment of glutamine – dependent tumors with ER stress inducing chemotherapy may be enhanced by modulation of glutamine metabolism. Indeed, the combination of ER stress inducing agents with glutaminase (GLS) inhibitors demonstrated strong antitumor potential in vitro and in vivo. Within our project, we investigate the role of glutamine metabolism in response to ER stress in PCM cell lines. Moreover, we aim to evaluate the potential of glutamine metabolism inhibition to sensitize PCM cells to ER stress-inducing agents.
Our results show that PCM cell lines rely on glutamine metabolism for their growth and survival. These cell lines are characterized by upregulated basal levels of ER stress markers such as BIP, p-eIF2a or XBP1s. Moreover, in these cells, the ER stress – related mechanism of ASCT2 degradation is also observed. Finally, the inhibition of glutamine metabolism combined with ER stress induction decreased proliferation and survival of PCM cell lines.

Fidyt Klaudyna1,2, Pastorczak Agata3, Szczygiel Kacper2, Goral Agnieszka2, Madzio Joanna3,1, Bartlomiejczyk Marcin3, Jansen Eugene4, Pal Deepali5, Blair Helen5, Graczyk-Jarzynka Agnieszka2, Muchowicz Angelika2, Patkowska Elzbieta6,5, Lech-Maranda ewa6,5, Mlynarski Wojciech3, Golab Jakub2, Firczuk Malgorzata2
1 Postgraduate School of Molecular Medicine, Medical University of Warsaw (Żwirki i Wigury 61 Str., 02-091 Warsaw, Poland)
2 Department of Immunology, Medical University of Warsaw (Banacha 1a Str., Building F, 02-097 Warsaw, Poland)
3 Department of Pediatrics, Oncology, Hematology and Diabetology, Medical University of Lodz (Sporna 36/50, Lodz, 91-738 Lodz, Poland)
4 Centre for Health Protection, National Institute for Public Health and the Environment (Antonie van Leeuwenhoeklaan 9 , 3721 Bilthoven, Netherlands)
5 Newcastle Cancer Centre at the Northern Institute for Cancer Research, Newcastle University (Brewery Lane, NE1 7RU Newcastle upon Tyne, United Kingdom)
6 Hematology Department, Institute of Hematology and Transfusion Medicine (Chocimska 5 Str., 00-001 Warsaw, Poland)
Presenting author: Klaudyna Fidyt, M.Sc. (klaudyna.fidyt@gmail.com)
B cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogeneous disease characterized by abnormal expansion of B cell precursors. Although treatment regimens and hence cure rates have improved significantly over past years (>90% for pediatric patients), some genetic subtypes, e.g. breakpoint cluster region-abelson kinase (BCR-ABL1) ALL, or mixed lineage leukemia (MLL)-rearranged ALL, are still associated with poor prognosis. The therapy of patients with unfavorable outcome and BCP-ALL relapses are still challenging, therefore novel targeted treatments with possibly low side effects are needed. In this study we aimed to validate antioxidant enzymes of the thioredoxin (TXN) system as potential targets in BCP-ALL. We observed elevated oxidative stress in serum of BCP-ALL patients as compared to age-related healthy subjects (HS), accompanied by upregulation of antioxidant enzymes of TXN system in BCP-ALL blasts. Subsequently, targeting TXN enzymes with small molecule inhibitors, auranofin (AUR) and adenanthin (ADE) resulted in BCP-ALL cells death. Primary leukemic cells were effectively killed not only in monoculture but also in the co-culture with bone marrow mesenchymal stem cells (BM-MSC), which were shown to provide chemoprotection for ALL. Finally, we observed that AUR delayed the progression of the disease in vivo in patient-derived xenograft model of MLL-rearranged ALL. In summary, our results show that targeting TXN system may be a novel strategy for the treatment of BCP-ALL and encourage further studies evaluating the efficacy of therapy combining TXN system inhibitors with other anti-leukemic drugs.
This work was supported by the National Science Centre grant 2015/18/E/ NZ5/00723

Małek Natalia, Mazur Antonina
Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw (Joliot-Curie 14a, 50-383 Wroclaw, Poland)
Presenting author: Natalia Małek, Ph.D. (en.malek@gmail.com)
INTRODUCTION: The majority of deaths due to the skin neoplastic changes is caused by melanoma, which is one of the most invasive type of tumours. Metastasis of melanoma is based on alterations in cells’adhesion and motility, in which actin is one of key players. The data on actbl2 – newly discovered actin isoform in tumorigenesis is limited, thus our studies focused on the role of actbl2 in melanoma cells’motility and invasiveness.
METHODS: In our studies we derived stable clones from a melanoma cell line (A375) deprived of ACTBL2 (actbl2) expression by the means of CRISPR/ Cas9(D10A) technique. Further we analyzed clones’motility in 2D and 3D conditions and proliferation ratio using BrdU assay, as all of those may influence melanoma progression. We also analyzed formation of stress fibers and motility structures, i.e. lamelipodia and invadopodia.
RESULTS: We observed changes in morphology and behavior of cells when deprived of expression of actbl2. Lack of ACTBL2 expression increased proliferation potential of tested cells. Additionally knock-out of ACTBL2 resulted in higher number of stress fibers. Affected 3D-migration pararelled with differences in invadopodia formation and may indicated that actbl2 has a role in invasive propagation of melanoma cells.
CONCLUSIONS: Summarizing, presented data shed new light on the functional role of actbl2 in melanoma cells. Studies may contribute to better understanding of the processes underlying the changes in the cytoskeleton during metastasis. Presented data is a result of an ongoing project.
Supported by the National Center for Science (2015/17/B/NZ3/03604) and statutory funds.

Pańczyszyn-Trzewik Patrycja , Sowa-Kućma Magdalena1, Sołek Przemysław , Misztak Paulina1, Papp Mariusz , Gruca Piotr3, Nowak Gabriel3
of chronic mild stress (CMS), as well as chronic antidepressant treatment on the p-S421-MeCP2 protein level in frontal cortex (FCx) and hippocampus (Hp) of rats.
METHODS: Rats were subjected to CMS procedure according to Pochwat et al [2]. The following drugs: imipramine (10mg/kg), escitalopram (10mg/kg), venlafaxine (10mg/kg) or olanzapine (2mg/kg) were given (i.p.) for 35 days. 24 h after the last dosage, animals were perfused transcardially with 4% paraphormaldehyde in 0.1M PBS. p-S421-MeCP2 protein levels was determined using immunofluorescence staining in paraffin-embedded brain sections.
RESULTS: CMS procedure induced a significantly decreased p-S421-MeCP2 protein level (by 22%) in FCx (but not in Hp). In FCx, chronic treatment of imipramine and escitalopram (but not venlafaxine and olanzapine) reversed CMS-induce changes. In Hp, imipramine, escitalopram and olanzapine (but not venlafaxine) normalized p-S421-MeCP2 level.
CONCLUSIONS: Obtained results, confirm the role of MeCP2 in development of CMS-induced depressive-like behavior. p-S421-MeCP2 protein level, seems to be important in the regulation of response to antidepressants treatment, however observed effects can vary depending on the brain structure or pharmacological profile of antidepressants.
This study was supported by National Science Centre grant No: 2013/09/D/ NZ7/02520.
[1] JNeurosci. 2012; 32(41): 14355–1436.
[2] Int JNeuropsychopharmacol. 2014; 17(3): 393–405.

Ludwig-Słomczyńska Agnieszka H.1, Seweryn Michał T.1, Kapusta Przemysław1, Pitera Ewelina1, Cyganek Katarzyna2, Mantaj Urszula3, Dobrucka Łucja2, Wender-Ożegowska Ewa3, Małecki Maciej T.2, Wołkow Paweł1
1 Center for Medical Genomics OMICRON, Jagiellonian University Medical College (ul. Kopernika 7c, 31-034 Krakow, Poland)
2 Department of Metabolic Diseases, Jagiellonian University Medical College (ul. Kopernika 15, 30-001 Kraków, Poland)
3 Division of Reproduction, Poznan University of Medical Sciences (ul. Polna 33, 60-535 Poznań, Poland)
Presenting author: Agnieszka H. Ludwig-Słomczyńska, Ph.D. (agnieszka. ludwig-galezowska@uj.edu.pl)
INTRODUCTION: Obesity results from an imbalance between energy intake and its expenditure. Since it was shown that interaction and communication between nuclear and mitochondrial genome are indispensable for normal cell function we have looked for epistatic interactions between the two genomes to find their correlation with BMI.
METHODS: The analysis was performed on 366 T1DM patients using Illumina Infinium OmniExpressExome-8 chip. Gene expression analysis was performed on GTex data. Association analysis between genetic variants and BMI was performed with the use of Linear Mixed. Analysis of association between mRNA expression and BMI was performed with the use of linear models and standard significance tests in R.
RESULTS: Among genes involved in epistasis we have identified mitochondrial transcription factor TFB2M. It interacted with mitochondrial variants localized to MT-RNR1, MT-ND2 and MT-ND4. Analysis of the interaction between nuclear variant rs6701836 localized to TFB2M and rs3021088 localized to MT-ND2 has shown that the combination of the two led to BMI decrease (p = 0.02411025). Each of the variants on its own does not correlate with higher BMI. Although rs6701836 is intronic it influences gene expression in thyroid (p = 0.000037). rs3021088 is a missense variant that leads to Alanine to Threonine substitution in the MT-ND2 gene. The analysis of the influence of genetic variant on gene expression has confirmed the trend explained above.
CONCLUSIONS: Our results show that nuclear-mitochondrial epistasis can influence BMI in T1DM patients. The correlation between transcription factor expression and existence of genetic variants will be subject of further analysis.
This study was supported by the grant from National Science Centre (NCN) no. UMO-2013/11/D/NZ5/03219.

