Science for you bulletin by scienceforyou

SCIENCE FOR YOU

synthetic molecules plants

biology

technology

genetics

pcr

transdifferentiation expression

receptors

qtl

lipids

histone

listeriolysin

infection

interactions

nevrons protein

yeast

bacteria stem cell

industry

bioinformatics

purification

NOVEMBER 2015

cancer

medicine

cells

science

biotechnology

phospholipase

biofilm

sequence

dna

inteins

research

Science for You [editors Vesna Kokondoska Grgič, Saša Rezelj]; [graphical design Larisa Tomšič]; [proofreading Petar Jankuloski]; [photography Irenej Šraml] 1st issue – Ljubljana – 2015 Circulation: 600 Free issue Copyright © 2015, Biotehnološko študentsko društvo, Ljubljana ISSN 2463-7742

Science for you, November 2015

PREFACE Nowadays, there is an enormous amount of data produced every day in life-science. Thus, one has to spend a lot of time searching for the right one. As students, we are bored of consuming knowledge in classrooms, we don’t know where else could we use this knowledge except for the exams. While searching in vastly amount of science articles is even more confusing. Being aware of those problems, we decided to make “Science for You” bulletin which comprises the results of the latest research in various fields of science (from medicine to IT). It is composed in such a way that anybody who is interested in life-science can find his topic. Furthermore, students can see the possibilities and get the inspiration for their future career. This bulletin also gives an inside of how different fields of science are connected to each other and only such connections bring the best research results. In addition, integration between disciplines is also important for international networking and sharing of knowledge. “Science for you” can be obtained via the website of the non-profit center for sharing knowledge ScienceMIX (www.science-mix.com). Further guidance documents are already published on this website. We hope this bulletin will be an inspiration for students and reaserchers and will inspire new connections for even more sucessful colaborations for future science research.

Vesna Kokondoska Grgič & Saša Rezelj

Sci

f e c en

ou y or

Science for you, November 2015

DNA & PROTEINS 4

Saša Rezelj and colleagues: Molecular Interactions

Urban Bezeljak: Inteins

10 Daniela Dukovska: The CD94/NKG2 “short” (hi)story

Tamara Šutuš Temovski: Association of genetic variants in 13 promoter region of DEPTOR gene with obesity and insulin resistance development 14 Jane Spirkoski: Pathways of deposition of H3.3 into MSC

chromatin

PLANT BIOTECHNOLOGY 27 Branka Javornik and colleagues: Presentation

of work at the Centre for Plant Biotechnology

Science for you, November 2015

CELLS Vesna Kokondoska Grgič: Transdifferentiation of 16 canine mesenchymal stem cells into nevrons Mojca Jež and colleagues: Stem cells in advanced cell 19 therapies at Blood Transfusion Centre of Slovenia Jernej Horvat: Development of an efficient feeding- 22 strategy for an industrial yeast strain Marina Pekmezović: Detection and image analysis of 25 clinical fungal biofilms

YOUTH & ENTREPRENEURSHIP Luka Ausec: Bioinformatics – Beginners’ 31 guide Sciencemix 33 Larisa Lara Tomšič: Non- formal science 34 Sabrina Lakotta: A young scientist’s 35 journey

DNA & PROTEINS: Molecular interactions

MOLECULAR INTERACTONS S R , aša

Laboratory

ezelj and colleagues for

Molecular Biology

and

Nanobiotechnology, National Institute

Chemistry Slovenia

In the Laboratory for Molecular Biology and Nanobiotechnology, we are performing top level research of biological processes, focusing on understanding the mechanism of proteins action and molecular interactions. We create new basic knowledge as well as introduce modern methodologies in the field of Life Sciences. We develop applications for solving genuine problems in biotechnology and the pharmaceutical industry, particularly in the development of biological drugs. Laboratory L11 is remarkably equipped with six smaller laboratories for the following research activities: Molecular Biology, Production and Purification of Proteins, Biophysical Characterization of Proteins, Crystallization of Biological Molecules, Cell Biology. We are open for research cooperation and offer students to make their diplomas in our laboratories. Contact: Head of the Laboratory Prof. Dr. Gregor Anderluh: gregor.anderluh@ki.si Research in the Laboratory for Molecular Biology and Nanobiotechnology is performed within the frames of the “Molecular Interactions” Program Group, including the following topics:

