Chembio-Abstract Boo..

International Conference
Chemical Biology:
Methods and Progress
Programme and Abstracts
Vienna, Austria
11 - 12 February 2013
1
Organizing Committee
Local Organizing Committee
International Organizing Committee
Prof. A. Touraev (President of Vienna
International Plant Conference
Association (VIPCA))
Andreas Marx (Co-Chair)
Jurgen Bajorath
Benoit Deprez
Eric Marechal
Mark Bradley
Lisa A. Marcaurelle
Ho Jeong Kwon
Edward Zanders
SPONSORS AND EXHIBITORS OF THE CONFERENCE
2
3
Dear Friends and Colleagues,
Welcome to the International Conference “Chemical Biology: Methods and Progress”!
Welcome to Vienna!
Chemical Biology is a scientific discipline at the interface of the life sciences and physical sciences
integrating a wide series of experimental techniques ranging from synthetic organic chemistry and
biochemistry to structural, molecular, and cellular biology. It is one of the fastest growing areas of
science today. By combining powerful techniques from organic synthesis, biophysical chemistry,
molecular biology and genetics, researchers all over the world are developing new molecules and
new strategies to combat cancer, inflammation, autoimmune diseases, as well as developing new
frontiers and applications in non-human science.
Vienna is located in the heart of Europe on the banks of the Danube River, and considered one of
the most important economic, cultural and touristic large cities of central Europe. Apart from
providing top science, the Conference will capture the spirit of the city thanks to the central
location of the venue offering a multitude of cultural events just minutes away.
International Conference "Chemical Biology: Methods and Progress" to be held in Vienna on
February 11-12th , 2013 will provide leading academy and industry scientists a platform to
communicate recent advances in Chemical Biology and an opportunity to define new frontiers and
establish multilateral collaborations.
Prof. Andreas Marx, Chair of the International Organizing Committee
Prof. Alisher Touraev, President of VIPCA, Chair of the Local Organizing Committee
4
5
Table of Contents
Programme at a Glance …………………………………………………………
8
Scientific Programme ……………………………………………………………
9
Abstracts of Oral Presentations …………………………………………….
11
Abstracts of Posters Presentations ……………………………………….
24
List of Poster Presentations ………………………………………………….
46
List of Participants ………………………………………………………………..
49
6
LemnaTec
Dr. Joerg Vandenhirtz
18 Schumanstr.
Wuerselen 52416
Germany
Tel.: +49 2405 4126-12
Fax: +49 2405 4126-26
Mob: +49 179 4576 321
Email: [email protected]
Web: http://www.lemnatec.com
LemnaTec’s team of scientists develops hard- and software solutions for Plant Phenomics, highthroughput and high-content screening of plants, seedlings, insects and other organisms and for
the automated evaluation of bio tests in ecotoxicology. Digital images are primarily taken by the
Scanalyzer systems PL, HTS and 3D, all set up in a modular design. Using advanced LemnaTec image
processing algorithms, every visible parameter (e. g. colour, shape, size, and architecture) is
subsequently measured and correlated with experimental records (e. g. genetic data). Our aim: to
visualise and analyse the biology beyond human vision.
7
Programme at a Glance
Monday
February 11
Tuesday
February 12
07.30 - 17.00
Registration
08.00 - 17.00
Registration
09.00 - 09.30
Welcome address by
A. Touraev,
Prof. Andreas Marx
09.00 - 10.30
Session IV
Chemoinformatics and Computational
Chemical Biology
09.30 - 10.30
Keynote Lecture:
Zanders E D
10.30 - 11.00
Coffee Break
10.30 - 11.00
Coffee Break
11.00 - 12.35
Session V
Molecular Imaging and Switching Tools
11.00 - 12.30
Session I
Strategies for Bioactive Small Molecule
Discovery
12.35 - 14.00
Lunch
12.30 - 14.00
Lunch
14.00 - 15.30
Session VI
Chemical Biology for the Control of Complex
Biological Systems
14.00 - 15.30
Session II
High-Content and High-Throughput Assay
Design
15.30 - 16.00
Coffee Break
15.30 - 16.00
Coffee Break
16.00 - 17.10
Session VII
Chemical Biology for Deciphering Cell
Signaling Pathways
16.00 - 17.20
Session III
Target Identification for Small Molecules
17.10 - 17.40
Closing Ceremony, Conference Photo
17.20 - 18.30
Poster Session (Odd Numbers)
17.40 - 19.00
Poster Session (Even Numbers)
18.30 - 20.00
Welcome Reception
19.00 - 22.00
Conference Dinner Party
8
SCIENTIFIC PROGRAMME
February 11 (Monday)
10.30 - 11.00
Registration
Opening
Welcome address by Touraev A (VIPCA)
Welcome address by Marx A (Germany)
Keynote Lecture
Zanders E D (UK): Chemical biology: successes and challenges
Coffee break
11.00 - 12.30
Session I: Strategies for Bioactive Small Molecule Discovery
Chairs
11.00 - 11.20 (+5)
12.30 - 14.00
Marcaurelle LA and Kwon HJ
Marcaurelle LA (USA): Diversity-oriented synthesis for chemical biology and drug
discovery
Kwon HJ (South Korea): Phenotypic screeening and multi-omics-based target
identification and validation (MOTIV) of bioactive small molecule
Thinon E (UK): Using chemical tools to validate a new target for cancer therapy
Gerwick L (USA): The anti - inflammatory honaucin natural products and synthetic
analogs: mechanism of action and in vivo results
Lunch
14.00 - 15.30
Session II: High-Content and High-Throughput Assay Design
Chairs
14.00 - 14.30 (+5)
15.30 - 16.00
Deprez B and Hoffmann C
Bradley M (UK): Polymer microarrays - high-throughput screening for materials to
control and modulate cells
Deprez B (France): Chemical control of the bacterial response to antibiotics: a new
antibacterial strategy
Dvorak Z (Czech Rep.): Transgenic reporter cell lines as the tool for identification of
candidate drugs: the case of AhR and GR
Coffee break
16.00 - 17.20
Session III: Target Identification for Small Molecules
Chairs
16.00 - 16.25 (+5)
16.30 - 16.55 (+5)
17.00 - 17.15 (+5)
Famulok M and Crews CM
Crews CM (USA): Small molecule control of intracellular protein levels
Famulok M (Germany): Chemical biology for deciphering cell signaling pathways
Ki Hyun Kim (South Korea): A small molecule targeting an ion channel inhibits
angiogenesis via the suppression of HIF-1signal transduction
Poster Session (Odd Numbers)
Welcome Reception
07.30 - 17.00
09.00 - 09.30
09.30 - 10.30
11.25 - 11.45 (+5)
11.50 - 12.05 (+5)
12.10 - 12.25 (+5)
14.35 - 15.05 (+5)
15.10 - 15.25 (+5)
17.20 - 18.30
18.30 - 20.00
9
February 12 (Tuesday)
08.00 - 17.00
Registration
09.00 - 10.30
Session IV: Chemoinformatics and Computational Chemical Biology
Chairs
09.00 - 09.30 (+5)
10.30 - 11.00
Bajorath J and Marx A
Bajorath J (Germany): Computational chemical biology: methods for the
identification of small molecular probes with target differentiation potential
Rognan D (France): Computational approaches to activity profiling of bioactive
compounds
Ocasio C (USA): Targeted greatwall kinase blockade offers a novel strategy for
chemoprevention
Coffee break
11.00 - 12.35
Session V: Molecular Imaging and Switching Tools
Chairs
11.00 - 11.30 (+5)
11.35 - 12.00 (+5)
12.30 - 14.00
Specht A and Urano Y
Specht A (France): Two-photon sensitive photo-triggers, design and applications
Urano Y (Japan): In vivo rapid cancer detection by topically spraying a novel gammaglutamyltranspeptidase-activated fluorescence probe
Hoffmann C (Germany): Site specific labelling with FlAsH or ReAsH - applications to
GPCRs
Lunch
14.00 - 15.30
Session VI: Chemical Biology for the Control of Complex Biological Systems
Chairs
14.00 - 14.30 (+5)
15.30 - 16.00
Marechal E and Woscholski R
Marechal E (France): A novel class of small molecules targeting the glycerolipid
metabolism system in various biological models
Woscholski R (UK): Pleckstrin homology (PH) domain mimicry: chemical biology of
phosphoinositide signalling
Ziegler S (Germany): Identification of the targets of natural product–inspired mitosis
modulators
Coffee break
16.00 - 17.10
Session VII: Chemical Biology for Deciphering Cell Signaling Pathways
Chairs
16.00 - 16.30 (+5)
16.35 - 17.05 (+5)
Zanders ED and Dell A
Marx A (Germany): Chemical biology of DNA replication
Dell A (UK): Glycosylation of mouse and human immune cells: insights emerging from
N-glycomics analyses
Closing Ceremony, Conference Photo
Poster Session (Even Numbers)
Conference Dinner Party
-Traditional Austrian food, wine and entertainment (professional singers and
musicians), located in one of Vienna's famous 'Heurigen'
-Cost: 40,- EUR
09.35 - 10.05 (+5)
10.10 - 10.25 (+5)
12.05 - 12.25 (+5)
14.35 - 15.05 (+5)
15.10 - 15.25 (+5)
17.10 - 17.40
17.40 - 19.00
19.00 - 22.00
10
Abstracts of
Oral Presentations
11
Chemical Biology: successes and challenges
Edward D. Zanders
PharmaGuide Ltd, Cambridge, United Kingdom
Chemical biology has a venerable history going back to the times when alchemy was giving way to modern
chemistry. Later on, biochemistry came into the ascendant, until the end of the last century. Since then, the
“omics” revolution, in the form of chemical genomics and proteomics etc, has rejuvenated the study of the
interaction between small organic molecules and macromolecules, particularly proteins.
The talk will start with a brief historical background to this wide-ranging field, followed by a description of
the main features of chemical biology. These features will be understood by practitioners in the growing
number of academic centres with the title, as well as by the pharmaceutical and agrochemical scientists
working in industry. Examples of successful research in chemical biology will then be given, followed by a
consideration of the challenges facing the field, including accessing multiple targets and modulating
protein-protein interactions. Finally there will be a brief description of some of the technologies under
development today that may have a significant impact upon the field in the future.
Diversity-oriented synthesis for chemical biology and drug discovery
Lisa A. Marcaurelle
H3 Biomedicine, Cambridge, MA USA
Recent evidence demonstrating the importance of structural complexity in advancing compounds to the
clinic points to the need for enhancing small molecule screening collections with sp3-rich compounds. The
development of diversity-oriented synthesis (DOS) strategies for accessing libraries of sp3-rich compounds
containing one or more stereogenic centers will be presented in two parts, including an overview of library
synthesis efforts at the Broad Institute and H3 Biomedicine. The DOS libraries span a broad range of
molecular frameworks, including macrocycles and medium-sized rings, as well as fused-, bridged- and
spirocyclic- ring systems. Cheminformatic design principles will be discussed as well as results of highthroughput screening.
12
Phenotypic screeening and multi-omics-based target identification and validation (MOTIV) of bioactive
small molecule
Ho Jeong Kwon
Chemical Genomics National Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function
Control, College of Life Science & Biotechnology, Yonsei University
A systematic screening and target protein identification of small molecule is pivotal for the discovery of new
bioactive small molecule and development into new therapeutic drug. Here, the efficient screening and
target protein identification methods for new bioactive small molecules with the potential for development
as new drugs will be proposed. This two-step procedure first employs phenotypic screening to discover and
develop new bioactive small molecules. Secondly, MOTIV provides systemic approach to discover the target
protein of bioactive small molecule. With the chemical genomics and proteomics approach of target
identification methods, various target protein candidates are identified. Then network analysis and
validations of these candidates result in identifying the biologically relevant target protein and cellular
mechanism. Collectively, the combination of phenotypic screening and MOTIV will provide an effective
approach to discover new bioactive small molecules and their target protein and mechanism identification.
In this presentation, our recent results from the application of this system will be also introduced.
Using chemical tools to validate a new target for cancer therapy
Emmanuelle Thinon, Remigiusz Serwa, David Mann, Edward W. Tate
Chemistry Department, Imperial College London, England
N-myristoyltransferase (NMT) catalyses the irreversible transfer of a C14 fatty acid to the N-terminal glycine
of proteins. Previous studies have shown that NMT is upregulated in several cancers and it has been
suggested that NMT could be a therapeutic target for cancer. Our aims are to use chemical tools to
demonstrate the on-target activity of an inhibitor and to understand all the downstream effects of NMT
inhibition in cells. We first selected the best human NMT inhibitor by screening a small library of
compounds. The toxicity of the most potent inhibitor was assessed in cancer cell lines. Cells were
completely killed after 7 days. Further analysis by flow cytometry of the cell cycle distribution showed a cell
cycle arrest in G1 after 1 day. To asses if the inhibitor was acting on-target in cells, we used a chemical
labelling technology developed in our group. An alkyne-tagged analogue of myristoyl-CoA was synthesised.