Donskow-Łysoniewska Katarzyna1, Doligalska Maria1, Kozłowska Ewa2, Krawczak Katarzyna1
1 Parasitology Department, University of Warsaw, Faculty of Biology (Miecznikowa 1, 02-096 Warsaw, Poland)
2 Immunology Department, University of Warsaw, Faculty of Biology (Miecznikowa 1, 02-096 Warsaw, Poland)
Presenting author: Katarzyna Krawczak, M.Sc. (kkrawczak@wihe.waw.pl)
Epidemiological data suggests the negative correlation between occurrence of autoimmune diseases and helminth infections. Nematode infection sometimes can have beneficial effect on patients with Multiple Sclerosis (MS). Heligmosomoides polygyrus infection is an established model of immunomodulation, reduces experimental autoimmune encephalomyelitis (EAE) symptoms, T regulator cells number is increased significantly in cerebrospinal fluid of EAE mice in 6th day post infection. In this study we tested and compared if nematode induced CD4+ or CD8+ Tregs were able to inhibit autoimmune induced CNS inflammation and reduce demyelisation of neurons.
We sensitized C57BL/6 female mice with MOG35-55 (myelin oligodendrocyte glycoprotein )peptide in CFA with Mycobacterium tuberculosis H37RA and Bordetella pertussis toxin. On the 21st day after sensitization animals received adoptive transfer of CD8+CD25hi lymphocytes from two donors: (i) uninfected
MOGp35–55-immunized mice for 27 days and (ii) MOGp35–55-immunized mice and infected with 300 L3 H. polygyrus for six days at 21 days post immunization. After 10 days post transfer changes in EAE clinical score and the nervous tissue structure were evaluated. The number of fluorescent dye-labelled transferred T cells was evaluated in the tissue cross sections of the brain.
CD8+CD25+ cells transfer leaded to EAE symptoms reduction. In both groups we obtained migration to cerebrospinal fluid and nervous tissue of high percentage of transferred T regulatory cells. CD8+ Treg transferred cells influenced the activity of donors’lymphocytes in cerebrospinal fluid. Structure of the brain tissue improved and recovered to the control level.
This work was supported by National Science Center grant No. 2014/15/N/ NZ6/025025.

Małachowska Beata1,2, Tomasik Bartłomiej1,2, Stawiski Konrad2, Fendler Wojciech3,2
1 Postgraduate School of Molecular Medicine, Medical University of Warsaw (61 Żwirki i Wigury St., 02-091 Warsaw, Poland)
2 Department of Biostatistics and Translational Medicine, Medical University of Lodz (15 Mazowiecka St., 92-215 Lodz, Poland)
3 Department of Radiation Oncology, Harvard Medical School, Dana-Farber Cancer Institute (4 Blackfan Circle, 02115 Boston, MA, United States)
Presenting author: Bartłomiej Tomasik, M.D. (bartlomiej.tomasik@umed.lodz.pl)
INTRODUCTION: The hunt for identification of easy to measure biomarkers of irradiation led scientists to the field of microRNA studies. We aimed at identification of evolutionary conserved, radiation-induced circulating miRNAs.
METHODS: The systematic review was registered in the PROSPERO database (ID: 81701). Three databases were searched: MEDLINE , Embase and Cochrane
Database of Systematic Reviews. We downloaded the list of studies with usage of the query: (circulating OR plasma OR serum) AND (miRNA or microRNA) AND (radiat* OR radiotherapy OR irradiati*). We selected 467 studies – 103 from MEDLINE and 364 from EMBASE. After deleting 116 duplicates, remaining 351 abstracts were then reviewed. Inclusion criteria were as followed: experimental study; human, mice, rat or non-human primates study; serum or plasma microRNA expression measured after irradiation exposure. Duplicates were excluded.
RESULTS: Screening procedure yielded 62 research studies. After verification, 31 articles were found to contain data on significant expression change after irradiation. Thus, we obtained database of 82 significant records from 31 articles reporting 350 significant changes (160 up-regulations and 190 down-regulations) of 129 microRNAs. The top 5 most commonly reported changes were: decrease of miR-150 (FC (fold change) = 0.39 (95%CI 0.33–0.45), miR-342 (FC = 0.57 (95%CI 0.38–0.86)) and miR-142 (FC = 0.51 (95%CI 0.41–0.64) and increase of miR-30b (FC = 2.22 (95%CI 1.66–2.98)) and miR-30c (FC = 2.63 (95%CI 1.86–3.72)).
CONCLUSIONS: Circulating microRNAs reflect biological impact of ionizing radiation and this effect remains stable irrespectively of the studied species. This makes circulating microRNAs as promising candidates for biodosimetry. The study is supported by FNP First TEAM Programme.

Prelowska Monika1, Mehlich Dawid1, Lazniewski Michal2, Kaminska Klaudia1, Gorczynski Adam3, Korwat Aleksandra3, sokolowska Olga1, Golab Jakub4, Biernat Wojciech3, Brognard John5, Plewczynski Dariusz2, Nowis Dominika1,4, Marusiak Anna1
1 Laboratory of Experimental Medicine, Centre of New Technologies (Banacha 2C, 02-097 Warsaw, Poland)
2 Laboratory of Functional and Structural Genomics, Centre of New Technologies (Banacha 2C, 02-097 Warsaw, Poland)
3 Department of Pathomorphology, Medical University of Gdansk (Smoluchowskiego 17, 80-214 Gdansk, Poland)
4 Genomic Medicine and the Department of Immunology, Medical University of Warsaw (Banahca 1A, 02-097 Warsaw, Poland)
5 Signaling Group, National Cancer Institute (Boyle Street, MD 21702 Frederic, United States)
Presenting author: Anna Marusiak, Ph.D. (a.marusiak@cent.uw.edu.pl)
INTRODUCTION: Breast cancer is a highly heterogeneous disease classified into several subgroups varying on molecular subtypes, treatment responses and clinical outcomes. Recent cancer genomic data revealed amplification or mRNA upregulation of Mixed-lineage Kinase 4 (MLK4) at 23% in invasive breast carcinoma. In this study, we examined the impact of MLK4 upregulation on the progression of breast cancer and its contribution to aggressive phenotype of breast cancer cells.
METHODS: To assess the functional role of MLK4, we used several phenotypic assays including proliferation, migration, invasion and 3D assays. We also performed transcryptomics analysis and immunohistochemial staining of samples obtained from breast cancer patients.
RESULTS: We first discovered that MLK4 is highly expressed in triple-negative breast cancer patients comparing to other subtypes. The knock-down of MLK4 in TNBC cell lines with high endogenous expression level of this kinase decreased cell proliferation and anchorage-dependent colony formation. Moreover, MLK4 depletion led to a dramatic reduction of migratory and invasive capacity of cells, reduction of spheroid formation in 3D and slower tumor growth in vivo. We also established that MLK4 activates NF-κB pathway and promotes mesenchymal phenotype of breast cancer cells. Furthermore, immunohistochemical staining of samples obtained from TNBC patients revealed positive correlation between high expression of MLK4 and metastatic potential of primary tumors.
CONCLUSIONS: Our data point out that MLK4 is important for acquisition of invasive phenotype of triple-negative breast cancer cells and it may represent a druggable target for a subset of breast cancer patients.