Integrity of lipid membranes Lipid membranes are crucial for normal cellular function. They enable selective transfer of molecules and protect the content of cells against environmental stress. We study peptides and proteins that damage lipid membranes via formation of transmembrane pores. Organisms can use these proteins as mode of attack or as defense tools that enable the immune system to remove unwanted or damaged cells. Understanding of the membrane-damaging processes at the molecular level represents an important basis for the development of new anti-microbial substances as well as a design of new strategies for the uptake of biological drugs by cells. Furthermore, pore-forming proteins can also be used as efficient molecular tools to study structure and function of cellular membranes. In our studies, we mainly focus on proteins from the MACPF/CDC superfamily, e.g. perforin from the immune system and listeriolysin O (LLO) from bacterium Listeria monocytogenes, as well as proteins similar to sea anemone toxins (actinoporines).

Fig.1: Giant unilamellar vesicles (GUVs) are used as a model membrane system to study membrane - protein interactions. Pore – forming protein LLO was added to GUVs (membranes are colored red due to rhodamine). Fluorescent dextrans (green) are in the surrounding solution. If the dextrans are small enough to flow through the pores then the inside of GUVs also becomes green (left part).

References:

Gilbert RJ, Dalla Serra M, Froelich CJ, Wallace MI, Anderluh G (2014) Membrane pore formation at protein-lipid interfaces. Trends Biochem Sci. 39: 510-516. Gilbert RJ, Mikelj M, Dalla Serra M, Froelich CJ, Anderluh G (2013) Effects of MACPF/CDC proteins on lipid membranes. Cell Mol Life Sci. 70:2083-2098. Praper T, Sonnen AF, Kladnik A, Andrighetti AO, Viero G, Morris KJ, Volpi E, Lunelli L, Dalla Serra M, Froelich CJ, Gilbert RJ, Anderluh G (2011) Perforin activity at membranes leads to invaginations and vesicle formation. Proc Natl Acad Sci U S A. 108: 21016-21021. Bakrač B, Kladnik A, Maček P, McHaffie G, Werner A, Lakey JH, Anderluh G (2010) A toxin-based probe reveals cytoplasmic exposure of Golgi sphingomyelin. Journal of Biological Chemistry 285: 22186-22195.

Mechanism of action of proteins from intracellular bacteria and their potential role in pathogenesis Studies of molecular mechanisms of infection and development of diseases are ultimately necessary in order to successfully design new efficient drugs. In the case of bacterial pathogenesis, our research interest is mainly focused on the host cell signaling following the bacterial invasion. We have been studying proteins involved in cyclic nucleotide mediated cell signaling, localized either in the cytosol or cellular envelope of Mycobacteria, as well as cytotoxic bacterial proteins (e.g. from Listeria), that form pores in the host cell membranes. We study the interactomes of these proteins, which helps us understand their biological roles. In addition, we also look for new potential virulence factors from these bacteria. Molecules of interest are characterized at genetic, bioinformatic, biochemical, biophysical and structural level, and biological effects of these proteins are also tested on mammalian cell cultures.

Science for you, November 2015

References:

Podobnik M, Siddiqui N, Rebolj K, Nambi S, Merzel F, Visweswariah SS. (2014) Allostery and conformational dynamics in cAMP binding acyltransferases. J Biol Chem 289: 16588-16600. Matange N, Podobnik M, Visweswariah SS (2014) The non-catalytic “cap domain” of a mycobacterial metallophosphoesterase regulates its expression and localization in the cell. J Biol Chem 289: 22470-22481 Podobnik M, Marchioretto M, Zanetti M, Bavdek A, Kisovec M, Cajnko MM, Lunelli L, Dalla Serra M, Anderluh G (2015) Plasticity of Lysteriolysin O Pores and its Regulation by pH and Unique Histidine. Sci Rep, Apr 8;5:9623.

Nanobiotechnology and development of new biological drugs Recombinant biological drugs are one of the fastest developing areas of the pharmaceutical industry. Our research group is involved in the development of protein therapeutics at several developmental levels. Up to now, we have prepared many production clones of E. coli, P. pastoris and A. niger, helped in optimization of bioproduction processes, developed protocols for purification and biophysical characterization of protein products as well as protocols for their biochemical modification (pegylation, lipophilization etc.) in order to achieve the desired features of these molecules in the relevant biological environments. For this purpose, we have also developed several cell models for quantification of in vitro potency as well as epithelial models for studying trans-epithelial delivery of therapeutics in various (nano) formulations. In addition to work on recombinant therapeutic proteins, we have also established a baculovirus expression system and procedures for preparation of virus-like particles based on recombinant proteins for development of model vaccines.