Cells were treated with this analogue which is transferred to the N-terminal glycine of NMT substrates. After
cell lysis, subsequent azide-alkyne “click” cycloaddition, with a capture reagent bearing a fluorophore,
allowed visualisation of the myristoylated substrates using in gel fluorescence. When the NMT inhibitor was
added to the cells simultaneously to YnC12, no protein was modified with an alkyne moiety and we saw no
fluorescence on the gel. The labelling technology was also combined with proteomics. NMT substrates can
be isolated from cell lysates using the affinity between the biotin moiety on the capture reagent and
streptavidin beads, and identified using LC-MS/MS. We were able to identify around 80 substrates of NMT.
As we now know that NMT inhibition can kill cancer cells, future work will focus on understanding which
substrates are more important to the cells and lead to cell death when they are not myristoylated.
13
The anti - inflammatory honaucin natural products and synthetic analogs: mechanism of action and in
vivo results
Lena Gerwick, Samantha Mascuch, Hyukjae Choi, Joseph Campanale, Mary Hensler,Josua Olson,
AmroHamdoun, Victor Nizet, William Gerwick
University of California San Diego
The honaucins are small lactone molecules isolated from a marine cyanobacterium found overgrowing a
coral reef off the coast of Hawaii. Honaucin A and several analogs have been shown to inhibit nitric oxide
production as well as production of proinflammatory cytokines interleukin 1, interleukin 6 and TNF alpha in
murine macrophages. In vivo, honaucin A was able to reduce phorbol ester-induced mouse ear edema. To
further study the mechanism of action of honaucin A, a fluorescently labeled probe was synthesized,
introduced to murine macrophages and found to rapidly localize to cellular organelles. Co-localization
studies were performed with the aim of identifying these organelles and examining the kinetics of probe
uptake. In addition, protein extracts from honaucin A-treated and untreated cells were probed with
antibodies to proteins involved in apoptosis and NF-κB signaling in an effort to further characterize the
compounds cellular effects.
Polymer microarrays - High-throughput screening for materials to control and modulate cells
Prof. Mark Bradley
School of Chemistry, EaStChem, University of Edinburgh
I will demonstrate a variety of approaches for at the preparation and high-content screening of polymer
microarray platform and their application in a number of cell based screens. Fabrication methods, including
direct inkjet based polymer synthesis and analysis with fixed and live cells on over 10,000 features will be
described.
Using polymer technology I will show how polymers have been identified and then developed for a myriad
of applications, including control of stem cells fate, corneal bandages, bacterial capture and thermally
responsive surfaces. This includes:
(i). Polymer blends that can find use as implants which support cell attachment, growth and differentiation,
and tissue regeneration in vivo and can be used in bone repair: This has led, for example, to polymers that
are able to bind Sto+ cells and promote bone regeneration with materials now entering large animal models
the first step on the road to human application.
(ii). Polymers able to support long term highly functional hESC-derived hepatocyte like cells (showing high
levels of both CYP3A4 and CYP1A2 expression) and which are now being explored as a coating on an extracorporeal support for trials in bio-artificial liver devices.
(iii). Polymers displaying binding or inhibition of binding of bacteria.
(iv). Polymers that bind human stem cells, maintain them in a highly controlled state, yet allow mild thermal
release, while maintaining full pluripotency.
14
Antagonists of the bacterial transcriptional repressor ETHR
Benoit Déprez
1
2
INSERM, U761, Lille, Faculté des Sciences Pharmaceutiques et Biologiques de Lille, Université Lille 2 Droit et Santé, Univ Lille Nord
3
de France, Institut Pasteur de Lille; 4IFR 142; 9Pôle de Recherche Interdisciplinaire pour le Médicament
Tuberculosis remains a major cause of mortality and morbidity killing each year more than two million
people. The emergence of multidrug-resistant strains of Mycobacterium tuberculosis, stresses the need for
alternative therapies. Ethionamide (ETH), a second-line antibiotic, is used to treat multidrug-resistant
tuberculosis. ETH is a prodrug and needs to be activated by the mycobacterial enzyme EthA in order to
inhibit InhA, the enoyl-acyl ACP reductase involved in mycolic acid biosynthesis. The negative regulation of
ethA, exerted by the bacterial transcriptional repressor EthR, limits the effectiveness of this prodrug[1].
We recently reported that drug-like EthR inhibitors are able to boost the antimycobacterial efficacy of
Ethionamide both in vitro and in vivo[2]. We report here the different strategies used to develop powerful
ligands of EthR. Structural and functional features of our repressor antagonists are presented.
Transgenic reporter cell lines as the tool for identification of candidate drugs: the case of AhR and GR
Aneta Novotna1, Radim Vrzal1, Petr Pavek2, Zdenek Dvorak1
1
Department of Cell Biology and Genetics, Faculty of Science,Palacky University, Slechtitelu 11, 783 71 Olomouc, Czech Republic
Department of Pharmacology and Toxicology, Charles University inPrague, Faculty of Pharmacy in Hradec Kralove, Heyrovskeho
1203, Hradec Kralove 50005, Czech Republic
2
The expression of genes involved in endogenous metabolism and in metabolism of drugs and other
xenobiotics is regulated at multiple levels. The key transcriptional regulators of drug metabolizing enzymes
and endogenous pathways are aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR). The
ligands for AhR and GR may cause drug-drug interactions. On the other hand, the ligands for these receptors
may be of therapeutic value. Therefore, the identification of new AhR and GR ligands is a highly relevant
issue. We present here two novel stable gene reporter cell lines, allowing high-throughput, specific and
selective identification and characterization of AhR and GR ligands [1, 2]. Acknowledgements: Our
laboratories are supported by the grants from the Czech Scientific Agency GACR 303/12/G163 and
303/12/0472. 1. Novotna, A., P. Pavek, and Z. Dvorak, Novel stably transfected gene reporter human
hepatoma cell line for assessment of aryl hydrocarbon receptor transcriptional activity: construction and
characterization. Environ SciTechnol, 2011. 45(23): p. 10133-9. 2. Novotna, A., P. Pavek, and Z. Dvorak,
Construction and characterization of a reporter gene cell line for assessment of human glucocorticoid
receptor activation. Eur J Pharm Sci, 2012. 47(5): p. 842-7.
15
Controlling intracellular protein levels using small molecules
Craig M. Crews
Yale University
Less than 20% of the proteome has enzymatic activity. However, current drug development efforts rely
heavily on the identification of enzymatic inhibitors, thus ignoring a significant fraction of the
proteome. The Crews lab has developed two technologies known as Hydrophobic Tagging and PROteolysis
TArgeting Chimera molecules (PROTACs) that can selectively knock down intracellular levels of a specific
protein. These cell permeable hetero-bifunctional molecules utilize the cells’ own ubiquitin/proteasome
protein degradation pathway to selectively destroy a target protein of choice. These approaches have been
validated by targeting various intracellular and membrane proteins for degradation in Proof-of-Concept
experiments. By pharmaceutically controlling protein function via a novel mechanism that does not rely
upon enzymatic inhibition, this approach may allow one to target those proteins that are currently not
‘pharmacologically vulnerable’.
Chemical biology for deciphering cell signaling pathways
Michael Famulok
Life and Medical Sciences (LIMES) Institute, University of Bonn, Germany
Small molecule inhibitors of proteins are invaluable tools in Chemical Biology. Their identification can be
tedious, because most screening methods have to be tailored to the corresponding drug target. We have
developed modular assays based on aptamer displacement or protein-dependent reporter ribozymes for
the screening of small-molecule inhibitors. As aptamers can be generated for virtually any protein, the assay
potentially identifies inhibitors for targets or individual protein domains for which no functional screen is
available. Thereby, chemical space is explored in a rapid, focused, and modular manner, by indirectly taking
advantage of the highest molecular diversity currently amenable to screening, namely that of 1016 different
nucleic acid sequences. I will discuss the application of these approaches to find new inhibitors for target
proteins, in particular for the small guaninenucleotide exchange factors (GEFs) of the cytohesin family.
Examples showing that these modulators can be used as tools for gaining novel biological insight are
provided.
16
A small molecule targeting an ion channel inhibits angiogenesis via the suppression of HIF-1signal
transduction
Ki Hyun Kim, JuYeol Park, Hye Jin Jung, Ho Jeong Kwon
Yonsei University
Small molecules have been highlighted as powerful probes to explore biological questions. To identify new
bioactive small molecules perturbing mitochondrial respiration in cells, a galactose/glucose media growth
inhibition screening was conducted using the LOPAC1280 library (Sigma #LO3300) and 46 small molecules
were selected as hit small molecules. Among 46 small molecules, LP19, ion channel protein inhibitor,
suppressed mitochondrial ROS and HIF-1α expression level in a dose dependent manner. As the result, LP19
was identified as the candidate that inhibited angiogenesis via the suppression of mitochondrial ROS
signaling of HIF-1α expression. Moreover, LP19 inhibited tumor induced angiogenesis including tube
formation and chemoinvasion in vitro. Collectively, a new small molecule, LP19, targeting an ion channel
protein was identified from the systemic cell-based assay and revealed to have anti-angiogenic activity via
the suppression of HIF-1α signal transduction. These results provide new insight into the biological network
between ion channel and mitochondrial ROS mediated HIF-1α signal transduction. Furthermore, LP19 will
be a useful small molecule probe to explore the underlying mechanism.
Computational methods for the identification of small molecular probes with target differentiation
potential
Jürgen Bajorath
Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, Rheinische FriedrichWilhelms-Universität Bonn, Germany.
Currently, many opportunities exist for the design of new computational approaches at the interface
between chemistry and biology. Among others, these include the exploration of molecular selectivity or
polypharmacology and the identification of small molecular probe candidates that interfere with specific
biological functions or differentiate between members of given target families. This presentation focuses on
the latter aspect. In order to identify probe candidates with target differentiation potential, computational
approaches are typically applied to mine compound profiling data. If large numbers of targets are studied,
the navigation and mining of high-dimensional activity spaces can quickly become complicated. For
example, when compound collections are profiled against subsets of the kinome, often including more than
100 kinases, high-dimensional activity spaces are obtained that are difficult to de-convolute and represent.
In this case, computational analysis schemes must be investigated that depart from conventional chemical
and activity space modeling. This contribution introduces new computational concepts for the identification
of individual compounds or pairs of compounds with high target differentiation potential. These approaches
are designed to reduce the complexity of high-dimensional activity spaces and quickly focus on key
compounds.
17
Computational approaches to activity profiling of bioactive compounds
Didier Rognan
University of Strasbourg - CNRS
Computational chemists have traditionally been focusing their efforts in designing and synthesizing
compounds targeting a single protein. Significant advances in miniaturizing chemical and biological
experiments led the pharmaceutical industry to massively invest in high-throughput screening robots able
to screen several million compounds. Since this key information was obviously not publicly available, drug
designers have been in the paradoxical situation where academic scientists had enough time to mine a huge
amount of data that they were lacking, whereas their industrial colleagues were in possession of the data
but not of the intellectual freedom to efficiently analyze it. Public initiatives to release previously hidden
data, archive published information or even to generate them have significantly changed the situation.
Million of bioactivity data points are now available for learning and prediction. Scientific and economic
pressure to design drugs with controlled selectivity profiles as well as the recent boost of drug repurposing
led to the development of in silico ligand profiling methods aimed at
(i) predicting potential targets (and thus a mechanism of action) for orphan bioactive ligands,
(ii)
identifying
off-targets
responsible
for
side
effects
and
adverse
reactions,
(iii) propose novel targets for existing drugs. From a conceptual point of view, three main approaches with
different starting points are possible. They will be reviewed from here on with an emphasis on success
stories.