Mazur Karolina1, Wolczyk Agata1, Nowak Barbara1, Kowalak Karol1, Niezbecka Weronika1, Wainer Irving W.2,3, Jóźwiak Krzysztof1, Wnorowski Artur1
1 Department of Biopharmacy, Medical University of Lublin (Chodźki 4A/01A, 20-093 Lublin, Poland)
2 Mitchell Woods , Pharmaceuticals (4 Corporate Dr, 06484 Shelton, United States)
3 Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health (251 Bayview Blvd, , 21224 Baltimore, United States)
Presenting author: Karolina Mazur, Student (mazurkarolina8@gmail.com)
(R,R’)-4′-Methoxy-1-naphthylfenoterol, (R,R’)-MNF, inhibits growth of 1321N1 astrocytoma and PANC-1 pancreatic carcinoma cells through different mechanisms. Attenuation of 1321N1 proliferation occurs via activation of β2-adrenergic receptor (β2-AR) whereas suppression of PANC-1 depends on the blockage of oncogenic receptor GPR55. Both β2-AR and GPR55 belong to G protein-coupled receptors (GPCRs), however they differ in respect to the downstream pattern of kinase activity. The first receptor interact predominantly with Gαs and Gαi to modulate the activity of protein kinase A (PKA), the latter acts through Gαq and Gα13 leading to the activation of protein kinase C (PKC). Moreover, both receptors interact with β-arrestin to elicit additional signaling events, including the activation of mitogenactivated protein kinases (MAPKs). Activation pattern of kinases in 1321N1 and PANC1 cells in response to (R,R’)-MNF remains to be deciphered. Thus, the purpose of this study was to map the activity of key cellular kinases using SDS-PAGE, western blotting and motif antibodies (i.e. antibodies that recognize phosphorylated epitopes characteristic for substrates of targeted kinase). This approach enabled us to cover a significant part of the human kinome. We identified PKA (EC50 = 9.26 nM), MAPK/CDK (IC50 = 5.14 nM) and AKT (EC50 = 11.39 nM) as main kinases affected by (R,R’)-MNF in PANC-1 cells, whereas ATM/ATR and AMPK were not affected. In 1321N1 cells, PKA and AKT were detected to undergo activation in response to (R,R’)-MNF treatment; however, further studies are needed to obtain a full picture of (R,R’)-MNFdependent kinome modulation in 1321N1 and PANC 1 cells.

Maj Maciej1, Wilk Małgorzata1, Tedesco Daniele2, Bartolini Manuela2, Jóźwiak Krzysztof1
1 Biopharmacy Department, Medical University of Lublin (ul. Chodźki 4A, 20-093 Lublin, Poland)
2 Department of Pharmacy and Biotechnology, University of Bologna (Via Belmeloro 6, 40126 Bologna, Italy)
Presenting author: Małgorzata Wilk (malgwilk07@gmail.com)
INTRODUCTION: It is estimated that 40% of marketed drugs produce their clinical effects via modulation of G protein-coupled receptors (GPCRs). One of the main obstacles in studying membrane receptors is the inherent difficulty of obtaining a fully functional, structurally stable and water-soluble form of GPCRs to be used for drug binding studies in solution. The advances in membrane protein research allowed to develop new approaches for the solubilization of GPCRs inside fragments of native cell membrane surrounded by an amphiphilic polymer. The most promising polymer for this application is the styrene-maleic acid co-polymer (SMA), which can be used to produce polymer-embedded membrane nanoparticles called SMA lipid particles (SMALPs).
METHODS: Plasmid construct encoding for human β2AR based on pcDNA3.1 vector was created. The receptor was tagged with a peptide FLAG tag at the N-terminus and at the C-terminus with attachment of oligohistidine tags, containing double oligo histidine sequences separated by a number of amino acids. HEK293T cells were transiently transfected using cationic polymer method. Cells were harvested and membrane fraction isolated. Correct expression of receptor was monitored with immunostaining. Cells were subjected to solution of SMA polymer and purified using immobilized metal ion affinity chromatography.
RESULTS: β2AR receptors were expressed correctly in membranes, exhibiting both aforementioned peptide tags with immunostaining methods. β2AR receptors were solubilized correctly with SMA based on turbidity measurement and size-exclusion chromatography results.
CONCLUSIONS: Embedding membrane particles in a SMALPs proves to be reliable method of obtaining functional, water soluble G-protein coupled receptor for downstream applications.

Sroka-Bartnicka Anna , , Borkowski Leszek , Polkowska Izabela , Pawlowska Marta , Palka Krzysztof , Zieba Emil , Slosarczyk Anna , Jozwiak Krzysztof1, Ginalska Grazyna3
Presenting author: Anna Sroka-Bartnicka, Ph.D. (anna.sroka@umlub.pl)
INTRODUCTION: Nowadays the challenge for the Medical engineering technologies is designing bone substitute materials. The increasing number of accidents, injuries and bone tumours along with developments in medical sciences result in growing demand for bone substitute materials.
METHODS: Regeneration of bone defects was promoted by a novel β-glucan/carbonate hydroxyapatite composite and characterized by Raman spectroscopy, microCT and electron microscopy. The elastic biomaterial with an apatite-forming ability was developed for bone tissue engineering and implanted into the critical-size defects of rabbits tibiae. The bone repair process was analyzed on non-decalcified bone/ implant sections during a 6-month regeneration period.
RESULTS: Using spectroscopic methods, we were able to determine the presence of amides, lipids and assign the areas of newly formed bone tissue. Raman spectroscopy was also used to assess the chemical changes in the composite before and after the implantation process. SEM analyses showed the mineralization degree in the defect area and that the gap size decreased significantly. Microscopic images revealed that the implant debris were interconnected to the poorly mineralized inner side of a new bone tissue.
CONCLUSIONS: Our study demonstrated that the composite may serve as a biocompatible background for collagen ingrowth and exhibits the advantages of applying Raman spectroscopy, SEM and microCT in studying these samples.

Ziemba Barbara1,2, Sikorska Hanna3,2, Jander Magdalena4,2, Appelhans Dietmar5, Bryszewska Maria4, Borowiec Maciej1, Franiak-Pietryga Ida6,7,1,2
1 Department of Clinical and Laboratory Genetics, Medical University of Lodz (251 Pomorska Str., 92-213 Lodz, Poland)
2 GeneaMed LTD (16/18/904 Kopcinskiego Str., 90-232 Lodz, Poland)
3 Bio-Assistance (705-801 Rue de la Commune Est, H2L 0A3 Montreal, QC, Canada)
4 Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz (141/143 Pomorska Str., 90-236 Lodz, Poland)
5 Bioactive and responsive Polymers, Leibniz Institute of Polymer Research Dresden (Hohe Str. 6, D-01069 Dresden, Germany)
6 Laboratory of Clinical and Transplant Immunology and Genetics, Copernicus Memorial Hospital (62 Pabianicka Str., 93-513 Lodz, Poland)
7 Department of Ophthalmology, University of California San Diego (9500 Gillman Dr, La Jolla, CA 92093-0718 San Diego, United States)
Presenting author: Barbara Ziemba, Ph.D. (barbara.ziemba@umed.lodz.pl)
INTRODUCTION: Dendrimers are well-defined, monodisperse, three-dimensional structures with bonds emerging radially from a core and functional terminal groups allowing attachment of various molecules. Because of their unique structure and properties, dendrimers have attracted great interest in biomedical applications. Our studies are based on dense shell (80–90% surface modification; DS) and open shell (35–50% surface modification, OS) maltotriose-modified fourth-generation poly(propylene imine) dendrimers as polymeric drugs in chronic lymphocytic leukemia (CLL). They demonstrate higher cytotoxicity to CLL than to normal cells and influence the expression of genes responsible for apoptosis and cancer cell survival.
METHODS: In vitro studies are not sufficient to confirm the effectiveness of the dendrimers as anti-leukemic agents that is why the in vivo studies were conducted. We used subcutaneous MEC-1 xenograft model of human CLL in NSG™ mice treated with OS, DS, fludarabine (FA) or 0.9% NaCl.
RESULTS: Results provided a preliminary assessment of the clinical value of treating CLL patients with glycodendrimers by proving, that they do have anticancer properties. OS dendrimer not only inhibited the subcutaneous tumour growth more efficiently than FA (TGI value 88.7% vs. 54.8% for FA and TCR 0.35 vs. 0.59 for FA) but also prevented/inhibited spread of CLL to the brain and its transformation into diffuse large B cell lymphoma (DLBCL) associated with a bad prognosis.
CONCLUSIONS: Although preclinical studies provided only a limited indication of the potential clinical efficacy, the results of our studies have a potential important impact for the design of the future personalized therapies as well as clinical trials.