Structural and functional characterisation of listeriolysin o Y406A mutant Saša Rezelj, Matic Kisovec, Marjetka Podobnik, Gregor Anderluh Listeriolysin O (LLO) is the most important virulence factor of the intracellular bacterium Listeria monocytogenes, which causes the foodborne disease listeriosis. LLO is a member of the family of cholesterol dependent cytolysins (CDCs), which form transmembrane pores in target cells. LLO enables bacteria to escape from the host phagolysosome via rapture of its membrane. Furthermore, it is also involved in the spread and reproduction of bacteria inside host cells, thereby leaving the host plasma membrane intact. LLO is unique among CDCs in its pH-dependent stability. It is stable at acidic pH and body temperature but at neutral pH and temperatures over 30°C its activity is reduced. In a heterologous expression system, we have produced and characterized the LLO Y406A mutant. We discovered that LLO Y406A has a different elution volume on gel filtration chromatography compared to LLO wild-type (WT) and they display significantly different elution volumes at different pH values (Fig. 2).

References:

Cegnar, Mateja, Podobnik, Barbara, Gaberc-Porekar, Vladka, Novak Štagoj, Mateja, Jalen, Špela, Komel, Radovan, Caserman, Simon. Erythropoietin conjugates having oral bioavailability: international publication number: WO 2015/032981 A1: international publication date: 12 March 2015 international application number: PCT/EP2014/069198. München: World Intellectual Property Organization, 2015. 27 pp. Kraševec, Nada, Milulnović, Tatjana, Anžur-Lasnik, Marija, Lukančič, Irena, Komel, Radovan, Gaberc-Porekar, Vladka. Human granulocyte colony stimulating factor (G-CSF) produced in the filamentous fungus Aspergillus niger. Acta chimica slovenica, ISSN 1318-0207. Marušić, Maja, Zupančič, Tina, Heibar, Gorazd, Komel Radovan, Anderluh, Gregor, Caserman, Simon. The Caco-2 cell culture model enables sensitive detection of enhanced protein permeability in the presence of N-decyl-[beta]-d-maltopyranoside. New biotechnology, ISSN 18716784, 25 Jun. 2013, vol. 30, iss. 5, pp. 507-515.

Fig. 2: Gel filtration chromatographs

DNA & PROTEINS: Molecular interactions

Interestingly, LLO Y406A was able to bind to the cholesterol-containing membranes in acidic and alkaline environment. Moreover, if LLO Y406A was bound at alkaline pH, when LLO Y406A pore formation is inhibited, the mutant could be (re-) activated upon acidification of the solution. We showed this by the surface plasmon resonance method (SPR), where large unilamellar vesicles (LUVs) containing fluorescent calcein were immobilized to the chip surface. We collected the fractions at each step (Fig. 4) and measured fluorescence upon calcein release from vesicles.

Fig. 3: Hemolytic activity (1/protein concentration at half of maximal hemolytic velocity) is shown in columns for proteins LLO WT and LLO Y406A at different pH values. Higher column means better hemolytic activity.

It is very interesting that both eluted out of the column later than what is expected for proteins of this size (e.g. perfringolysin O (PFO WT), MW 56 kDa). This indicates that the mutation probably effects the conformation of the protein or changes the interactions between the protein and gel matrix. A special feature of LLO Y406A is that it shows normal hemolytic activity (red blood cell lysis) at acidic pH, whereas the activity is almost lost at neutral pH (Fig. 3).

Fig. 4: Fluorescent signal of the fractions collected on SPR machine at each step. LUVs containing calcein inside were bound to chip surface with running buffer pH 8.0. Negative control is the injection of buffer pH 5.7 prior to protein injection. The remaining LUVs, which weren’t perforated due to protein activity at pH 8.0 (LLO WT) or after acidification with buffer pH 5.7 (LLO Y406A), released the calcein after detergent injection.