Targeted greatwall kinase blockade offers a novel strategy for chemoprevention
Cory Ocasio
University of Sussex
The proper orchestration of mitotic progression is controlled by the balanced activity of mitotic kinases and
counteracting phosphatases. Misregulation of these controls can be detrimental causing loss of entire
chromosomes and aneuploidy. Considering that mitotic kinases are only expressed in actively dividing cells,
targeting these kinases for cancer management is highly desirable. To this end, we are validating the mitotic
kinase Greatwall Kinase (Greatwall) as a target for cancer therapy. Initial studies show that siRNA
knockdown of Greatwall leads to rapid cell death in a number of transformed cell lines. Surprisingly, we
found that untransformed RPE cells with low Greatwall protein and mRNA levels remained unaffected after
Greatwall knock down. Moreover, Greatwall levels fluctuate dramatically among different cancer cell lines
and are generally low in non- transformed tissues. Seminal studies using Xenopus egg extracts and
drosophila larvae show that Greatwall plays an integral role in the cell cycle control of rapidly dividing
embryonic cells and it is possible that some tumor cells have reverted back to this state of dependency on
Greatwall. This makes Greatwall even more promising as an anti-cancer agent as its activity is dispensable in
somatic cells but critical to the survival and proliferation of cancer cells. In light of these findings, I aim to
pursue target validation studies with Greatwall, elucidate synergistic effects using known mitotic kinase
inhibitors, and work closely with a team of medicinal chemists to characterize novel small-molecule
Greatwall modulators. Nonspecific interactions between known mitotic kinase inhibitors and normally
dividing cells often lead to unwanted side-effects. Targeting Greatwall provides an opportunity to
circumvent this undesirable outcome and advance translational research.
18
Two-photon sensitive photo-triggers : design and applications
Alexandre Specht
Laboratoire de Conception et Application de Molécules Bioactives
Control of cellular processes, which comply with a spatial and temporal organization, can be attained using
triggering methods. Photochemical activation of an inert biological precursor offers a way to attain this
spatiotemporal control. Photoactivatable precursors of biological effectors called “caged compounds” 1
were applied to investigate several biological involving light-activatable neurotransmitters, second
messengers, enzyme or receptor binders as well as small molecule gene regulators.2 Recent efforts
prompted the development of photoremovable groups that have increased photochemical efficiencies for
two photon excitation to allow more sophisticated applications.3 Two-photon excitation produces excited
states identical to standard UV excitation while overcoming major limitations when dealing with biological
materials, like 3D spatial resolution, tissue penetration and toxicity. We have recently developed a new
series of photoremovable groups: an o-nitrophenethyl derivative, which exhibits the necessary properties
for a satisfactory caging group including a fast and efficient photolytic reaction at near-UV irradiation4.
Molecular engineering of the two-photon uncaging cross-sections was performed on this platforms leading
to photoremovable groups with ultra-efficient uncaging cross-section5.
The development and the new opportunities of our new two-photon sensitive photoremovable groups will
be discussed here, including applications in neuro5 and cellular- biology6 using two-photon uncaging.
In Vivo Rapid Cancer Detection by Topically Spraying a Novel γ-Glutamyltranspeptidase-activated
Fluorescence Probe
Yasuteru Urano
Graduate School of Medicine, The University of Tokyo (Japan)
Basic Research Program, Japan Science and Technology Agency (Japan)
Fluorescence imaging is one of the most powerful techniques currently available for continuous observation
of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical
importance for fluorescence imaging, and we have succeeded to construct several versatile rational design
strategies for novel fluorescence probes based on the concept of photoinduced electron transfer1),2) and
intramolecular spirocyclization3),4).
Very recently, we have succeeded to develop various novel protease probes which were applicable for living
cell system4). For example, gGlu-HMRG, a novel spirocyclized rhodamine-based fluorescence probe for glutamyltranspeptidase (GGT), which is well-known to be upregulated in various cancer cells, was
successfully developed (Fig. 1a). By applying gGlu-HMRG to various cancerous cell lines whose GGT activity
is upregulated, fast enzymatic reaction of gGlu-HMRG with GGT occurs on the plasma membrane to yield
highly fluorescent product HMRG (Fig. 1b), which led us to establish a novel and highly activatable strategy
for sensitive and fast-responding fluorescence imaging of tiny tumors in vivo. In mouse models of
disseminated human peritoneal ovarian cancer, activation of gGlu-HMRG occurred within 1 min of topically
spraying onto tissue surfaces that are suspected of harboring tumors, creating high signal contrast between
the tumor and the background (Fig. 1c)5). We believe gGlu-HMRG probe could aid surgeons in detecting
tiny cancerous nodules for accurate biopsy and tumor resection, delineating the borders of tumors for
complete removal and confirming no residual tumor.
19
Site specific labelling with FlAsH or ReAsH – Applications to GPCRs
Carsten Hoffmann
Bio-Imaging-Center, Rudolf-Virchow-Zentrum, University of Wuerzburg,
Site specific labeling of proteins in living cells is an important requirement to permit imaging of cellular
signaling pathways. We have optimized a selective labeling strategy that uses a small six amino acid motif
(CCPGCC) which tightly binds the fluorescein arsenical hairpin binder (FlAsH). Currently, we combined FlAsH
with the cyan fluoresecent protein (CFP) to permit fluorescence resonance energy transfer (FRET). This
approach can be used to investigate conformational changes of GPCRs (G protein-coupled receptor) and
thus permits to study receptor activation in real-time and living cells. Using two different labeling sites for
FlAsH within the third intracellular loop of the muscarinic acetylcholine receptor subtype 3 (M3-AChR), we
can demonstrate distinct conformational changes of this important regulatory protein for different ligands.
The amplitude of the conformational change within one particular receptor domain is strongly correlated
with the onset of β-arrestin-2 recruitment to the M3-AChR.
A novel class of small molecules targeting the glycerolipid metabolism system in various biological models
Laurence Boudière, Eric Maréchal
Membrane Lipidome Team, Plant & Cell Physiology Lab, CEA Grenoble, Grenoble, France
A piperidinyl-benzimidazolidinone scaffold has been found in independent studies, in the chemical structure
of inhibitors of membrane glycerolipid metabolism. These inhibitors act on enzymes manipulating either
diacylglycerol (i.e. galvestine-1, an inhibitor of monogalactosyldiacylglycerol synthase from plants, identified
from a screen developed by our team) or phosphatidic acid (i.e. halopemide, an inhibitor of phospholipases
D from mammals). Blocking agents of membrane biogenesis might therefore be screened in focus libraries
of piperidinyl-benzimidazolidinone analogs. This presentation describes how valuable this novel chemotype
can be in various models of organisms and in different contexts, from plant development to cancer or
infectious diseases.
20
Pleckstrin homology (PH) domain mimicry: Chemical biology of phosphoinositide signaling
Rüdiger Woscholski
Imperial College London
Phosphoinositides are important lipid second messengers that control many signalling pathways such as cell
survival, growth and differentiation. Changes in phosphorylation of these lipids are recognised by specific
protein domains, which in turn will change the cellular function of the proteins harbouring these domains.
The lipid recognition domains have been therefore employed to probe and disturb phosphoinositide
dependent signalling, with the PtdIns(4,5)P2 binding PH domain of the phospholipase C being one of the
more specific biological tools at our disposal. We present here the creation and cellular validation of a
chemical PH domain mimetic (PHDM) that has the ability to disrupt selectively PtdIns(4,5)P2 dependent
processes such as transferrin uptake, cytoskeleton remodelling and mitochondrial survival. The latter results
in the autophagy of mitochondria, a process that has so far not been associated with PtdIns(4,5)P2 and
seems to be mediated by protein kinase C.
Identification of the targets of natural product–inspired mitosis modulators
Verena Pries, Claas Gerding-Reimers, Heiko Dueckert, Tobias Vogt, Andreas Brockmeyer, Petra Janning,
Kamal Kumar, Slava Ziegler, Herbert Waldmann
Max-Planck-Institut fur molekulare Physiologie Abt. Chemische Biologie Otto-Hahn-Strasse 11, 44227 Dortmund
Technische Universitat Dortmund, Fakultat Chemie Lehrbereich Chem. BiologieOtto-Hahn-Strasse 6, 44227 Dortmund (Germany)
The scaffolds of natural products define the areas of chemical space explored by nature in evolution.
Natural product–inspired compound collections are expected to be biologically relevant and therefore
enriched in bioactivity. Given the responsiveness of mitosis to perturbation by small molecules, we
investigated our focused natural product based compound collections by means of a phenotypic screen for
mitosis modulators in mammalian cells. Several small molecules were identified to induce an accumulation
of round-shaped cells with condensed DNA which is indicative of mitotic arrest. Characterization of the
mitotic phenotypes revealed novel tubulin modulators and compounds which impair the centrosomal
integrity without directly interefering with the microtubule cytoskeleton. The structure-activity relationship
guided the design and synthesis of probes which were employed for affinity chromatography based
proteomics to identify the cellular targets and elucidate the mode of action.
21
Chemical Biology of DNA Replication
Andreas Marx
Department of Chemistry and Konstanz Research School Chemical Biology University of Konstanz
DNA is the storage of genetic information in Nature. Transmission of the genetic information from the
parental DNA strand to the offspring is crucial for the survival of any living species. The entire DNA synthesis
in DNA replication is catalyzed by DNA polymerases and depends on their ability to select the canonical
nucleotide from a pool of structurally similar building blocks. Besides the crucial biological role of DNA
polymerases, these enzymes are the workhorses in numerous important molecular biological core
technologies such as the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing,
and nucleic acids based diagnostics.
We will report results on insights into how DNA polymerases faithfully recognize their template, how
chemically modified nucleotides with potential in biotechnological applications are processed, and finally,
new approaches to study the orchestration of human DNA polymerases by mimicry of posttranslational
protein modification.
Glycosylation of mouse and human immune cells: insights emerging from N-glycomics analyses.
Anne Dell
Department of Life Sciences, Imperial College London, SW7 2AZ, UK
Ultra-high sensitivity mass spectrometric strategies incorporating MALDI-MS/MS and nanoelectrospray(ES)-MS/MS enable very complex mixtures of glycans and glycopeptides from biological extracts
of cells and tissues to be studied thereby revealing the types of glycans present and, importantly, providing
clues to structures that are likely to be functionally important. Glycomic methodologies seek to define the
total N-glycan and/or O-glycan repertoire in a biological sample. Data emerging from our glycomic
programmes of collaborative research, which are helping to provide new insights into the functions of
glycans in the immune system, will be described. These glycomic methodologies have been exploited by the
NIH Consortium for Functional Glycomics whose Analytical Core (based at Imperial College) was funded
from 2001 to 2011 to carry out high throughput analyses of murine and human haematopoietic cell
populations in order to provide a public glycomics data resource for the scientific community. Key outputs
of this programme will be described together with progress on the development of informatic tools to
manage large volumes of glycomics data.
22
23
Abstracts of
Poster Presentations
24
N 1. Phosphoinositides (PIPs) as probes for studying and targeting the metastasis-promoting phosphatase
PRL-3
Sven Stadlbauer, Maja Koehn
European Molecular Biology Laboratory Heidelberg
The phosphatase of regenerating liver (PRL) family is a subclass of dual specificity phosphatases (DSPs). One
of its members, PRL-3, is a cancer biomarker and a promising therapeutic target in cancer. It is known to
promote cancer metastasis and was also reported to enhance proliferation in many primary cancers. In
addition, its high abundance correlates with poor prognoses in cancer patients. Therefore, inhibiting PRL-3
can improve malignant tumour and metastasis therapy. PRL-3 dephosphorylates phosphoinositides (PIPs), in
particular PI(4,5)P2, which are integral building blocks of cell membranes and important signaling lipids.
PIPs interact with numerous proteins, which can bind and/or modify the PIPs through enzymatic reactions
that involve phosphorylation and dephosphorylation, as well as lipid hydrolysis. However, due to the highly
dynamic character of these interactions, direct detection is complicated and often impossible. A more
detailed understanding of the interactions with phosphatases would allow for the targeted synthesis of
inhibitors for PIP-phosphatases, such as PRL-3. Therefore, we are in the process to synthesize modified PIPs
by solid-phase synthesis to provide previously unavailable tools to probe inositol signaling. Furthermore
other proteins interacting with PRL-3, while binding to PIPs can be additionally identified. A future aim is to
apply these newly designed probes to study PRL-3–PIP interactions in living cells. This will significantly
contribute to the understanding of PRL-3 molecular mechanisms and signaling pathways.
N 2. Analyzing the regulations on agricultural genetic resources
EunMi Kim1, Jung Mi Num1, Sejin Kim1, Duraisamy Kalpana2
1
Department of Commerce and Trade, Chonbuk National University,
National University.