Szopa Iwona, Bujak Joanna K., Łabędź Agata, Majchrzak Kinga
Department of Physiological Sciences, Warsaw University of Life Sciences (ul. Nowoursynowska 159, 02-776 Warsaw, Poland)
Presenting author: Iwona Szopa, M.Sc. (iwonaszopa93@gmail.com)
INTRODUCTION: Domestic dog is an attractive model for immunological studies. Major subsets of the dog immune system were characterized with significant homology to humans. However, culture of large amounts of canine T cells for the purpose of adoptive cellular immunotherapy still requires optimization.
METHODS: We used nano-sized beads coated with anti-canine CD3 antibody to trigger the signal mediated by TCR. Moreover we coated beads with anticanine CD28 antibody to provide co-stimulatory signal ensuring proliferation and cytokine production. We evaluated T cells activation status based on phenotypic features of T cells and expression of CD25 molecule (alpha chain of IL2 receptor), which is upregulated upon activation. In addition, we used bead at different concentrations, at either 1:1, 1:2 or 1:0.5 T cell:magnetic bead ratio. We also determined the impact of temperature range from 370C to 410C on T lymphocytes activation and proliferation.
RESULTS: Our research shown that low-strength activation signal (1:0.5 ratio) caused increased expression of CD25 molecule on canine T cells, 24 and 72h post-stimulation. Lower beads concentration made T lymphocytes to create multiple aggregates, which are the sign of cells activation. We found that 38.50C is optimal temperature for canine T cell activation and expansion.
CONCLUSIONS: Overall our research revealed the optimal conditions for canine T cells expansion for further immunological assessment and most importantly for adoptive T cell transfer, which is a very promising therapy to treat cancer in humans, as well as, in canine patients.
Research funded by the European Union and the Foundation for Polish Science.

Wysocka Marta, Pietraszek-Gremplewicz Katarzyna, Nowak Dorota
Department of Cell Pathology, University of Wroclaw (Joliot-Curie 14 A, 50-383 Wroclaw, Poland)
Presenting author: Marta Wysocka, M.D. (marta.wysocka@uwr.edu.pl)
Apelin belongs to the family of adipokines – hormones released by adipose tissue. It is secreted peptide derived from 77-amino acid precursor – preproapelin, that is cleaved and produces a family of apelin-derived fragments, including apelin-36, -17, -13 and its pyroglutamyl form [Pyr-1]apelin-13. Apelin is a ligand for APJ receptor, that belongs to G protein-coupled receptor family. Presence of apelin in different tissues indicates, that it may play crucial role in many physiological processes, including cardiovascular system regulation, angiogenesis and metabolism regulation. Moreover, apelin is connected with several pathologies, such as heart diseases, obesity and cancer. Accumulating evidence suggests that this peptide can enhance migration and invasion of cancer cells.
Our data indicated that apelin-36 may affect migration abilities of colon cancer cells. However, the antagonist of the APJ receptor, ML221, which inhibits apelin-APJ complex formation, could show opposite effect. We observed the increased ability to creating migratory protrusions – blebs – in cells incubated with apelin-36 and opposite effect with ML221, what was confirmed by examination of level of proteins connected with regulation of actin polymerization. Therefore, we checked the migration and invasion abilities of colon cancer cells using TranswellTM assay. Apelin-36 increased ability to migrate and invade, whereas APJ antagonist decreased these facilities. Furthermore, we examined the proteolytic capabilities to degrade the gelatin using fluorescent-substrate degradation assay. The cells incubated with apelin-36 demonstrated increased activity of metalloproteases. Summarizing, our data shows, that apelin-36 stimulates migration, invasion and increases proteolytic abilities, whereas APJ antagonist ML221 acts in opposite way.

Miksa Beata, Sierant Małgorzata, Skorupska Ewa
Chemistry, Centre of Molecular and Macromolecular Studies Polish Academy of Science (Sienkiewicza, 90-363 Łódź, Poland)
Presenting author: Beata Miksa, Ph.D. (miksa@cbmm.lodz.pl)
Here we report the first of the phenosafranin-chlorambucil conjugate as a new type of a chemotherapeutic agent suitable for dual detection methods (spectrophotometric and fluorescence) in imaging systems and cancer treatment. The synthetic cationic dye (3,7-diamino-5-phenylphenazinium chloride) is used as a fluorescent light-triggered scaffold that acts as a carrier for an anti-cancer drug. The chlorambucil was attached covalently via amide bonds to the bifunctional fluorophore, which facilitates tracking with visible light. Our studies revealed that the new photosensitive compound exhibits improved intrinsic activity in vitro in HeLa cells culture experiments; thus it could be a potential anti-cancer candidate in theranostic drug-delivery systems. In light of the urgent need for in vivo monitoring of the biodistribution of anti-cancer drugs, this strategy for the synthesis of innovative conjugates based on the phenosafranin backbone offers a promising possibility for drug control in anti-cancer therapy and diagnosis. This aspect makes the phenosafranin-chlorambucil conjugate unique among currently available biomarkers.

Zareba Ilona1, Surazynski Arkadiusz1, Miltyk Wojciech2, Palka Jerzy1
1 Department of Medicinal Chemistry, Medical University of Bialystok, Bialystok, Poland
2 Department of Pharmaceutical Analysis, Medical University of Bialystok, Bialystok, Poland
Presenting author: Ilona Zareba (ilona.zareba@gmail.com)
Metformin, the first-line drug in the treatment of type II diabetes evokes anti-cancer activity. However, the molecular mechanism of this process is unknown. One of the effects of metformin is activation of AMP kinase (AMPK), which activates PRODH/POX, the mitochondrial enzyme converting proline to pyrroline-5-carboxylate. During this process ATP is produced for survival or ROS for apoptosis. It has been suggested that the proline availability for this process may represent switching mechanism between apoptosis/autophagy. The main process for proline utilization is collagen biosynthesis that limit proline availability to degradation in mitochondria. The aim of the studies was to evaluate the role of PRODH/POX in metformin-induced apoptosis/ autophagy in MCF-7 cells.
We found that metformin added to cultured MCF-7 cells contributed to dosedependent decrease in cell viability. Cell viability and DNA biosynthesis was decreased to about 60% of control after incubation with metformin. Collagen biosynthesis was similarly decreased to about 40% of control, simultaneously. Inhibition of collagen biosynthesis by metformin suggests inhibition of free proline utilization in cytoplasm and availability of proline to mitochondrial degradation by PRODH/POX. Metformin in dose-dependent manner increased expression of PRODH/POX and AMPKβ in MCF-7 cells, as well as increased expression of beclin-1 (autophagy marker) and cleaved caspase-9 (mitochondrial-dependent apoptosis marker). The data suggests metformin induces PRODH/POX apoptosis/autophagy in MCF-7 cells.
The link between AMPK, PRODH/POX and proline with apoptosis/autophagy in tumor cells and stimulating the effect of metformin on AMPK allows to present a hypothesis on the mechanism of PRODH/POX-dependent apoptosis/autophagy.

Janyst Karolina1, , Janyst Michał1,2, Siernicka Marta1,2, Lasek Witold2
1 Postgraduate School of Molecular Medicine, Medical University of Warsaw (Żwirki i Wigury 61, 02-091 Warsaw, Poland)
METHODS: Cytotoxic /cytostatic effect of the agents was tested in a 72-h MTT assay. Apoptosis assay by flow cytometry was performed using annexin V-FITC/PI assay. Apoptotic proteins: caspase-3, caspase-9 and the key marker of cell cycle arrest protein p21 were determined by Western blotting.
RESULTS: Incubation of ovarian cancer cells with scriptaid and bortezomib (or doxorubicin) led to synergistic antitumor effect resulting from both induction of apoptosis and inhibition of proliferation. In contrast, combination of paclitaxel or carboplatin and scriptaid presented additive antitumor effects against ovarian cancer cells. Etoposide did not significantly affect cell viability. Additionally, treatment with scriptaid and bortezomib resulted in a marked increase in p21, suggesting that cell cycle arrest mechanisms significantly contributed to the cytotoxic/cytostatic effects of this combination. Contrary to, did not change expression of caspase 3 and caspase 9.
CONCLUSIONS: Our data suggest that the use of scriptaid may enhance effectiveness of conventional chemotherapy of ovarian cancer and that the new combination: scriptaid and bortezomib could be used as a treatment option of heavily pretreated patients.