LLO Y406A showed its pore forming activity to be pH dependent. This unique feature that allows regulation of the protein’s activity by pH, makes LLO Y406A very appealing for pharmaceutical and biotechnological applications. For this reason we patented this mutant: Anderluh, Gregor, Podobnik, Marjetka, Rezelj, Saša, Kisovec, Matic, Cajnko, Miša Mojca. Slovenian patent application ‘OD pH ODVISEN MUTANT LISTERIOLIZINA O’, Ljubljana: Urad Republike Slovenije za intelektualno lastnino, 2014. Reference:

Rezelj, S. 2014. Structural and functional characterization of listeriolysin O Y406A mutant. Master thesis, Ljubljana, University in Ljubljana, Biotechnical faculty: 101 pp.

Fig. 5: Photos from Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry Slovenia

Science for you, November 2015

INTEINS , urban bezeljak Laboratory

Biotechnology, National Institute

Chemistry Slovenia

The National Institute of Chemistry in Ljubljana is one of the leading research institutions in Slovenia. The institute’s fourteen laboratories cover wide range of chemistry-related fields, from inorganic chemistry to biotechnology and life sciences. For the past three years, I have been working as an intern in the Laboratory of Biotechnology, a renowned laboratory with diverse interests in immunology, microbiology and the up-and-coming field of Synthetic Biology, where we try to apply engineering principles to construct and assemble novel useful biological parts from existing natural or completely synthetic repertoire of macromolecules. The field’s ambition is to drive next big (synbio) revolution that will tackle the challenges of tomorrow: from pollution and agriculture to medicine and new materials. My master’s thesis project, under the supervision of Prof. Dr. Roman Jerala, was focused on an interesting group of proteins, called inteins. These peptides, found in some unicellular organisms, have a unique ability to catalyze protein splicing, a process similar to intron splicing during the maturation of mRNA molecules. We set off to better characterize these fascinating peptides and to improve the selectivity of the splicing reaction in mammalian cell cultures by introducing point mutations into selected intein moieties.

Introduction Inteins are self-processing parasitic protein domains that catalyze their own post-translational excision from the host proteins and subsequent ligation of the two peptide fragments (called exteins) into reconstituted and fully functional host protein. This particular feature makes inteins a valuable tool in protein engineering, molecular biology and synthetic biology. We distinguish two kinds of protein splicing: a continuous intein domain catalyzes its excision from its host in cis-splicing reaction, whereas split inteins can reconstitute protein fragments that are transcribed from different genes in protein transsplicing (Figure 1). The first example of protein splicing with intein domains was discovered 25 years ago in baker’s yeast Saccharomyces cerevisiae (1). To date, several hundred inteins were discovered in all three domains domains of life – archaea, bacteria and eukarya. Their origin is still shrouded in mystery, however it is assumed that they share

a common ancestor with other regulatory selfprocessing proteins (2). Inteins are regarded as parasitic genetic elements that invade genes of different proteins and get transcribed as well as translated alongside their hosts (exteins). After the protein synthesis, the parasitic intein domain excises itself from the rest of protein and joins the ends of the extein, which regains its function as if nothing had happened. Researchers around the globe utilize these peptides to purify proteins of interest without any additional tags, to perform protein semi-synthesis by joining different peptide fragments or ligating various modifiers to the protein of interest in vitro and in vivo (3). Inteins have also been proven a valuable tool in NMR spectroscopy, where they allow to segmentally label larger proteins with 15N for easier spectra interpretation (3). Lastly, inteins can be used for protein cyclization to produce circular proteins with enhanced stability and to control protein activity with conditional protein splicing (3). In the latter application, splicing in trans- is controlled by an outside signal – small

Figure 1: a schematic representation of protein splicing in cis- (A) and in trans- (B).