2
Department of Forest Science and Technology, Chonbuk
With the tremendous technological advances made in genetics and biology in recent years, the economic
and commercial value of genetic resources has increased steadily. While the South possessing genetic
resources advocates conservation of genetic resource, the North part using the resources has a preference
for public use of genetic resources and private property right for bio- industrial products. These contradict
interests resulted in the establishment of Convention on Biological Diversity (CBD) and the Trade Related
Intellectual Property Rights (TRIPs). The CBD negotiations originally focused on conservation alone. Soon,
however, the negotiators included national sovereignty to genetic resources and a threefold objectiveConservation, Sustainable, Access and Benefit Sharing (ABS)-to prevent unfair exploitation of the rich
genetic wealth and traditional knowledge by the developed countries. TRIPs agreement stimulates
technological innovation through harmonization and strengthening of domestic patent legislation by all
WTO members. The industrialized countries advocated it in order to ensure revenue from innovations in all
technological fields, including biotechnology. According to many studies a conflict relation exists between
CBD and TRIPs with regard to ABS, as the two regimes operate under different assumptions with regard to
property rights pertaining to genetic resources-hence, the two treaties further distinctly different
objectives. This paper explores the reason for the conflict relationship between the CBD and the TRIPs
agreement by reflecting the two theoretical perspectives: international political view and international
economic view.
25
N 3. Modulation of branchial CFTR expression in sea bream encountering abrupt salinity changes
Norman Y. S. Woo, Teresa W. S. Yuen
The Chinese University of Hong Kong
A cystic fibrosis transmembrane conductance regulator (CFTR) homolog has been identified in the
osmoregulatory organs, including gill and posterior intestine of the euryhaline sea bream, Sparussarba. The
CFTR gene is generally accepted to play important roles in chloride transport, the expression of which will
be crucial for survival of fish in different salinities. In order to test the importance of branchial CFTR in
salinity adaptation, sea bream were subjected to long-term acclimation to different salinities, ranging from
0 to 70 ppt for four weeks. The partial CFTR gene sequence obtained from the gills of sea bream (GenBank
Accession no. EF017215) was used to obtain specific oligonucleotides for detection of CFTR mRNA
expression in osmoregulatory organs. Sea bream acclimated extremely well to a wide salinity gradient (0 –
70 ppt salinity) with no change in branchial CFTR mRNA expression. Whether gill CFTR is relevant to rapid
salinity changes was tested through a series of experiments involving abrupt transfer of fish from seawater
(33 ppt) to hypo- osmotic environment (6 ppt) and vice versa. There was a transient decrease in branchial
CFTR mRNA expression 12 h after hypo-osmotic transfer, but the expression rapidly returned to pre-transfer
level 24 h after transfer. The response in the reverse transfer was different as transfer from hypo-osmotic
environment to seawater upregulatedbranchial CFTR mRNA, first observable 6 h post-transfer, and elevated
CFTR expression was maintained until 24 h after transfer. These experiments suggest a differential role of
CFTR in allowing euryhaline fish to adapt to abrupt hypo- or hyper-osmotic challenges. [This work was
supported by a Hong Kong Research Grants Council General Research Fund CUHK 477111]
N 4. Nitric oxide production during human hydatidosis: relationship with protein levels and cystic fertility
Razika Zeghir-Bouteldja1, ChafiaTouil-Boukoffa2
1
2
UAMOB, USTHB
Nitric oxide (NO) and its stable metabolites (NO2 –and NO3 −) have been identified as major effector
molecules during the majority of parasitic infections. Production of NO has been shown to be induced by
interferon gamma (IFN-γ) during human hydatidosis, suggesting the relevant role of NO in the host defense.
Human hydatidosis is an endemic parasitic disease caused by the larval stage of the tapeworm of
Echinococcusgranulosus. It constitutes a major health problem in North Africa, particularly in Algeria. This
parasitosis is characterized by a prolonged coexistence of Echinococcusgranulosus and its host without
effective rejection of the parasite. In this study, we investigated in vivo production of nitrite (NO2− + NO3−)
in sera of Algerian patients carrying different cyst locations. Nitrite (NO2− + NO3−) levels were evaluated by
the Griess method. Our results indicated that the levels of nitrite were significantly higher in the sera of
hydatic patients than those of healthy controls supporting the involvement of nitric oxide (NO) in
antihydatic action. The levels of nitrite in sera of the patients with hepatic hydatidosis were significantly
higher than those with pulmonary infection. The lower serum (NO2− + NO3−) levels were observed in the
relapsing cases. In addition, (NO2− +NO3−) levels of fertile hydatic fluids were significantly higher compared
to infertile fluids. Our results suggest that the presence of NO products in hydatic fluids seems to be related
to the location and the fertility of hydatic cysts. The assessment of protein concentration in hydatic fluids
showed that the concentration of proteins was not exclusively dependent on the fertility but on the cyst
locations.
26
N 5. In utero and lactational exposure to dioxin disrupts the reproductive function in female rat offspring
before and after puberty
Eman Elsharkawy, Neveen El-Nisr
Faculty of Medicine, Cardiology Department. Alexandria University Alexandria, EGYPT
To evaluate effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo-rho-dioxin (TCDD) exposure on the
reproductive function in female rat offspring, before and after puberty. The pregnant Sprague Dawely rat
administered 0, or 1.0 microg TCDD/kg on Gestation Day (GD) 8 and 15. Female offspring were examined at
the post-weanling before puberty on post natal day (PND) 21 and in young adult stage of development on
PND42. Ovulation assessment, radioimmunoassay for serum gonadotropins and steroids and histomorphmetric analysis to the ovaries were evaluated. The analysis included a count, measurement and
classification of preantral and antral follicles throughout the entire ovary on PND 21. The results indicate
that TCDD treatment significantly reduced the ovulation rate, serum gonadotropins and steroids levels and
the number of antral and preantral follicles of certain size classes. The histopathological examination
revealed small preovulatory follicles displaying an atretic morphologic difference among the ovaries of rats
exposed to TCDD treatments. These data support the hypothesis that TCDD results in adverse effects on
female reproductive function. However, the age of animals before or after puberty play an important role in
the difference between results. Moreover, TCDD exposure on the GD 8 or 15 has a great concern in the
results observed
N 6. A Cellular Automata Model to Predict Lung Cancer Risk Indcued Radon Progeny Alpha Particles
SamanehBaradaran, NiazMaleknas, SaeedSetayeshi, Mohammad Reza Kardan
Amirkabir University of Technology, Tehran, Islamic Republic of Iran
Exposure to Radon and its decay products is one of the important risks of ionizing radiation from natural
sources. It is the second leading cause of lung cancer after smoking in the world. This special characteristic
makes an increase in methods and models of lung cancer risk prediction from Radon. In this paper, we
present a stochastic cellular automaton based on sugarscape to computational study complex biological
effect of radon progeny alpha particles in lung bronchial airways. Our major objective is an assessment of
lung cancer risk by following mechanism of cell action in different radiation doses. The model included
mechanism of DNA damage induced alpha particles hits and formation of transformation in the lung cells.
To achieve our goal, we follow the metabolism rate of infected cell induced alpha particles traversals in
sugarscape environment to reach oncogenic transformation. For the first time, a cellular automata model is
used to calculate transformation frequency in lung bronchial airways induced Radon and to predict lung
cancer risk. The results are validated by a comparison epidemiological data, dosimetric and biological
models. It has been shown that the cellular automata using sugarscape model could be a suitable method
for cancer risk prediction.
27
Conviron is a global supplier of controlled environment systems offering an extensive product portfolio
including single and multi-tier chambers and rooms, research growth houses, and related services.
At Conviron, we have a very strong record of achievement and a deeply held conviction that our primary role is to support the work and
mission of our clients. We recognize the value of a complete solution for our clients and appreciate that each client has unique needs
and challenges. In keeping with our goal, we see our role as far more than a supplier of products. A Conviron client immediately
becomes a life-time partner of our company, and as your
partner, our role is to help you achieve your goals and vision,
and to support you throughout the life of your equipment.
Conviron’s expertise in assuming full responsibility for a
project – from project consultation to design, manufacturing,
installation, commissioning, training and finally to the on-going
support of the equipment for its entire operating life – allows
our clients to focus on their important research while leaving
the equipment details to us. Our entire team follows a set of
core values which includes an ongoing commitment to:
•
•
•
•
Integrity, professionalism and passion;
Teamwork;
Producing innovative, high quality, reliable products; and
Building client empathy and trust.
Whether your plans involve a start-up operation with limited
equipment or a large multi-chamber complex, with our broad
portfolio of products, configurable options, and customengineering capabilities, we can handle projects of any size
and complexity. And because such a vast majority of Conviron
projects involve some level of customization, we employ an
in-house team of nearly 35 engineers, technologists and
controls experts. This is why we often refer to ourselves as a
‘design firm with manufacturing capabilities’.
Conviron has secured a sophisticated international client base by providing a strong value proposition and competitive advantage – that
being true, fully-integrated services and a technical knowledgebase with expertise in environmental control. With the ongoing
commitment of continuous and significant investment in research and development to sustain our position of leadership, this business
model has helped Conviron establish a brand image defined by trust, reliability, integrity, innovation and value.
28
N 7. Natriuretic effect of cardiotonic drug Adenocin in isolated rat kidneys involves activation of the Na+K+-ATPase-Src kinase pathway
Galina Sukoyan1, Edisher Tsivtsivadze2, Pavel Galenko-Iaroshevsky3, Dmitry Ionov3
1
2
Scientific Research Institute of General Pathology andPathophysiology of Russian Academy of Medical Science; Biotechpharm GE,
3
Ltd; Kuban State Medical University
Adenocin is a original combined medicine containing clinically relevant beta- avetyldigoxin (BD), and are
now considered as endogenous steroid hormones. Binding of BD to Na+-K+-ATPase has been associated, in
kidney cells, to the activation of the Src kinase pathway and Na+-K+-ATPase internalization. Nevertheless,
whether the activation of this cascade also occurs with other cardiotonic steroids and leads to diuresis and
natriuresis in the isolated intact kidney is still unknown. In the present work, we perfused rat kidneys for
120 min with adenocin (0,1, 0,5, or 1 microM for BD) and measured its vascular and tubular effects. We
probed the effect of 10 microM 3-(4-chlorophenyl)1- (1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin4amine (PP2), a Src family kinase inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]
butadiene (UO126), a highly selective inhibitor of both MEK1 and MEK2, on adenocin- and BD-induced renal
alterations. Adenocin at 0,1, 0,5 and 1,0 microM profoundly increased several parameters of renal function
in a time- and/or concentration-dependent fashion. At a concentration that produced similar inhibition of
the rat kidney Na+-K+-ATPase, BD alone had a much smaller diuretic and natriuretic effect. Although
Adenocin fully inhibited the rat kidney Na+-K+-ATPase in vitro, was threefold higher than the concentration
used ex vivo and all its renal effects were blunted by PP2 and UO126. Furthermore, the phosphorylated
(activated) ERK1/2 expression was increased after Adenocin perfusion and this effect was totally prevented
after PP2 pretreatment. The present study shows for the first time the direct diuretic, natriuretic, and
kaliuretic effects of Adenocin in isolated rat kidney and the relevance of Na+-K+-ATPase mediated signal
transduction.
N 8. Type III plant polyketide synthases in Gerbera hybrida
Juha Kontturi
University of Helsinki
Plants produce a large class of secondary products which have both biological and pharmaceutical use.
Plant polyketide derived compounds are the largest and most diverse group of plant secondary metabolites.
Polyketides have an important role in plants acting as defense and antimicrobial agents. They also have an
essential role in medicine due to their activities as antimicrobial, antiparasitic, antineoplastic and
immunosuppressive compounds. Our research is focusing on functionally characterizing type III polyketide
synthases in the ornamental plant Gerbera hybrida. The research is focused on the cloning of genes to
expression vectors and the use of heterologous expression systems for production of the enzymes. After
successful cloning and transformation to production host, the proteins will be over expressed, isolated and
the tagged proteins will be purified. Finally enzymatic assays will be done using wide range of starter
substrates and HPLC based system will be set up for analyzing the produced compounds.
29
N 9. Pharmacology correction of Myocardial Infarction Mediating Inflammatory Responses and
Ventricular Remodeling in Experiments
Nikoloz Gongadze1, Tamar Kezeli2, Tamar Bochorishvili1, Nino Dolidze1
1
2
Tbilisi State Medical University, Javakhishvili Tbilisi State University
Objective—The goal of this study was to investigate the role of platelets in systemic and cardiac
inflammatory responses and the development of postinfarct ventricular complications, as well as the
efficacy of antiplatelet interventions. Methods and Results—Using a dog myocardial infarction (MI) model,
we determined platelet accumulation and severity of inflammation within the infarcted myocardium by
immunohistochemistry and biochemical assays, analyzed peripheral blood platelet-leukocyte conjugation
using flow cytometry, and tested antiplatelet interventions, including thienopyridines and platelet
depletion. Platelets accumulated within the infarcted region early post-MI and colocalized with
inflammatory cells. MI evoked early increase in circulating platelet-leukocyte conjugation mediated by Pselectin/P-selectin glycoprotein ligand-1. Antiplatelet interventions inhibited platelet-leukocyte conjugation
in peripheral blood, inflammatory infiltration, content of matrix metalloproteinases or plasminogen
activation, and expression of inflammatory mediators in the infarcted myocardium and lowered rupture
incidence. Nadcin therapy alleviated the extent of chronic ventricular dilatation by serial echocardiography.