Goral Agnieszka1, Graczyk-Jarzynka Agnieszka1, Muchowicz Angelika1,
Zagozdzon Radoslaw1,2,3, Winiarska Magdalena1, Bajor Malgorzata1, Trzeciecka Anna1, Fidyt Klaudyna1,4, Krupka Joanna A.1, Cyran Julia1, Szczygiel Kacper1, Efremov Dimitar G,5, Gobessi Stefania5, Bobrowicz Malgorzata1, Barankiewicz Joanna6, Malenda Agata6, Lech-Maranda Ewa6, Miazek-Zapala Nina1,7, Golab Jakub1, Firczuk Malgorzata1
1 Department of Immunology, Medical University of Warsaw (1a Banacha Street, 02-097 Warsaw, Poland)
2 Department of Clinical Immunology, Transplantation Institute, Medical University of Warsaw (59 Nowogrodzka Street, 02-006 Warsaw, Poland)
3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences (5a Pawinskiego Street, 02-106 Warsaw, Poland)
4 Postgraduate School of Molecular Medicine, Medical University of Warsaw (61 Zwirki i Wigury Street, 02-091 Warsaw, Poland)
5 Molecular Hematology, International Centre for Genetic Engineering & Biotechnology (99 Loc. Padriciano, 34149 Trieste TS, Italy)
6 Department of Hematology, Institute of Hematology and Transfusion Medicine (14 Indiry Gandhi Street, 02-776 Warsaw, Poland)
7 Institute of Physiology and Pathology of Hearing, World Hearing Center (10 Mochnackiego Street, 02-042 Warsaw, Poland)
Presenting author: Agnieszka Goral, Ph.D. (agnieszkabozena.goral@gmail.com)
Malignant B-cells have elevated levels of ROS compared with healthy counterparts, and therefore are sensitive to ROS-inducing therapies, such as L-ascorbate (L-ASC), which generates H202. To maintain cellular homeostasis and protect from oxidative damage, malignant B cells upregulate antioxidant systems, mainly the peroxiredoxin (PRDX)-thioredoxin (TXN)-tioredoxin reductase (TXNRD) system and the glutathione (GSH) system, which undermine the efficacy of ROS-inducing therapies.
Our aim was to evaluate the cytotoxic effect of L-ASC in combination with inhibitors of antioxidant enzymes to malignant B cells.
We observed that the inhibition of TXN-TXNRD system with two inhibitors: SK053 and auranofin (AUR) greatly enhanced the cytotoxicity of L-ASC toward malignant B-cell lines. In turn, the treatment with L-ASC and buthionine sulfoximine, a glutathione system inhibitor, had no effect. Moreover, we showed that the L-ASC+AUR combination triggered massive increase of intracellular ROS level, which was abolished in the presence of catalase (CAT), a known H2O2 scavenger. The synergistic cytotoxicity of L-ASC and AUR combination we also observed in primary B-CLL cells cultured ex vivo. Importantly, there was almost no toxicity towards normal B-cells. Moreover, the mRNA level of PRDX1, TXN1 and TXNRD1, but not CAT or glutathione peroxidase 1, was upregulated in B-CLL cells treated with L-ASC+AUR, which suggest the key role of TXN-TXNRD system in ROS removal. Altogether, our results indicated that the L-ASC and AUR combination exhibits selective anti-cancer activity and has a potential as therapy for B-cell malignances.
This work was supported by the OPUS grant from National Science Center (2016/21/BNZ7/02041).

Kuska Mikołaj, Kutner Jan J. , Klimecka Maria M.1, Laskowska Anna A.1, Karolak Natalia N.1, Merski Matthew M.1, Górna Maria Wiktoria M.W.1
families were obtained by heterologous expression and chromatographic purification. The proteins will subsequently be characterized by their secondary structure and protein-protein or protein-ligand interactions, using molecular biophysics techniques like circular dichroism and differential scanning calorimetry. Such information may not only contribute to better understanding of the function of RNA-binding proteins, but also to the search for specific biotechnological applications of investigated proteins.

Janyst Michał1,2, Janyst Karolina1,2, Kaleta Beata3, Lasek Witold2
1 Postgraduate School of Molecular Medicine, Medical University of Warsaw (Żwirki i Wigury 61, 02-091 Warsaw, Poland)
2 Department of Immunology. Centre of Biostructure., Medical University of Warsaw (Banacha 1a, 02-097 Warsaw, Poland)
3 Department of Clinical Immunology, Medical University of Warsaw (Lindleya 4, 02-005 Warsaw, Poland)
Presenting author: Michał Janyst, M.D. (michal.janyst7@gmail.com)
INTRODUCTION: Regulatory T lymphocytes (Tregs) exert suppressive effects on other immune cells. The aim of the study was to compare the effect of immunomodulators: rapamycin and prednisolone on activity of Tregs. Rapamycin has been recognized as optimal inducer of Tregs, but the effect of prednisolone on Tregs has been less studied.
METHODS: In the first step of the study, drug-induced changes of Treg phenotype was assessed. Isolated CD4+ lymphocytes were cultured in the presence of anti-CD3/CD28 microbeads, TGF-β and different concentrations of immunomodulators for 5 days. Phenotype of Tregs was analysed using flow cytometry. Functionality of Tregs was measured in MLR tests. Following 5-day culture with microbeads, TGF-β, and either drug, Tregs were isolated and incubated with responding CD4+ T cells and allogenic peripheral blood mononuclear cells. Suppression of proliferation was measured using thymidine incorporation assay.
RESULTS: Induction of Foxp3 in CD4+ T cells incubated with rapamycin at a concentration of 2.5 mg/ml, and with prednisolone at a concentration of 25 mg/ml was higher compared to control samples – 2.69 ± 0.2 and 2.68 ± 0.16 fold increase, respectively. Tregs obtained from cultures with immunomodulators were characterized by high suppressive activity against responder cells. This effect was more significant in cultures with prednisolone-stimulated in comparison to rapamycin-stimulated Tregs (suppression of proliferation 30% vs 49%).
CONCLUSIONS: In this study, we showed that rapamycin and prednisolone promote expansion of T regulatory cells from naive CD4+ T cells. Prednisoloneinduced Tregs suppressed proliferation of responder lymphocytes to a greater extent than rapamycin-induced Tregs.

Pajer Mateusz1, Kulczycka Katarzyna1, Zawadzka Izabela1, Łochowski Mariusz2, Pietrzak Jacek1, Balcerczak Ewa1, Jeleń Agnieszka1
1 Faculty of Pharmacy, Department of Pharmaceutical Biochemistry and Molecular Diagnostics (Muszyńskiego 1, 90-151 Łódź, Poland)
2 Department of Thoracic Surgery, Memorial Copernicus Hospital (Pabianicka 62, 93-513 Łódź, Poland)
Presenting author: Mateusz Pajer (mateusz.pajer@stud.umed.lodz.pl)
INTRODUCTION: ABCB1 gene encodes MDR1- P-glycoprotein (P-gp). P-qp takes part in the transport of drugs, creates a barrier for xenobiotics, removes them from the inside of the cell preventing their accumulation in various organs. One of the most common polymorphisms present in the ABCB1 gene is C3435T. The aim of study was to asses C3435T SNPin the ABCB1 gene in the group of patients with lung cancer.
METHODS: The material for the study consisted of 40 blood samples collected from patients with diagnosed lung cancer; control group accounted for 96 blood samples taken from blood donors. For genotyping C3435T SNP in ABCB1 gene PCR-RFLP method was used.
RESULTS: Preliminary assessment of the C3435T SNP in the ABCB1 gene showed that CT genotype was dominant in both groups. However, TT genotype was more common in persons with lung cancer than healthy people (p = 0.02377). An analysis was also carried out that studied the connection between genotype and clinical parameters of cancer. It shows that the presence of allele T is associated with histological grading and occurs in G1 or G2 cases; that was statistically significant (p = 0.01492).
CONCLUSIONS: Conducted analysis reveal the existence of the relationship between C3435T SNP in the ABCB1 gene and the occurrence of lung cancer and degree of its malice.
Funds: 502-03/3-015-02/502-34-085, 503/3-015-02/503-31-001-17

Chrobak Olga M., Starosta Agata L.
Faculty of Biology and Biotechnology, Maria Curie Skłodowska University in Lublin (ul. Akademicka 19, 20-033 Lublin, Poland)
Presenting author: Olga M. Chrobak, Ph.D. (olga.chrobak@umcs.pl)
The ribosome profiling (RIBO-seq) is a powerful method allowing for direct monitoring of the exact position of the ribosome on transcripts – the socalled translatome [1][2]. Unlike RNA-seq (total mRNA sequencing), RIBO-seq not only provides information about the mRNA composition in the cell at a given time, but also tells us about the rate of translation of each mRNA, number of copies of a synthesized protein, unveils ribosome stalling events – regulatory mechanisms, evaluates the character of small-RNAs (coding versus non-coding) or reveals cryptic open reading frames, which may lead to discovery of novel proteins and pathways.
For this project, the original protocol published by the Weissmann lab [1] has been restructured. During translation, approximately 28–30 nucleotides of the mRNA are buried within the ribosomal small subunit. Since upon nuclease treatment these nucleotides are protected from degradation, such ribosome-protected mRNA fragments can be converted into a DNA library and characterized by deep sequencing (using next generation sequencing methods).
Introduced amendments resulted in a simple, robust, reliable and reproducible protocol generating good quality sequencing data. The protocol has been successfully applied to study translation in various bacteria including Escherichia coli, Bacillus subtilis and Streptomyces spp.
[1] Ingolia, N.T., Brar, G.A., Rouskin, S., McGeachy, A.M. and Weissman, J.S. (2012). Nature protocols, 7, 1534–1550.
[2] Ingolia, N.T. (2014). Nature reviews. Genetics, 15, 205–213.