Conclusion—Platelets play a pivotal role in promoting systemic and cardiac inflammatory responses postMI. Platelets accumulate within the infarcted myocardium, contributing to regional inflammation,
ventricular remodeling, and rupture. Treatment with Nadcin reduces the severity of inflammation and risk
of post-MI complications, demonstrating a previously unrecognized protective action.
N 10. Cross-regulation between the renin–angiotensin system and inflammatory mediators and efficacy
of therapy of chronic heart failure
Irina Kuzmina1, Donetskaya Olga2, Viktor Malikov3, Veronika Tulupova2,Margarita Fedorova2, Nataly
Shuldeshova2
1
2
3
N.V. Sklifosovsky Emergency Care Scientific-Research Institute, Clinic No 1, Moscow , Bakulev Research Centre of Cardiac Surgery
One of the major conceptual advances of the pathogenesis of congestive heart failure (CHF) has been the
insight that HF may progress as the result of the sustained overexpression of biologically active
‘‘neurohormones’’, which by virtue of their deleterious effects are sufficient to contribute to disease
progression by provoking worsening left ventricular (LV) remodeling and progressive LV dysfunction. A
second class of biologically active molecules, cytokines, has also been identified in the setting of CHF. The
overexpression of cytokines is sufficient to contribute to CHF progression. Although important interactions
between proinflammatory cytokines and the adrenergic system have been recognized in the heart, the
nature of the important interactions between proinflammatory cytokines and the renin–angiotensin system
(RAS) has become apparent only recently. Although speculative, one potential reason for the so-called
phenomenon of ‘‘neurohormonal escape’’, in which there is disease progression despite blockade of RAS,
may relate to the redundancy that exists between cross-regulated biological systems, such as the RAS and
proinflammatory cytokines. This statement notwithstanding it is also possible that a certain degree of crossregulation of these biologically active systems is necessary to maintain homeostasis. For example, excessive
blockade of cross- regulated biologically active systems, such as the RAS, the adrenergic system, endothelin
and inflammatory mediators, may explain some of the untoward outcomes of CHF. Thus efficacy therapeutic
action could be rich only under harmonize simultaneously action of multiple targets of CHF. According to
our clinical results one of the candidate for such treatment is cardioprotective drug Adenocin.
30
LGC Genomics
(www.lgcgenomics.com)
is the genomics division of the international science-based LGC Group. LGC Genomics has
recently merged with KBioscience, a UK-based technology company providing its own range of
SNP genotyping chemistry and novel instrumentation to the life science research and quality control
communities. The combined company now provides a full range of high quality genomics
products, services and solutions including, sample preparation (primarily, nucleic acid extraction),
nucleic acid sequencing, genotyping and biobanking. LGC Genomics has laboratories in the UK,
Germany and North America along with sales and support staff in over 15 locations in Europe,
America and the Far East.
The portfolio includes:
-
Genotyping services, assays and reagents
-
DNA sequencing services including next generation sequencing services on both the
Roche 454 & Illumina HiSeq2000 platforms
-
DNA and RNA extraction products and services
-
DNA/RNA biobanking including, storage, retrieval and analysis services
-
Reagents and consumables for molecular biology
-
Pharmacogenetics and diagnostic services
-
Laboratory instrumentation including plate sealers, liquid handling robots, highthroughput PCR thermocyclers, plate readers and a suite of laboratory automation
instruments
LGC (www.lgcgroup.com) is an international science-based company and market leader in
analytical, forensic and diagnostic services and reference standards. Operating internationally
through four divisions - LGC Forensics, LGC Genomics, LGC Standards and LGC Science &
Technology - the LGC Group employs 1,460 staff in 31 laboratories and centres across Europe as
well as in China, India and the US.
31
N 11. Synthesis of Novel Sulfanyl Aryl Azo Dyes as New Potent Tyrosinase Inhibitors
Hooshang Hamidian
Department of Chemistry, Payame Noor University (PNU), 19395-4697 Tehran, Iran
Six new sulfanyl aryl azo dyes (5a-5f) were prepared by diazotization of 4- aminohippuric acid, were coupled
with N, N-dimethylaniline, 1-naphthol and 2- naphthol, were condensed with 4-fluoro benzaldehyde or 4trifluoromethoxy benzaldehyde, then the compounds (4a-4f) reacted with 3,4-dithio-toluene. All
synthesized compounds (5a-5f) showed the most potent mushroom tyrosinase inhibition, comparable to
that of Kojic acid, as reference standard inhibitors. All the novel compounds were characterized by UV-Vis,
FT-IR, 1H NMR, 13C NMR, and elemental analysis.
N 12. Boletus edulis fraction exhibits antiproliferative and proapoptotic properties against colon cancer
cell lines
Marta Lemieszek1, Claudia Cardoso2, Fernando Nunes2, Ana Barros2, Piotr Pozarowski4, Wojciech Rzeski1,5
1
2
Department of Medical Biology, Institute of Agricultural Medicine,Jaczewskiego 2, 20-090 Lublin, Poland, CQ-Vila Real, Chemistry
3
Research Centre, Chemistry Department, University of Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal, CITAB - Centre
for the Research and Technology of Agro-Environment and Biological Sciences. Department of Agronomy, University of Trás-os4
Montes e Alto Douro, 5001-801 Vila Real, Portugal, Department of Clinical Immunology, Medical University, Chodźki 4A, 20-093
5
Lublin, Poland, Department of Virology and Immunology,UMCS, Akademicka 19, 20-033 Lublin, Poland
Edible mushrooms represent a valuable source of anticancer agents. Their application to cancer prevention
and treatment raises global interest because of high efficiency typed in multiple complex pharmacological
actions on different cellular and molecular targets with minimal unwanted side effects. An interesting and
promising source of the new anticancer compounds appear to be Boletus edulis. The presented study
proved anticancer potential of fractions isolated from B. edulis and describe the molecular mechanisms of
their chemopreventive activity in colon carcinoma cells. The antiproliferative activity of B. edulis fractions
was screened by MTT and BrdU assay in human colon carcinoma cells. Fraction with the greatest
therapeutic potential named BE3 was examined in detail. Flow cytometry and Western blotting were
applied to cell cycle analysis and protein expression involved in selected coumpaund anticancer activity.
Commercial kits were used to verified fraction ability to induce apoptosis. Fraction BE3 inhibited cancer cell
proliferation which was accompanied with cell cycle arrest in G0/G1 phase in case of LS180 cells, and in Sphase in case of HT- 29 cells. Growth inhibition was closely associated with modulation of cell cycle key
regulatory proteins expression. Further more, investigated fraction induced apoptosis in colon cancer cells.
Our results indicate that BE3 fraction possessantiproliferative and proapoptotic properties against colon
cancer cell lines. Obtained positive results will provide a rationele for future development of dietary
supplements as well as healthy dietary habits which could be beneficial in colon cancer chemoprevention.
This work was supported by the National Science Centre, Grant Number 2011/01/M/NZ7/02691.
32
N 13. Synthesis of silver nanoparticles using simulated microgravity grown K. pneumoniae culture filtrate
it’s antibacterial activity
Duraisamy Kalpana1, Ryu Su Min1 , Yang Soo Lee1,
1
2
Department of Forest Science and Technology, Chonbuk National University, Department of Commerce and Trade, Chonbuk
National University
K. pneumoniae was grown under modeled microgravity and the cultural filtrate was effectively used to
synthesize silver nanoparticles using silver nitrate salt solution as substrate. Silver nanoparticles were
successfully synthesized at the ratios of 3:7 and 4:6 of cultural filtrate and silver nitrate salt solution
respectively. Highly uniform and concentrated silver nanoparticles were produced at 4:6 ratio of cultural
filtrate and silver nitrate salt solution. Surface plasmon absorbance at 405-407 nm confirmed the synthesis
of silver nanoparticles. The size of silver nanoparticles were measured between 15-37 nm and found to
possess spherical shape by TEM analysis. The crystalline nature of synthesized silver nanoparticles was
studied using XRD analysis and XRD pattern was obtained corresponding to the planes (1 1 1), (2 0 0), (2 2
0), (3 1 1). The protein biomolecules present in the culture filtrate was found to stabilize the silver
nanoparticles as observed from FTIR analysis. Anitbactericidal activity was found to present in the
biosynthesized silver nanoparticles against Gram negative Escherichia coli and Salmonella enterica and
moderate antibacterial activity was seen against Gram positive Streptococcus pyogenes.
N 14. Melanoidins isolated from heated potato fiber (potex) affect human colon cancer cells growth via
modulation of cell cycle
Ewa Langner, Fernando M. Nunes, PiotrPożarowski, MartynaKandefer-Szerszeń, Stefan G. Pierzynowski,
WojciechRzeski
Department of Virology and Immunology, Maria Curie-Skłodowska University, Lublin, Poland; Department of Medical Biology,
Institute of Agricultural Medicine, Lublin, Poland; Department of Cell and Organism Biology, Lund University, Lund, Sweden
Dietary fiber serves as a nonnutritive element of regular diet proposed to maintain human health,
particularly in respect of colorectal diseases. Among others, potato fiber preparations (i.e. Potex), prepared
from potato cell wall material are broadly used in food industry. Heat processing of foods containing Potex is
very common, and this may lead to melanoidins formation from the potato fiber preparation due to its high
polysaccharides and proteins content. Our goal was to verify whether both heated potato fiber extract and
melanoidins isolated from the extract exerts growth-inhibiting activity in human colon cancer cells in vitro.
Methods and results: Model LS180 human colon adenocarcinoma cell line was used in the study. The cells
were tested upon treatment with roasted potato fiber extract (AM4) as well as with high (HMW) and low
(LMW) molecular weight fractions isolated from the extract. The activity of aforementioned compounds to
inhibit colon cancer cells growth was observed both at cellular (measured by MTT method) and molecular
level. Roasted potato fiber extract induced alternations in cell cycle regulatory proteins i.e. cyclin D1,
CDK4/6, p21, p27 and p53 and arrested cells in G0 phase of the cell cycle. Moreover, LMW compounds
revealed markedly stronger potential to alter specific molecular targets comparing to HMW compounds.
Conclusion: The results emphasize that both high and low molecular weight melanoidins contribute to
growth inhibiting activity of heated potato fiber preparation in LS180 colon cancer cells in vitro. This work
was supported by Grant 2011/01/N/NZ5/04242 from National Science Center, Poland.
33
N 15. Proliferation and harvest of human mesenchymal stem cells using new thermoresponsive nanocomposite gels
Kazutoshi Haraguchi, Noriko Kotobuki
Kawamura Institute of Chemical Research
For tissue engineering and regenerative medicine, stem cells should be effectively cultured in vitro. New
thermoresponsivenanocomposite gels (MD-NC gels), consisting of inorganic clay (hectorite) and copolymers
composed of hydrophobic 2-methoxyethyl acrylate (MEA) and hydrophilic N,N-dimethylacrylamide (DMAA)
units, could be applied in cell culture and cell harvesting without trypsinization, specifically using
mesenchymal stem cells (MSCs). The composition of the MD-NC gel (the ratio of the two monomer types
and the clay content) was found to determine its swelling properties in the culture medium,
thermosensitivity, protein adsorption, and cell attachment and proliferation. Various kinds of human cells,
including MSCs, osteoblast (HOS) cells, fibroblast (NHDF) cells, and epithelial cells could be effectively
cultured on MD-NC gels. In particular, on an MD10-NC2 gel with relatively low DMAA and clay content, the
cells could be harvested by decreasing the temperature, either as a cell sheet (MSCs or NHDF cells) or as a
population of suspension cells (HOS cells). Further, it was found that the MD10- NC2 gel is suitable for stem
cell differentiation. Because of their thermosensitivity, controllable modulus, and surface properties, MDNC gels are promising cell culture substrates useful for tissue engineering and regenerative medicine. (1) J.