Jura Roksana S., Starosta Agata L.
Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University (Plac Marii Curie-Skłodowskiej 5, 20-400 Lublin, Poland)
Presenting author: Roksana S. Jura, M.Sc. (roksanajura@gmail.com)
INTRODUCTION: Sporulation is a way for bacteria to respond to hostile conditions like nutrient deprivation. It’s highly organized process were mother cell divides asymmetrically to give a rise to a spore. Unlike vegetative division, where cells divide every 20–40 min, sporulation takes hours and can be divided into highly trackable stages. Sporulation in Bacillus subtilis – a non–pathogenic relative of such bacteria like B. anthraxis or B. cereus – is well described on the level of transcriptional regulation, but almost nothing is known about translation regulation of this process. Intriguingly, as early as 1970s, it was reported that ribosomes isolated from vegetative and sporulating cells translate mRNA differently. We use sporulation as a model to study specialization of ribosomes – a way to regulate gene expression on the translational level.
METHODS: Sporulation can be easily induced and synchronized. We look into translational machinery of B. subtilis applying high-throughput approaches, like ribosome profiling, combined with genetic approaches and microscopy. We expect to find ribosomes carrying different sets of ribosomal proteins (still unexplained role of paralogues of r-proteins) showing selective properties towards specific mRNAs.
RESULTS: Our preliminary data shows huge global rearrangements of the translatome during sporulation. Moreover, we have identified translation of previously unannotated regions and 5’UTRs occupied by ribosomes – indicating translation regulation. Currently, we are selecting and testing factors which may have a role in ribosome specialization and translational remodeling.

Zakrocka Izabela1, Targowska-Duda Katarzyna M.2, Wnorowski Artur2, Kocki Tomasz1, Jóźwiak Krzysztof 2, Turski Waldemar A.1
1 Department of Experimental and Clinical Pharmacology, Medical University of Lublin (Jaczewskiego Street 8b (Collegium Pathologicum), 20-090 Lublin, Poland)
2 Department of Biopharmacy, Medical University of Lublin (Chodźki Street 4a (Collegium Pharmaceuticum), 20-093 Lublin, Poland)
Presenting author: Izabela Zakrocka, Ph.D. (izabela.zakrocka@umlub.pl)
Overactivity of renin-angiotensin system (RAS) is an essential trigger of cardiovascular disorders. Among antihypertensive agents, angiotensin II type 1 receptor blockers (ARBs) play significant role in improving patients’survival. Beyond hypotensive properties, ARBs were shown to abolish glutamate’s toxicity to provide additional level of organ protection, however the exact mechanism of this action is not well understood.
An endogenous ionotropic glutamate receptors antagonist, kynurenic acid (KYNA), is formed from kynurenine (KYN) by kynurenine aminotransferases (KATs). Out of four described KAT isoenzymes, KAT II is the predominant enzyme found in the brain, whereas in kidney KAT III and KAT IV. High KYNA level has been linked with negative symptoms of schizophrenia and Alzheimer’s disease progression. Elevated KYNA serum concentration was reported in end stage kidney failure.
The goal of our study was to analyze the effect of ARBs on KYNA production in rat brain cortex and kidney in vitro. All tested ARBs, irbesartan, losartan and telmisartan dose-dependently decreased KYNA production in both organs. Molecular docking suggested that the ARBs interact with residues within the active site of KAT II, mirroring the interactions of native substrate.
This novel mechanism of ARBs action can explain protective effects of the drugs in the context of glutamate toxicity and suggest repurposing ARBs towards application in the management cognitive deficits and in kidney function control.
This study was supported by the grant from National Science Centre (NCN) PRELUDIUM 4, UMO-2012/07/N/NZ4/02088 and grant for young scientists from Ministry of Science and Higher Education MNmb515/2016.

Pieklarz Katarzyna, Modrzejewska Zofia, Tylman Michał
Faculty of Process and Environmental Engineering, Lodz University of Technology (Wolczanska 213, 90-924 Lodz, Poland)
Presenting author: Katarzyna Pieklarz, M.Sc. (katarzyna.pieklarz@edu.p.lodz.pl)
INTRODUCTION: Connections of biopolymers with nanostructured carbon materials are recently the subject of intensive research. The nanocomposites containing chitosan and graphene oxide are particularly important. These complexes have many interesting properties, such as biocompatibility, nontoxicity and antibacterial, so can be safely used in medicine. The aim of this study was prepared a new generation of hybrid system for used in bone tissue engineering. A subject of research were thermosensitive chitosan hydrogels containing graphene oxide prepared from β – sodium glycerophosphate.
METHODS: Chemical structure of CS-GO nanocomposites was analyzed by FTIR spectroscopy. The crystallinity of hydrogel structure was determined by X-ray diffraction analysis. Within biological tests there has been an evaluation of survival of osteoblast cells the Saos-2 line. A cell culture was carried out under aseptic conditions, maintaining constant temperature parameters – 37°C, humidity – 95% and carbon dioxide content – 5%.
RESULTS: The obtained diffraction patterns have shown that the chitosan chloride gels contain crystalline phases due to presence of β – sodium glycerophosphate and precipitation of NaCl during drying, while the lactate gels are partially amorphous. In turn, the biological research has shown that osteoblast cultures on the carrier with GO were characterized by a significantly higher number of viable cells than in the case of a pure hydrogel.
CONCLUSIONS: Based on the results of the study, it was found that chitosan hydrogels with GO can be a potential material used as injectable scaffolds for the regeneration of bone tissue.

Koj Natalia , Sobalska-Kwapis Marta ,1, Słomka Marcin2,1, Marciniak Błażej2,1, Strapagiel Dominik2,1
and mechanical functioning. In this study we are focusing on the gene KCNK10 coding potassium channel which belongs to the subfamily of two pore domain K+ channels. The objective of present study is to determine in what manner the polymorphic risk variants in KCNK10 gene affect susceptibility to occurrence the myocardial infraction in the Polish population.
METHODS: All participants were from Poland and were registered in a POPULOUS collection. 118 adults who have experienced myocardial infraction were consider as a case group. Control group consisted of 150 people over 65 years old without incidents of myocardial infraction. Genomic DNA were genotyped using microarrays and 23 SNPs in gene KCNK10 were analyzed.
RESULTS: Our results showed statistically significant difference between presence of a heterozygous missense mutation (rs17762463) in the gene KCNK10. This genetic variant found on microarrays is significantly related to myocardial infraction in Polish population. Therefore we tried to predict the effect of this variant on structure and function of KCNK10 using a tool PredictSNP. PolyPhen-2 and SIFT indicated that this mutation was likely to damage the structure and function of KCNK10.
CONCLUSIONS: Our result suggests that the heterozygous missense mutation (rs17762463) in the gene KCNK10 may be involved in determining the susceptibility to myocardial infraction in Polish population.
Project No. POPC.02.03.01-00-0012/17

Walczak Katarzyna, Marciniak Sebastian
Department of Pharmacology, Faculty of Nursing and Health Sciences, Medical University of Lublin (al. Racławickie 1, 20-059 Lublin , Poland)
Presenting author: Sebastian Marciniak (sebastian.marciniak@umlub.pl)
INTRODUCTION: The latest research on the peripheral effect of kynurenic acid (KYNA) show that it reduces the development of oxidative stress and has an anti-inflammatory and cytoprotective effect, so it causes biological actions which may inhibit acute liver failure (ALF) development. The aim of this work was to assess the influence of KYNA on the inflammatory process and oxidative stress in thioacetamide (TAA) induced ALF in rats.
METHODS: The research was conducted on male Wistar rats. The level of liver damage was estimated based on histopathological image analysis as well as alanine and aspartate aminotransferase (ALT, AST) activity. The influence of KYNA on the synthesis of interleukin 10 (IL-10) and cachectin (TNF-α) as immunological activation markers was also investigated. The level of oxidative stress was assessed based on the measurement of concentration of heme oxygenate induced form (HO-1), the level of lipids peroxidation products – malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and myeloperoxidase activity (MPO). Additionally, oxygen radical absorbance capacity (ORAC) was quantified based on the decrease in fluorescence of the so-called molecular probe. The level of oxidative protein damage was assessed based on measurement of concentration of thiol groups (-SH) in the liver homogenates. The nitric oxide (NO) synthesis was estimated basing on concentration of nitrates and nitrites in the plasma. The concentration of KYNA in tissue homogenates was assessed with HPLC methods.
RESULTS: KYNA had positive effect on all studied biochemical parameters and decreased level of proinflammatory TNF-α.
CONCLUSIONS: KYNA shows a protective effect, inhibiting development of TAA-induced ALF.

Starosta Agata L.
Ecotech-COMPLEX / Laboratory of Gene Expression, Maria Curie-Skłodowska University (Głęboka 39, 20-612 Lublin, Poland)
Presenting author: Agata L Starosta, Ph.D. (agata.starosta@umcs.pl)
Ribosomes are a centre of every living cell. For decades, they were perceived as homogeneous macromolecules carrying constant set of ribosomal RNAs and proteins. Consequently, they were not considered to actively participate in the regulatory role of gene expression. The hypothesis of specialized ribosomes assumes the existence of a subpopulation of ribosomes carrying unique structural properties allowing fast and precise response to environmental stimuli throughout selectivity for distinct mRNAs.
We use sporulation process in Bacillus subtilis bacteria as a model to study regulation of gene expression on translational level. Using combination of ribosome profiling, genetics, biochemistry and microscopy, we aim to identify factors modulating translation and accounting for ribosomal selectivity towards mRNAs. Initial data shows massive global rearrangements in proteins synthesis profile and unveils interesting events, like expression of previously unannotated genes, occurrence of paralogues of ribosomal proteins or rearrangements in the ribosomal structure – implying a presence of distinct sub-sets of ribosomes.
Spores, widely found in environment, can survive most of the processes used for sterilization of bacteria, including heat, radiation, chemical treatment, high pressure, they are an increasing burden in food processing and in hospitals.
This work will shed more light on how translation contributes to the gene expression regulation during sporulation. Finding specialised ribosomes will add a new level of regulation of gene expression with a ribosome as an active element. Moreover, within the result, we may identify critical elements suitable for the rational design of new drugs, leading to discovery of novel potential targets for antimicrobials.

Stear Michael1, Krawczak Katarzyna2, Donskow-Łysoniewska Katarzyna2
1 Department of Animal, Plant and Soil Sciences, La Trobe University (Victoria, 3086 Melbourne, Austria)
2 Laboratory of Parasitology, Military Institute of Hygiene and Epidemiology (Kozielska 4, 01-163 Warszawa, Poland)
Presenting author: Katarzyna Krawczak, M.Sc. (kkrawczak@wihe.waw.pl)
In long-lived gastrointestinal nematode infections, immunosuppression is obviously beneficial for the parasite: it prevents parasite killing and expulsion, improves the fitness of the parasite and benefits the host through inhibition of inflammatory reactions and prevention of pathology. Nematodes produce and release factors that may actively modulate immune reactions, mimic host molecules or deactivate/neutralize immune factors and cells directly. There is increasing evidence supporting a role for helminth surface glycoproteins as well as excretory-secretory products which interact with host glycan-binding proteins and shift the host response toward Th2 responses, and possibly regulatory responses. Nematode galectins bind to β-galactosides (such as lactose and N-acetyllactosamine) and can be involved in modulation of host responses via an unknown mechanism. As we observed nematode galectin it either binds to or is bound by IgE. This possible antibody neutralization/ deactivation during infection could explain incomplete immunity despite the high level of IgE during nematode infection in high responder host.
The study was supported by National Science Center grant No.2016/23/B/ NZ6/03464 (ID352771).

Kędzierska Magdalena1, Tyliszczak Bożena2, Drabczyk Anna§, Kudłaczyk-Kramarczyk Sonia3, Potemski Piotr1
1 Department of Chemotherapy, Copernicus Hospital, Lodz, Poland, Medical University of Lodz (Pabianicka 62, 93-515 Lodz, Poland)
2 Department of Chemistry and Technology of Polymers, Cracow University of Technology (Warszawska 22, 31-155 Cracow, Poland)
3 Institute of Inorganic Chemistry and Technology, Cracow University of Technology (Warszawska 24, 31-155 Kraków, Poland)
Presenting author: Magdalena Kędzierska, Ph.D. (kameleonmagda6@gmail. com)
Nowadays, application of nanotechnology in many areas is becoming an interesting alternative for scientists. Diversity of potential use of nano-sized materials results in the advanced development of the mentioned scientific discipline. Among nanomaterials magnetic nanoparticles exhibit a wide range of application possibilities. These particles can significantly affect the development of medicine and pharmacy [1][2]. In presented research synthesis of magnetic nanoparticles coated with gold nanoparticles was conducted. First step of the synthesis involved preparation of magnetic nanoparticles by Massart synthesis, i.e. co-precipitation of chlorides in alkaline environment. Subsequently, nanogold shell was formed on obtained nanoparticles by means of gold-containing reagent and suitable reducing agent. The procedure was carried out in the presence of stabilizing agent to prevent agglomeration of received nanoparticles. Prepared nanomaterials were subsequently subjected to the studies using DLS (Dynamic Light Scattering) analysis and microscopic techniques.
The authors would like to thank the Ministry of Science and Higher Education (Grant no: 0489/IP3/2015/73) for providing financial support to this project.
[1] Mohammed L., Gomaa H.G., Ragab D., Zhu J., Magnetic nanoparticles for enironmental and biomedical applications: A review, Particuology, 2017, 30, 1–14.
[2] Zare T., Sattarahmady N., A Mini-Review of Magnetic Nanoparticles: Applications in Biomedicine, Basic & Cinical Cancer Research, 2015, 7(4), 29–39.

Maruszewska-Cheruiyot Marta, Donskow-Łysoniewska Katarzyna, Doligalska Maria
Department of Parasitology, Institute of Zoology, Faculty of Biology, University of Warsaw (Miecznikowa 1, 02-096 Warsaw, Poland)
Presenting author: Marta Maruszewska-Cheruiyot, M.Sc. (mmaruszewska@biol. uw.edu.pl)
Autoimmune disorders are an increasing problem especially in developed countries. There is lack of effective therapy against diseases consequent to inflammatory reactions and current treatment based on corticosteroids has many side effects, hence new cure methods are needed. The use of tolerogenic dendritic cells (DC) is a very promising strategy in autoimmunological disorders treatment. DC as the main group of antigen presenting cells plays a crucial role in developing immune response. Exposing DC to immunomodulatory factors can define tolerogenic reaction, subsequently inhibit inflammation accompanying autoimmune problems. One of the immunoregulatory agents can be nematodes. The immunosuppression induced by worms prevents parasite extermination hence support their survival in the host as a result of prolonged immunoregulation. It has been confirmed already that infection by these multicellular parasites limits symptoms of autoimmune disorders such inflammatory bowel disease and multiple sclerosis on animals as well as humans in clinical studies. However, helminth therapy has many disadvantages. Elaboration of DC exposed to nematodes then using them as vaccine against autoimmune disorders is a promising strategy that avoids parasitic infection.
Supported by Polish National Science Centre Grants No. 2017/25/N/NZ6/01523

Niebudek Katarzyna1, Wtorek Karol1, Wodziński Damian1, Jesionek-Kupnicka Dorota2, Balcerczak Ewa1, Mirowski Marek1, Żebrowska Marta1
† Laboratory of Molecular Diagnostics and Pharmacogenomics, Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Interfaculty Cathedral of Laboratory and Molecular Diagnostics, Medical University of Lodz (Muszyńskiego 1, 90-151 Łódź, Poland)
2 Department of Patology, Medical University of Lodz (Pomorska 251, 92-213 Łódź, Poland)
Presenting author: Katarzyna Niebudek, M.D. (katarzyna-niebudek@wp.pl)
INTRODUCTION: The possible interaction between gene polymorphism and cancers is very interesting issue. SMAD3, also known as mothers against decapentaplegic homolog 3, is a member of SMAD family and it is encoded by the SMAD3gene located on chromosome 15q21-22. SMAD3 is the one of the most important agent in transforming growth factor-β (TGF-β) pathway. It is supposed that the abnormal function or expression of SMAD3gene may be one of the genetic factors affecting colorectal cancer risk. Purpose: Evaluation of C20917T polymorphism in the SMAD3 gene in patients with colorectal cancer.
METHODS: DNA isolated from frozen tissue from patients diagnosed with colorectal cancer (N = 53) and from healthy people (N = 51). The evaluation of the polymorphism was conducted with applying the PCR-RFLP technique. [Consent of Bioethics Committee of Medical University of Lodz No: RNN/8/ 08/KE’].
RESULTS: The frequency of particular genotypes in both groups was consistent with the Hardy-Wineberg equilibrium. There was no significant statistical difference between colorectal cancer cohort and healthy individuals (p = 0.9310). In the case of the assessment of the relationship between genotypes and age, gender, TNM classification and histological malignancy in the colorectal cancer cohort, also no statistically significant differences were found (p = 0.9155; p = 0.2243; p = 0.0784; p = 0.9678, respectively).
CONCLUSIONS: The examined polymorphism seems not to correlate with the risk of colorectal cancer development. However obtained results require confirmation in further researches on the greatest group of patients.
Acknowledgments: Funded by No. 503/3-015-02/503-31-001and No. 502 -03/3-015-02/502-34-073 of Medical University of Lodz.