Biomedical Mater. Res.A in press (2012). (2) J. Biomaterials Sci., 22, 2389 (2011) (3) Biomacromolecules, 7,
3267 (2006)
N 16. Reverse Virtual Screening and Molecular Docking Approach in Studies on OxindolePentacyclic
Alkaloids of Uncariatomentosa
Pawel Kozielewicz1, Katarzyna Paradowska1, Mire Zloh2
1
2
Department of Physical Chemistry, Medical University of Warsaw, Department of Pharmaceutical and Biological Chemistry, UCL
School of Pharmacy
Uncariatomentosa (eng. Cat’s claw) is a liana that grows in Latin America. Extract from this plant has been
reported to cause apoptosis in cancer cells. Pentacyclicoxindolestereoisomeric alkaloids are the agents
responsible for this effect, yet their mechanism of action remains unknown. In this work 6 alkaloids:
isomitraphylline, isopteropodine, mitraphylline, pteropodine, speciophylline and uncarine F were studied
on. The aim of this work is to examine whether reverse screening and molecular docking can explain
mechanism of action of biomolecules with limited experimental data Reverse screening with TarFisDock and
ReverseScreen3D servers was used to find potential macromolecular targets. 54 proteins were selected for
docking based on the results of screening. In the next step the alkaloids were docked into these targets
using AutoDock. Among various macromolecules, Protein Kinase C alpha has been shown to be the most
likely target for the alkaloids. This finding was based on both binding energies and the correlation between
activity and docking scores. The proposed interaction mechanism established by docking is that alkaloids
anchor to the binding pocket of PKC alpha and create hydrogen bonds with Leu345 and Val420. The
hydrophobic interactions are exhibited between ligands’ atoms and Phe350, Val353 and Met470. Other
proteins, like dihydrofolatereductase or Protein Kinase B may also be involved in pro-apoptotic action of the
studied ligands. It was established that the alkaloids interact with specific amino acids in the binding sites of
the proteins. These findings support the opinion that reverse screening and molecular docking are valuable
tools in studies of biologically active compounds. However, the results obtained in this study have to be
verified by flexible docking and in vitro studies.
34
35
N 17. Co-expression of Neoagarobiose Transporter and Neoagarobiose Hydrolase in E. coli
Hye Ran Jang, Sun Bok Lee
Department of Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang 790-784, Korea
Agarose, composed of D-galactose and 3,6-anhydro-L-galactose (L-AHG), is hydrolyzed to neoagarooligosaccharide by beta-agarase catalyzed depolymerization. Neoagarobiose (NAB), a disaccharide produced
by beta- agarase hydrolysis, has linear structure composed of D-galactose and L-AHG. E.coli can utilize Dgalactose for growth; however, it can neither transport nor utilize NAB into cells. In this study, we newly
constructed NAB-utilizing E. coli by inserting NAB transporter and NAB hydrolase genes, both isolated from
marine Pseudomonas sp. Due to toxicity of transporter overexpression, the expression of two genes was
controlled by using different cloning vectors which have different expression levels. NAB transporter was
cloned into the low copy vector for reducing expression levels, whereas NAB hydrolase was cloned into the
high copy vector and overexpressed for NAB hydrolysis. The expression level of NAB transporter, which is
fused with GFP protein to detect membrane proteins in E. coli, was directly monitored by a fluorescence
microscope. Constructed E. coli with two genes was expressed in LB medium containing NAB as a substrate.
Substrate changes were observed using TLC and HPLC analysis of fermentation broth. The experimental data
obtained in this work indicate that NAB was successfully transported into E. coli and that the D-galactose
produced by NAB hydrolase was utilized for cell growth.
N 18. Multiple Structural Isomers of 3,6-Anhydro-L-galactose
Shin Yup Lee, Hyun Seung Lim, Sun Bok Lee
Department of Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang 790-784, Korea
3,6-Anhydro-L-galactose (L-AHG) is accounted for approximately half of the components of agarose, which is
polymer of L-AHG and D-galactose. It is characterized by including a bridged bicyclic system containing fused
five- and six-membered rings, unlike any other sugars found in the nature. Considering the structural and
chemical properties of L-AHG, there are possibilities of being used in various ways. However, so far, only a
few studies have been done about the chemical characteristics and structures of L-AHG. In particular,
currently revealed information on the biological function or metabolic pathway of L-AHG is insufficient. We
firstly utilized acid or enzymatic treatment to produce L-AHG from agarose, and then separated them by
adsorption chromatography. With the results of the TLC and HPLC analysis, we have found that L-AHG
contains multiple kinds of structures; we suggest those preferred structures as open- alpha, open-beta,
closed-alpha and closed-beta. We have also observed the preferred structures exist in a certain ratio and it
could be altered based on the environment of pH and temperature. The results have shown that L-AHG
prefers open forms in the acid condition and low temperature and closed forms in the alkaline condition
and high temperature. In order to understand which form of L- AHG mainly utilized in biological system, we
conducted enzyme-substrate docking simulation with L-AHG dehydrogenase whose structure was recently
revealed. From the calculation results, open forms of L-AHG showed lower binding energy and shorter
distance between substrate L-AHG and cofactor NAD+ than closed forms of L-AHG. This result means that LAHG dehydrogenase prefers open forms of L-AHG rather than closed forms. These researches are expected
to be used to understand how L-AHG is metabolized in vivo, and to produce various L- AHG derivatives.
36
N 19. Synthesis and anti-inflammatory activity of some 1, 2, 3-benzotriazine derivatives using 2-(4-oxo-6phenylbenzo] d [ [1, 2, 3[triazine -3(4, 4)-y1) acetohydrazide as a starting material
Yasser Selim
Faculty of Specific Education, Zagazig University, Zagazig, Egypt
In continuation to our search for new heterocyclic system based anti-inflammatory, the suggestion and
synthesis of some 1, 2, 3-Benzotriazine derivatives, were herein realizing. The pharmacological screening
showed that many of these compounds have good anti-inflammatory activity. The structure assignments of
the new compounds are based on chemical and spectroscopic data and pharmacological properties are
reported.
N 20. Development of cardiac specific troponin Spiegelmer
Zsuzsanna Szeitner1, Szilvia Nagy1, Gergely Lautner2, Robert Gyurcsanyi2, Tamas Meszaros1
1
2
Semmelweis University Department of Medical Chemistry, MolecularBiology and Pathobiochemistry, Department of Inorganic and
Analytical Chemistry, BudapestUniversity of Technology and Economics
Cardiac specific troponins (cTnI and cTnT) are amongst the most preferred diagnostic biomarkers of
myocardial infarction; therefore, several methods have been developed for detection of cTnI and cTnT.
Although diverse technical approaches are applied in the presently available troponin detecting devices,
their selectivity always relies on application of specific antibodies. We aimed at producing cTnI specific
Spiegelmers to provide alternative receptors for biosensor development. These nuclease resistant
oligonucleotide ligands can recognize the target molecule with high selectivity and affinity, which makes
them attractive molecules for in vivo application. A stable, 9 amino acid composed domain of cTnI was
chosen as selection target and synthesized from D-amino acids. The peptide was immobilized on magnetic
beads, challenged with the initial pool of random DNA sequences and selection of candidate
oligonucleotides were carried out by the SELEX procedure. The selection cycle was repeated nine times with
gradually decreasing peptid concentration and more vigorous washing conditions to increase the affinity of
oligonucleotides. The selectivity of oligonucleotides was further enhanced by negative-selection steps steps.
According to the sequencing result, the selection procedure resulted in isolation of few oligonucleotides.
The L-oligonucleotide variant of the most abundant sequence was synthesized and its selectivity and
binding characteristics was examined with AlphaScreen, surface plasmon resonance and fluorescence
anisotropy techniques. The obtained data confirmed the success of selection procedure, since the studied
Spiegelmer selectively binds to cTnI with a dissociation constant in the nanomolar range.
37
N 21. Associations of polymorphic variants of VDR FokI, cyp2R1 and cyp24A1 genes with of bone turnover
parameters and related cardiovascular disease.
Joanna Sajkowska, Jacek Lukaszkiewicz
Department of Biochemistry and Clinical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Poland
Vitamin D plays an important role in regulating plasma, calcium and phosphate homeostasis and, according
to current reports, has a significant impact on the circulatory system. The main aims of this study were to
assess the frequency of the nuclear receptor of vitamin D (VDR) gene polymorphisms FokI and two enzymes
which are involved in the metabolism of vitamin D – cyp2R1 and cyp24A1 among Polish women (n=154) and
to search for correlations between these polymorphic variants and bone turnover parameters and related
cardiovascular disease. DNA was purified from whole blood samples using the automatic technique on
magnetic particles. Genetic polymorphisms were determined by Real-time polymerase chain reaction (PCR).
It was demonstrated that a number of dependencies exist between polymorphic variants of the genes and
pathological phenomena in the body, which are the development of obesity and vitamin D deficiency. The
newly discovered link between energy metabolism and bone metabolism was supported by present
findings. It was demonstrated that the polymorphisms VDR FokI and cyp2R1 do not have a simple impact on
values of systolic blood pressure, diastolic blood pressure and the heartrate. The results also point to the
alarmingly low levels of vitamin D among Polish women (the concentration of 25(OH)D3 was below 20
ng/ml in 68% subjects), which as shown in this study, may be associated with weight gain and an increase in
diastolic blood pressure. This research is in line with current aspirations to develop the future routine
molecular diagnostics, and therefore take action to implement prevention and treatment for each patient
(on his “genetic measure”) and may represent a broad introduction to the study of individual metabolic
profiles of vitamin D.
N 22. Circumventing the misparing in bispecific antibodies
Justyna Iwaszkiewicz1, Vincent Zoete1, Olivier Michielin1, Jean-Pierre Mach2
1
2
Swiss Institute of Bioinformatics, University of Lausanne
The bispecific antibodies are a popular tool of cancer immunotherapy offering much higher effector
functions than traditional monoclonal antibodies. One of the problems in the bispecific antibody production
is the mispairing of the light and heavy chains of the different antibody specificity. The objective of this
study was to propose the complementary mutations at the interface between the constant domains of the
light (CL) and heavy chain (CH1) of the bispecific antibody that would lead to a proper pairing of chains
sharing the same antibody specificity. The modification spots were chosen using with computational tools:
Designer and FoldX programs and MM-GBSA method. The efficiency of mutations was tested by comparison
of free energy of interaction of properly paired modified light and heavy chains with the mispaired ones
using MM-GBSA method. The most promising sets of modifications were chosen and tested experimentally.
38
SYSTAT Software GmbH
Systat Software Inc.(SSI), headquartered in San Jose, California, is a leading provider of specialized
software and solutions for the scientific community. Systat Software GmbH, Erkrath, Germany handles
European operations of Systat Software Inc. UK operations are handled from Systat Software Inc UK
Branch, London.
Products
• SigmaPlot - graphing and statistical analysis software
• SigmaStat - permanently integrated into SigmaPlot with Version 11
• SYSTAT – powerful statistical analysis and graphics software package
• SigmaScan Pro – image analysis
• TableCurve 2D – automated curve fitting
• TableCurve 3D – automated surface fitting
• PeakFit – automated peak separation and analysis
• AutoSignal – automated signal analysis
• SigmaCERF – collaborative electronic notebook system
• Enzyme Kinetics Module – enzyme kinetics analysis, now integrated in SigmaPlot
• Electrophysiology Module – SigmaPlot add-on, for direct import of electrophysiology data
Experience
• Systat Software GmbH, established in the European market for more than 20 years now,
provides specialized scientific software products and services for life sciences, environmental
sciences, physical sciences, biomedical & pharmaceutical research, medical research, and
engineering
• More than 300.000 users worldwide, including government organizations, academic institutions
and leading corporations
For more information, please contact
39
N 23. Precise targeting, knock out and replacement of asdA gene of Salmonella typhimurium by λ REDmediated recombination
Ke ZHANG, Bin YU, Bo-jian ZHENG
Microbiology Department, the University of Hong Kong
In order to construct a heterologous antigen delivery vehicle, the λ RED- mediated recombination method
was applied to precisely target, knock out and replace the asdA gene from a Salmonella typhimurium (S.
typhimurium) named S129. Briefly, the asdA gene sequence of S. yphimurium and kanamycin resistence
gene sequence of plasmid pL452 were used as template to design PCR primers. Forward primer (FP)
sequentially contained a SalI digesting site, 5’ beginning open reading frame (ORF) of asdA and 5’ beginning
ORF of kanamycin resistence gene of pL452. Reverse primer (RP) sequentially included 3’ end ORF of
kanamycin resistence gene of pL452, 3’ end ORF of asdA and a SalI digesting site. PCR was conducted by the
FP and RP primers and the PCR product which contained the SalI enzyme site, partial asdA beginning ORF,
the whole kanamycin resistence gene, partial asdA end ORF and SalI enzyme site was generated as linear
dsDNA. The λ RED proteins (exo, beta and gum) expression plasmid pSim6 was electroporated into S129.