Spodzieja Marta1, Kalejta Katarzyna1, Bojko Magdalena1, Iwaszkiewicz Justyna2, Sieradzan Adam1, Karczyńska Agnieszka1, Speiser Daniel E.3, Magiera Małgorzata4, Holak Ted4, Dubin Grzegorz5, Pikuła Michał6, Zoete Vincent2, Michielin Olivier2, Parys Maciej7, Hupp Ted , Derre Laurent , Rodziewicz-Motowidło Sylwia1
1 Faculty of Chemistry, University of Gdańsk (Wita Stwosza 63, 80-308 Gdańsk, Poland)
2 The Swiss Institute of Bioinformatics (Quartier Sorge, Batiment Genopode, CH-1015 Lausanne, Switzerland)
3 Department of Oncology, Ludwig Cancer Research (Biopole 3, Rte Corniche 9A, CH-1066 Epalinges, Switzerland)
4 Faculty of Chemistry, Jagiellonian University (Gronostajowa 2, 30 – 387 Kraków, Poland)
5 Małopolska Centre of Biotechnology, Jagiellonian University (Gronostajowa 7A , 30-387 Kraków , Poland)
6 Department of Clinical Immunology and Transplantology, Medical University of Gdańsk (M. Skłodowskiej-Curie 3a, 80-210 Gdańsk, Poland)
7 Hospital for Small Animals, The University of Edinburgh (Easter Bush Campus, EH25 9RG Roslin, Midlothian, United Kingdom)
expressed on the T cell with their ligands expressed on the antigen-presenting cell (APC) or tumor cells. Co-receptors (immune checkpoints) expressed on the T cell can lead to activation or inhibition of immune response. PD-1 (Programmed death-1) and PD-L1 (Programmed death-ligand 1) or HVEM (Herpes virus entry mediator) and BTLA (B- and T-lymphocyte attenuator) or HVEM and CD160 (Cluster of differentiation 160) are one of immune checkpoint molecules responsible for inhibition of the immune system. It was shown that is involved in the negative regulation of T cell responses in cancer patients and can be targeted by immunotherapy.
Based on the 3D structures of checkpoint complexes the series of peptides, potential inhibitors, were designed and synthesized. To study the proteinpeptide interactions the immunoenzymatic assay (ELISA), Nuclear Magnetic Resonance (NMR), mass spectrometry (MS) and cellular assay were carried out. Synthesized peptides can be the groundwork for the future design of the anticancer therapies.

Pankanin Dominika , Santarem Nuno , Kong Thoo Lin Paul , Cordeiro da Silva Anabela
a high toxicity with significant side effect and are associated with growing resistance. Therefore, in the absence of vaccines, there is an increasing need for alternative treatment options. A set of new hydroxyamino and mononapthalimido derivatives was tested against the protozoans Leishmania infantumand Trypanosoma bruceithe causative agents of Leishmanisis and HAT, respectively. The toxicity of the tested compounds was determined by growth inhibition of THP-1 cells. The results suggest that chemical changes in the structure of hydroxamic derivatives as well as changes in the carbon chain linking the naphthalamide and substituting groups, affect their anti-parasitic activity. The NA compound proved to be very effective (EC50 equal 3.55 µM) on L. infantumparasites. Although effective in L. infantumpromastigotes itself was less effective in the Leishmaniaintracellular amastigotes. This compound may be used as a reference for the future drug development. In the case of T. bruceiAS1, AS4, APN, NI and NA compounds showed great effectivity and low toxicity at the same time. The results can provide some basis for the further in vivo testing of the effectivity and toxicity in mice. This work provides new insights into the design and optimization of more potent and parasite specific drugs.

Gołąbek Ilona, Jóźwiak Krzysztof
Biopharmacy Department, Medical University of Lublin, ul. Chodźki 4a, 20-093 Lublin
Presenting author: Ilona Gołąbek (ilonag9333@gmail.com)
INTRODUCTION: Epibatidine (EPI), an alkaloid found in many poisonous frogs of South America is highly potent agonist of nicotinic acetylcholine receptor (nAChR) and important tool to study the receptor pharmacology. The fact that the source animals are not sensitive to EPI toxic effects is very intriguing and very recent work identified several SNP modifi cations of genes encoding nAChR subtypes originating from different species of these poisonous frogs. The aim of this student project is to evaluate by molecular simulations how nAChR variants carrying these modifications interact with EPI and other agonists in comparison with the WT receptor.
METHODS: Molecular models of nAChR variants were prepared by homology modeling using 5kxi.pdb template. Docking simulations of EPI, nicotine and acetylcholine were performed by comparison of several different programs and algorithms.
RESULTS: The most important modification is an exchange of Ser108 into Cys of nAChR beta subunit, the residue provides very important interaction with the hydrogen bond acceptor atom of the agonist. Four other modifications seems to play auxiliary effects in diminishing the binding affinity to EPI in relation to other agonists.
CONCLUSIONS: Resolving of molecular mechanism responsible for nAChR relative insensitivity to EPI is an important step in understanding of the receptor chemistry and may result in better strategies for development of new drug candidates with optimized pharmacological profile.

Wójtowicz Sylwia, Cieślik Magdalena, Czapski Grzegorz A., Strosznajder Joanna B.
Department of Cellular Signalling, Mossakowski Medical Research Centre Polish Academy of Sciences (5 Pawinskiego Street, 02-106 Warsaw, Poland)
Presenting author: Sylwia Wójtowicz (swojtowicz@imdik.pan.pl)
INTRODUCTION: The interaction between neuronal and glial cells is crucial for the pathomechanism of Alzheimer’s disease (AD). Microglia activation could be involved in neuroprotection, however, could also enhance amyloid beta (Aβ) neurotoxicity. In this study we focused on the impact of Aβ oligomers (AβO) on transcription of genes for anti-oxidative enzymes, including sirtuins, and other mitochondria-related genes in neuronal SH-SY5Y and microglial BV2 cells. Moreover, we investigated the consequence of pharmacological modification poly(ADP-ribose)polymerase-1 (PARP-1) and Sirtuin-1 in AβO toxicity.
METHODS: In this study the biochemical and molecular biology methods were applied.
RESULTS: Our data indicated significant differences in the effect of AβO on expression of several genes related to antioxidative defence and to mitochondrial function in neuronal and glial cells. In neuronal cells downregulation of Sod2, Sirt5 and Sdha expression by AβO was observed. Moreover, AβO increased transcription of Sod1, Cat and mt-Nd1 in these cells. However, in microglial cells AβO significantly downregulated transcription of Gpx4, Sirt1, Sirt3, mt-Nd1, Sdha, Mfn2 and significantly enhanced expression of Sod2 and Dnm1l. AβO decreased viability of both cell types, but increased oxidative stress and reduced mitochondrial membrane potential only in SH-SY5Y. Activator of Sirtuin 1 (SRT1720) and inhibitor of PARP-1 (Olaparib) efficiently protected neuronal but not microglial cells against AβO toxicity.
CONCLUSIONS: Our results suggested that cell type-specific alterations of gene expression in neurons and microglial cells could play a key role in different susceptibility of these cells to AβO-evoked apoptotic signaling in neurodegenerative disorders.

Borzych Michalina K., Kułaga Marta, Ogłodek Ewa, Grzesińska Anna
Klinika Psychiatrii, Collegium Medicum Uniwersytetu Mikołaja Kopernika (ul. Skłodowskiej-Curie 9, 85-094 Bydgoszcz , Poland)
Presenting author: Michalina K. Borzych (michalina.borzych@wp.pl)
Physical activity is an important part of a healthy lifestyle. It is relevant how it affects the mood, feeling of self-satisfaction and accepting your own body. The purpose of the study is to estimate how physical activity impacts on occurrence of depressive disorders. The study was conducted with an anonymous questionnaire, sent out electronically via social networking sites. It contained Beck Depression Inventory and questions about physical activity. The questionnaire estimates the correlation between physical activity of questioned people and the occurrence of various degrees of depressive disorders among them. It also focuses on motivation to exercise and how it affects the mood.
Among people declaring themselves as physically inactive we observed a higher percentage of the occurrence of depressive disorders than among people declaring as themselves physically active. It is important, that in the group of physically inactive people the most common disorder was severe depression, while in the group of physically active people it was mild depression. The main motivation of questioned people to physical activity was the desire to improve the appearance and well- being, as well as the positive impact on health.