The pSim6-S129 was cultured in 32 degrees and OD600 value was required to 0.3 from the subculture
pSim6-S129 in a 1/50 (v/v). Culture the OD 0.3 pSim6- S129 in 42 degrees with15 minutes to induce the exo,
beta and gum expression, then apply cold sterilized deionized water to wash twice. Mix 50ul of this cell
pellet with 150ng PCR product that contained the SalI enzyme site, asdA sequences and whole kanamycin
resistence gene, then electroporate the mixture. The survival cells were confirmed by a asdA mutant
phenotype--2,6- diaminopimelic acid (DAP) growth dependent manner and dramatically virulence
decreasing in Balb/C mice challenge. The further experiments demonstrated the asdA mutant S129 which
anchored pIRES-eGFP could express eGFP in in vitro/in vivo infected Caco-2 cells and chicks.
N 24. Pyrrole-based mimics of LxxLL-like binding motif with antiproliferative activity
Marco Persico1, Anna Ramunno1, Vita Maglio1, Silvia Franceschelli1, Alfonso Carotenuto1, Diego
Brancaccio1, Nausicaa Orteca1, Luigi Michele Pavone2, Ettore Novellino1, Caterina Fattorusso1
1
2
Department of Pharmacy - University of Naples "Federico II" (Italy), Department of Biochemistry and Medical Biotechnologies University of Naples "Federico II" (Italy)
A recent and promising approach for the development of new anticancer agents is targeting key proteinprotein interactions involved in the regulation of the cell cycle. Typically, small molecules able to modulate
such interactions target the so-called “hot spots”, sub-regions endowed with a functional and structural
adaptivity [1]. It was reported that a particular spatial orientation of three hydrophobic moieties on a small
molecular scaffold is able to mime the hydrophobic side chains of the interacting residues of LxxLL
sequence, a conserved binding motifs that is widely used in transcriptional regulation [2]. In order to
identify new anticancer leads, we synthesized a small set of pyrrole-based mimics of this α-helical secondary
structure. All compounds were evaluated for their antiproliferative activity against several cancer cell lines
(MCF7, Huh7, M14, Jurkat), as well as, on mouse monocyte macrophages (Raw) cell line. Three of them
showed a selective cytotoxicity against M14 melanoma cell line at low micromolar concentration. Although
biological results suggested the involvement of p53 in their anticancer mechanism of action, NMR studies
were not able to detect any inhibition of the p53/MDM2 complex. With the aim of determining the
molecular mechanism of action of the new derivatives, 3-D pharmacophore models were generated and
used to perform 3-D searches in molecular databases. Overall, our findings indicate the ability of our
compounds to mime hydrophobic residues present in LxxLL-like and LxxLLxxL protein recognition motifs,
paving the way for the rational design of new optimized analogues. [1] Wells, J. A et al Nature. 2007, 450,
1001-1009. [2] Ferlini, C. et al Cancer Res 2009, 69, OF1-OF9.
40
N 25. Microscale, in vivo biodiscovery: Using zebrafish to identify neuroactive secondary metabolites
Alexander D. Crawford1, Soura Challal2, Nadine Bohni2, Olivia E. Buenafe3, Danielle Copmans3, Emerson
Ferreira-Queiroz2, Camila V. Esguerra3, Peter de Witte3, Jean-Luc Wolfender2
1
2
Chemical Biology Group, Luxembourg Centre for Systems Biomedicine, Esch-sur-Alzette, Luxembourg, Chemical Biology Group,
3
Luxembourg Centre for Systems Biomedicine, Esch-sur-Alzette, Luxembourg, Laboratory for Molecular Biodiscovery, Universiteit
Leuven, Leuven, Belgium
Zebrafish are increasingly accepted as a powerful in vivo system to identify and functionally analyze diseaserelevant genes and bioactive small molecules. We have recently established zebrafish as a platform for in
vivo bioassay-guided natural product (NP) discovery. Zebrafish offer the possibility of rapid in vivo
bioactivity analysis of small molecules at the microgram scale - an attractive feature for NP discovery when
combined with high-resolution fractionation technologies and analytical methods such as UHPLC-TOFMS
and microflow NMR. Numerous biomedically relevant assays are now available in zebrafish, encompassing
most indication areas. Our primary focus is on the discovery and functional elucidation of neuroactive NPs
using high-throughput zebrafish assays for the analysis of behavior and in vivo neural activity. Our
laboratories have recently combined advanced analytical methods with high-content screening in zebrafish
to create an integrated platform for microgram-scale, in vivo NP discovery. The key advantages of this
approach are (1) the microgram scale at which both biological and analytical experiments can be
performed; (2) the ability of UHPLC-TOFMS and microflow NMR data to enable dereplication early in the NP
discovery process; and (3) the ability to systematically perform NP discovery using in vivo assays, thereby
enabling the identification of NPs with novel bioactivities that to date could not be readily found using in
vitro screens. Together with new methods such as behavioral fingerprinting in zebrafish, we are also using
this in vivo NP discovery platform to pursue the rapid functional characterization of neuroactive secondary
metabolites.
N 26. Binding of Pregnane X receptor mutants to DR3 motif of human CYP3A4 gene promoter with the
use of nonradioactive electrophoretic mobility shift assay (EMSA)
Aneta Vavrova1, Aneta Novotna1, Radim Vrzal1,Petr Pavek2, Zdenek Dvorak1
1
Department of Cell Biology and Genetics, Faculty of Science,Palacky University, Slechtitelu 11, 783 71 Olomouc, Czech Republic,
Department of Pharmacology and Toxicology, Charles University inPrague, Faculty of Pharmacy in Hradec Kralove, Heyrovskeho
1203, Hradec Kralove 50005, Czech Republic
2
The pregnane X receptor (PXR) is a key xenobiotic receptor that regulates expression of numerous drugmetabolizing enzymes. Some posttranslational mechanisms modulate its transcriptional activity. In the
present work we examined T248, Y249 and T422 putative phosphorylation sites determined based on in
silico consensus kinase site prediction analysis. Site-directed mutagenesis was performed to generate
phospho-deficient and phospho-mimetic mutants. We examined binding of PXR mutants to the CYP3A4
gene response element DR3 by nonradioactive electrophoretic mobility shift assay (EMSA). To determine
the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting PXR-DNA interaction,
nuclear extracts from Hela cells transfected by PXR mutants or in vitro translated proteins (reticulocyte
lysate) were used for binding reaction. Double-stranded 5’-biotinylated oligonucleotides containing specific
DNA-binding sequence for PXR (DR3 motif from the XREM sequence of CYP3A4 gene promoter) were used
as probes. The results showed that the ability to bind to DNA in vitro was substantially decreased in case of
T248D, T422D and T248V mutants [1]. Moreover, we found that in vitro translation is not suitable source of
PXR protein for EMSA analysis compared to nuclear extracts due to the presence of biotinylated proteins
background in reticulocyte lysate. Acknowledgements: Our laboratories are supported by the grants from
the Czech Scientific Agency GACR 303/12/G163 and 303/12/0472. 1. Doricakova, A., et al., The role of residues
T248, Y249 and T422 in the function of human pregnane X receptor. Archives of toxicology, 2012.
41
N 27. Highly stable A-switch from artificial nucleic acid
Vipin Kumar Badarya, Venkitasamy Kesavan
Department of biotechnology, indian institute of technology, Madras (iitm), india
The self assembling property of nucleic acid through Watson-Crick hydrogen bonding in a precise manner
make these macromolecules in demand for constructing nanodevices [1]. These nanodevices are made up
of unusual structure of DNA motifs called i-motif, G-quadruplux and A-motif. Molecular properties of poly
r(A) were explored by Watson and Crick through X-ray studies [2]. Further it was utilized to develop A-switch
[3]. Here it is important to note that protons are the external stimuli for A-switch, therefore chemical
integrity is always questionable under acidic condition. There is a need to construct an artificial nucleic acid
which should be chemically more stable under drastic conditions with superior function. Herein, we report
a synthesis and physical studies of novel chiral artificial nucleic acid called Butyl Nucleic Acid (BNA). To
demonstrate its property as an A-switch, a stretch of poly (A) of BNA was studied by CD and UV melting
under neutral and acidic conditions. CD melting studies reveals that under neutral condition poly (A) was
weakly organized structure, but at low pH it gained highly organized and rigid structure. Based on pH
titration it was found that BNA based A-switch has conformational transition at pH 4.9 and its structural
conformation was highly reversible with acid-base change. In order to understand the physical stability of
BNA based A-motif, we studied the melting transition at pH range (2 to 11), which illustrates that BNA based
A-motif is more stable than DNA based A-motif. Since, BNA does not suffer from depurination,
thermodynamic parameters were calculated by melting curves. This is the first report where artificial
nucleic acid has been used to develop A-switch. References [1] Simmel, F. C et. al., Angew. Chem. Int. Ed.
2011, 50, 3124; [2] A. Rich et. al., J. Mol. Biol. 1961, 3, 71; [3] Y. Krishnan et. al., Nucleic Acids Res. 2009, 37,
2810.
N 28. Identification and Characterization of L-fuconatedehydratase from Chromohalobactersalexigens
DSM 3043
Jeong Ah Kim, Sun Bok Lee
Department of Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang 790-784, Korea
Seaweed is a third generation biomass and can be used as an alternative resource of fossil fuels and
chemicals. Compared to crops and wood, land-based biomass resources, seaweed is easy to grow and has
high productivity. Nevertheless it is difficult to use as the industrial resource, due to the unknown metabolic
pathway of seaweed-derived monosaccharides. L-fucose is a deoxy sugar that is enriched in some fractions
of seaweed biomass, such as fucoidan. Fucoidan is a sulfated polysaccharide found mainly in various species
of brown algae such as Laminariadigitata, Fucusvesiculosus and Ascophyllumnodosum. After hydrolysis of
this polysaccharide, it is separated into L-fucose. Some microorganisms are known to metabolize L-fucose
via oxidative pathway. They convert L-fucose to L-fuconate, and then L-fuconate is converted to 2-keto-3deoxy-L-fuconate by L-fuconatedehydratase. Chromohalobactersalexigens DSM 3043 is a marine gramnegative bacterium. The genome of C. salexigens contains four COG4948 aldonic-acid gene clusters and one
of four enzymes is Csal_1748. In this study, we have identified a gene encoding L-fuconatedehydratase
(Csal_1748). Csal_1748 was cloned and expressed in E. coli BL21 (DE3) and the recombinant protein showed
high reactivity toward L-fuconate (100%). The enzyme also showed reactivity toward D-arabonate (18.7%),
D-xylonate (17.8%), L-arabonate (17.0%), D-gluconate (12.2%), D-mannonate (11.1%) and D- galactonate
(6.8%). Our experimental data indicate that Csal_1748 has broad substrate specificity and may be employed
for the metabolism of various seaweed-derived monosaccharides.
42
N 29. 4-Hydroxy-5-Methylcoumarin biosynthesis in Gerbera hybrida
Juha Kontturi, Milla Pietiäinen, Teemu Teeri
University of Helsinki
Plants produce a large class of secondary products which have both biological and pharmaceutical
properties. Plant polyketide derived compounds are the largest and most diverse group of plant secondary
metabolites. Polyketides have an important role in plants acting as defense and antimicrobial agents. They
also have an essential role in medicine due to their activities as antimicrobial, antiparasitic and
immunosuppressive compounds. Our research is focusing on functionally characterizing type III polyketide
synthases in the ornamental plant Gerbera hybrida. The research is focused on coumarin biosynthesis.
Gerbera is known to contain 4- Hydroxy-5-Methylcoumarin which has been shown to have antifungal
properties. The biosynthesis of the coumarin is still unclear. Previous research indicates that it is derived
from the acetate-malonate pathway. The model suggests that the enzyme uses acetyl-CoA and four
malonyl-CoA molecules to produce a pentaketide which is further reduced by another enzyme to finally
produce the chemical of interest. The right protein itself remains still unknown but most likely it comes from
the already known type III polyketide synthases in Gerbera. We are using heterologous expression systems
for protein production and enzyme assays to screen different starter CoA:s. Finally the products are
analyzed using HPLC.
N 30. Fluorescent detection of DNA using new cyanine dyes LO
Yuliia Didan1, Valentina Negrutska2, Dmytro Kryvorotenko2, Igor Dubey2
1
2
Taras Shevchenko National University of Kyiv, Institute of Molecular Biology and Genetics of the National Academy of Sciences of
Ukraine
Fluorescent dyes are widely used in various biomedical applications. A series of new cyanine dyes LO
(Lepidine Orange derivatives), structural analogues of the classic intercalating cyanine dye Thiazole Orange,
have been investigated as fluorescent stains for detecting DNA in agarose gels. Electrophoretic mobility shift
assay was applied to characterize the binding of LO to DNA. LO dyes form highly fluorescent stable
complexes with nucleic acids. Decrease of DNA electrophoretic mobility was observed upon increasing the
dye/base pair ratio (dbpr) in pre-stained DNA samples up to 0.05, and the bands started to become diffuse
when the dbpr reached a value above 0.2. It may indicate the appearance of partially denatured DNA. LO-7
dye was found to be well-suited for high sensitivity DNA detection. The minimum amount of DNA detected
with laser gel scanner (with excitation wavelength 488 nm) is about 80 pg, and about 0.3 ng of DNA can be
detected in the gel using standard UV-translluminator. This sensitivity is at least 3 times higher than can be
achieved with ethidium bromide under the same conditions. Moreover, this sensitivity is comparable to that
of the commercial SYBR Green I DNA staining dye. The minimum amount of DNA that can be seen in SYBR
Green I–stained gel with UV- transillumination is 0.15 ng. The fluorescence of LO-7–DNA complexes
depends on DNA amount in the probe. Fluorescence intensity enhancement is observed upon DNA quantity
increase from 0.5 to 12.5 ng per band, and above 25 ng the emission remains stable. LO-7 allows the
quantitation of 0.5 to 4 ng DNA as in this range the fluorescence-concentration dependence is linear. SYBR
Green I has similar fluorescence–DNA quantity relationship profile. In conclusion, new easily available
cyanine dye LO-7 can be used for the detection of DNA in electrophoretic gels.
43
N 31. TI2BioP: Topological Indices to Biopolymers. Its practical use to unravel cryptic bacteriocin-like
domains
Guillermin Aguero-Chapin1, Gisselle Perez-Machado2, Reinaldo Molina-Ruiz2, Yunierkis Perez-Castillo2,
Aliuska Morales-Helguera2, Vitor Vasconcelos1, Agostinho Antunes1
1
2
CIIMAR, University of Porto, Portugal, Molecular Simulation and Drug Design (CBQ), Central University of Las Villas, Santa Clara,
54830, Cuba
Bacteriocins are proteinaceous toxins produced and exported by both gram-negative and gram-positive
bacteria as a defense mechanism. The bacteriocin protein family is highly diverse, which complicates the
identification of bacteriocin-like sequences using alignment approaches. The use of topological indices (TIs)
irrespective of sequence similarity can be a promising alternative to predict proteinaceous bacteriocins.
Thus, we present TI2BioP (Topological Indices to BioPolymers) as an alignment-free approach inspired in
both the TOPS-MODE (Topological Substructural Molecular Design) and MARCH-INSIDE (Markov Chain
Invariants for Network Selection & Design) methodology. TI2BioP allows the calculation of the spectral
moments as simple TIs to seek Quantitative Sequence-Function Relationships (QSFR) models. Since
hydrophobicity and basicity are major criteria for the bactericide activity of bacteriocins, the spectral
moments (HPμk) were derived for the first time from protein artificial secondary structures based on amino
acid clustering into a Cartesian system of hydrophobicity and polarity. Several orders of HPμk characterized
numerically 196 bacteriocin-like sequences and a control group made up of 200 representative CATH
domains. Subsequently, they were used to develop an alignment-free QSFR model allowing a 76.92%
discrimination of bacteriocin proteins from other domains. The model showed a prediction overall
performance of 72.16%, detecting specifically 66.7% of proteinaceous bacteriocins whereas the
InterProScan retrieved just 60.2%.
44
Weiss Technik UK
Weiss Technik UK has over 45 years’ experience in the design, manufacture and supply of Fitotron®
Plant Growth Chambers and Rooms, supplying over 85 countries world-wide.
Products:
• Fitotron® Standard Growth Chambers for Plant Growth, Arabidopsis, Insect Storage, Insect
Incubation, Seed Storage, Seed Germination, Tissue Culture and Constant Temperature.
• Fitotron® LED Plant Growth Chambers with research and production lighting modules.
• Fitotron® High Specification Plant Growth Chambers.
• Fitotron® High Specification Plant Growth Rooms.
• Fitotron® Multi-tier Cultivation Rooms.
For more information, please contact:
Weiss Technik UK Ltd
Außenbüro Königswinter
Bergstraße 35
53639 Königswinter, Germany
Tel: +49 2223 90 3305
Email: [email protected]
www.fitotron.de
Weiss Technik UK Ltd
Units 37-38 The Technology Centre
Epinal Way
Loughborough, Leicestershire
LE11 3GE, United Kingdom
Tel: +44 (0)1509 631590
Email: [email protected]
www.weiss-uk.com
45
List of Poster Presentations
Poster number. Author and title
Page in
abstract book
Poster № 1. Sven Stadlbauer. Phosphoinositides (PIPs) as probes for studying and targeting
the metastasis-promoting phosphatase PRL-3
25
Poster № 2. EunMi Kim. Analyzing the regulations on agricultural genetic resources
25
Poster № 3. Norman Y. S. Woo. Modulation of branchial CFTR expression in sea bream
encountering abrupt salinity changes
26
Poster № 4. Razika Zeghir-Bouteldja. Nitric oxide production during human hydatidosis:
relationship with protein levels and cystic fertility
26
Poster № 5. Eman Elsharkawy. In utero and lactational exposure to dioxin disrupts the
reproductive function in female rat offspring before and after puberty
27
Poster № 6. SamanehBaradaran. A Cellular Automata Model to Predict Lung Cancer Risk
Indcued Radon Progeny Alpha Particles
27
Poster № 7. Galina Sukoyan. Natriuretic effect of cardiotonic drug Adenocin in isolated rat
kidneys involves activation of the Na+-K+-ATPase-Src kinase pathway
29
Poster № 8. Juha Kontturi. Type III plant polyketide synthases in Gerbera hybrida
29
Poster № 9. Nikoloz Gongadze. Pharmacology correction of Myocardial Infarction
Mediating Inflammatory Responses and Ventricular Remodeling in Experiments
30
Poster № 10. Irina Kuzmina. Cross-regulation between the renin–angiotensin system and
inflammatory mediators and efficacy of therapy of chronic heart failure
30
Poster № 11. Hooshang Hamidian. Synthesis of Novel Sulfanyl Aryl Azo Dyes as New
Potent Tyrosinase Inhibitors
32
Poster № 12. Marta Lemieszek. Boletus edulis fraction exhibits antiproliferative and
proapoptotic properties against colon cancer cell lines
32
Poster № 13. Yang Soo Lee. Synthesis of silver nanoparticles using simulated microgravity
grown K. pneumoniae culture filtrate it’s antibacterial activity
33
Poster № 14. Ewa Langner. Melanoidins isolated from heated potato fiber (potex) affect
human colon cancer cells growth via modulation of cell cycle
33
Poster № 15. Kazutoshi Haraguchi. Proliferation and harvest of human mesenchymal stem
cells using new thermoresponsivenanocomposite gels
34
46
Poster № 16. Pawel Kozielewicz. Reverse Virtual Screening and Molecular Docking
Approach in Studies on OxindolePentacyclic Alkaloids of Uncariatomentosa
34
Poster № 17. Hye Ran Jang. Co-expression of Neoagarobiose Transporter and
Neoagarobiose Hydrolase in E. coli
36
Poster № 18. Hyun Seung Lim. Multiple Structural Isomers of 3,6-Anhydro-L-galactose
36
Poster № 19. Yasser Selim. Synthesis and anti-inflammatory activity of some 1, 2, 3benzotriazine derivatives using 2-(4-oxo-6-phenylbenzo] d [ [1, 2, 3[triazine -3(4, 4)-y1)
acetohydrazide as a starting material
37
Poster № 20. Zsuzsanna Szeitner. Development of cardiac specific troponin Spiegelmer
37
Poster № 21. Joanna Sajkowska. Associations of polymorphic variants of VDR FokI, cyp2R1
and cyp24A1 genes with of bone turnover parameters and related cardiovascular disease.
38
Poster № 22. Justyna Iwaszkiewicz. Circumventing the misparing in bispecific antibodies
38
Poster № 23. Ke ZHANG. Precise targeting, knock out and replacement of asdA gene of
Salmonella typhimurium by λ RED-mediated recombination
40
Poster № 24. Anna Ramunno. Pyrrole-based mimics of LxxLL-like binding motif with
antiproliferative activity
40
Poster № 25. Alexander D. Crawford. Microscale, in vivo biodiscovery: Using zebrafish to
identify neuroactive secondary metabolites
41
Poster № 26. Aneta Novotna. Binding of Pregnane X receptor mutants to DR3 motif of
human CYP3A4 gene promoter with the use of nonradioactive electrophoretic mobility
shift assay (EMSA)
41
Poster № 27. Vipin Kumar Badarya. Highly stable A-switch from artificial nucleic acid
42
Poster № 28. Jeong Ah Kim. Identification and Characterization of L-fuconatedehydratase
from Chromohalobactersalexigens DSM 3043
42
Poster № 29. Juha Kontturi. 4-Hydroxy-5-Methylcoumarin biosynthesis in Gerbera hybrida
43
Poster № 30. Yuliia Didan. Fluorescent detection of DNA using new cyanine dyes LO
43
Poster № 31. Agostinho Antunes. TI2BioP: Topological Indices to Biopolymers. Its practical
use to unravel cryptic bacteriocin-like domains
44
47
48
List of Participants
Antunes Agostinho
[email protected]
Atanassova Ana
Bayer CropScience NV
[email protected]
Athoumani Daroueche
Direction Nationale De L,Industrie
Union Des Comoros
[email protected]
Attipoe William Elikem
EASTBANK High School
[email protected]
Bajorath Jürgen
University of Bonn
[email protected]
Bradley Mark
University of Edinburgh
[email protected]
Crawford Alexander
University of Luxembourg
[email protected]
en.be
[email protected]
Crews Craig
Yale University
[email protected]
[email protected]
Dell Anne
Imperial College London
[email protected]
Deprez Benoit
U761 Biostructures and Drug
Discovery, Institut Pasteur de Lille
[email protected]
Didan Yuliia
[email protected]
Dvorak Zdenek
[email protected]
Famulok Michael
Rheinische Friedrich-Wilhelms
Universität Bonn / LIMES-Institut
[email protected]
Filipuzzi Ireos
Novartis NIBR
[email protected]
Gerwick Lena
University Of California San Diego
[email protected]
Haraguchi Kazutoshi
Kawamura Institute Of Chemical
Research
[email protected]
Hoffmann Carsten
University of Wuerzburg
[email protected]
Imaeda Takashi
Kyowa Hakko Kirin
[email protected]
Itävaara Merja
Vtt
[email protected]
Jang Hye Ran
Department Of Chemical
Engineering, POSTECH
[email protected]
Kim Ki Hyun
[email protected]
Kim Jeong Ah
Department Of Chemical
Engineering, Pohang University Of
Science And Technology
[email protected]
Kim Eun Mi
Chonbuk National University
[email protected]
Kontturi Juha
University Of Helsinki
[email protected]
Kwon Ho Jeong
Yonsei University
[email protected]
Lee Yang Soo
Chonbuk National University
[email protected]
Lim Hyun Seung
[email protected]
Marechal Eric
CNRS-CEA Grenoble
[email protected]
Marx Andreas
University Konstanz
[email protected]
McHagama Fatima
Direction Nationale De L,Industrie
Union Des Comoros
[email protected]
Merchant Andrew
University Of Sydney
[email protected]
Mészáros Tamás
Semmelweis University,
Department Of Medical Chemistry
[email protected]
Mohamed Echat
Direction Nationale De L,Industrie
Union Des Comoros
[email protected]
Ocasio Cory Antonio (Tony)
University Of Sussex
[email protected]
Panuschka Claudia
Springer Verlag
[email protected]
Patterson Lisa Marcaurelle
H3 Biomdicine
[email protected]
49
Ramunno Anna
Department of Pharmacy-University
of Salerno (Italy)
[email protected]
Razika Zeghir -Bouteldja
[email protected]
Rognan Didier
VMR 7200 CNRS - University of
Strasburg
[email protected]
Russinova Jenny
Vib
[email protected]
Specht Alexandre
UMR 7199 CWRS/UDS
[email protected]
Spichal Lukas
Institute of Experimental Botany, AS
CR
[email protected]
Stadlbauer Sven
European Molecular Biology
Laboratory
[email protected]
Szeitner Zsuzsanna
Semmelweis University,
Department Of Medical Chrmistry
[email protected]
eis
Thinon Emmanuelle
[email protected]
Urano Yasuteru
The University of Tokyo
[email protected]
Vavrova Aneta
[email protected]
Vrzal Radim
[email protected]
Woscholski Rüdiger
Imperial College London
[email protected]
Zanders Edward
Pharmaguide Ltd.
[email protected]
Ziegler Slava
Max-Planck-Institute Of Molecular
Physiology
[email protected]
50