DNA & PROTEINS: Inteins

molecule, reducing agent, light, temperature and pH value (4). By splitting a protein of interest in two with adjoined split inteins, proteinâ&#x20AC;&#x2122;s activity can be carefully regulated with presence or absence of the stimuli, thus promising faster responses than classical biosensors that require signal transduction, induction of transcription and translation. The biggest challenge for conditional protein splicing to overcome is its selectivity: how to reduce unwanted reactions in the absence of inducing signal. Recently, researchers had used artificially split inteins that, unlike naturally occurring split inteins, exhibit reduced affinity between the two halves. However, unspecific splicing remains significantly over background levels (5, 6). During our study, we prepared new mutants of commonly used split inteins and compared their selectivity against their unmodified counterparts. Results We prepared mutants of commonly used artificially split Sce VMA intein from Saccharomyces cerevisiae and naturally split intein Npu DnaE from cyanobacteria Nostoc punctiforme. To monitor protein splicing in HEK293T human cell line, we used a proved experimental design (6), where we fused the N- and C-intein coding sequence to N- and C-fragments of split firefly luciferase gene (fLuc), respectively, alongside with coiled-coil domains that provided selectivity for the dimerization of the protein constructs. The point mutations introduced into Sce VMA N-intein caused reversal of electrostatic charge on selected amino acid residues (K30E, R44E, K53E) and caused the N- and C-intein domains to repel each other. This led to reduced protein trans-splicing when we transfected the cells with constructs without complementary coiledcoil pairs. The splicing was detected by measuring the enzymatic activity of the reconstituted firefly luciferase with luminometer (Figure 2). Although the splicing reaction was not very efficient compared to constitutive production of active luciferase with degradation tag, the newly produced intein mutant boosted the intensity of the measured signal when we used complementary coiled-coils, and, at the same time, increased the selectivity of the splicing reaction. To confirm that the luciferase activity was due to protein splicing, we prepared the same constructs with inactive inteins (C1A and N246A;

VMA*) and observed reduced levels of signal intensity, hence confirming the measured luciferase activity as a result of protein splicing.

Figure 2: mutated VMA N-intein increases selectivity of protein splicing in HEK293T cells, measured 2 days after transfection. When we transfected constructs with compatible coiled-coil pairs (RR-EE), the level of protein splicing with mutated N-intein domain VMANmut remained high, whereas when we combined constructs with coiled-coils with lower affinity (EE-EE), the mutated VMAN contributed to lower levels of unwanted splicing reaction.

Figure 3: point mutations in DnaE N-intein domain have little effect on splicing specificity in HEK293T cells, measured 2 days after transfection.

Science for you, November 2015

Similar experiments were conducted with highly active and notoriously unspecific naturally split intein Npu DnaE (Figure 3). We used two mutated versions of N-intein domain with different degrees of mutated residues (DnaENmut1: E54K, E61K, D85K, E91K and DnaENmut2: E7K, E52K, E54K, E61K, D85K, E89K, E91K, respectively). However, despite the larger number of mutated residues, the introduced differences had little effect on specificity of Npu DnaE splicing. With increasing number of point mutations in N-intein domain, the efficiency of splicing decreased and specificity only slightly improved as revealed by reconstituted firefly luciferase activity.

the split intein. In conclusion, highly efficient and specific conditional protein splicing in transcould provide new biosensors that would rapidly detect splicing-inducing molecules or conditions in vivo and in vitro or advanced therapeutics, which would switch into active form precisely at the desired time or location.

Discussion Conditional protein splicing is one of the most exciting possible applications of inteins as it allows directly controlling the structure and activity of a desired protein by splitting it into two fragments and then reconstituting the two halves rapidly and dependably exactly when and where we want them to. We still seek the ideal split intein that would direct conditional trans-splicing only when the induction signal is present. The artificially split inteins as Sce VMA exhibit less intrinsic affinity between the split domains, but some background splicing activity nevertheless remains. By introducing three reverse charge point mutations, we successfully increased both fidelity and specificity of firefly luciferase reconstitution with Sce VMA split intein. However, naturally split inteins are far faster and more efficient catalysts of trans-splicing and are thus more desired parts for conditional splicing implementations. Inteins like Npu DnaE associate freely when there is no induction signal and are widely regarded as incompatible with conditional protein splicing. Here, we tried to increase the splicing specificity by introducing several point mutations into DnaE N-intein with very limited success. Despite marginally decreasing the splicing efficiency in conditions where the constructs should not form dimers, the mutations also caused an overall reduction in intein catalytic activity. Other methods to decrease the intrinsic affinity between N- and C-intein domains that causes low specificity may include changing the size of N- and C- intein in favour of one or the other, shortening the highly basic regions on Nintein domain that electrostatically interact with the C-intein or performing directed evolution of

5 X. Huang et al., DNA Cell Biol. 31 Suppl 1, S2–10 (2012).

References:

1 P. M. Kane et al., Science. 250, 651–657 (1990). 2 T. M. Tanaka Hall et al., Cell. 91, 85–97 (1997). 3 N. H. Shah, T. W. Muir, Chem. Sci. 5, 446–461 (2014). 4 N. I. Topilina, K. V Mills, Mob. DNA. 5, 5 (2014).

6 D. F. Selgrade, J. J. Lohmueller, F. Lienert, P. a Silver, J. Am. Chem. Soc. 135, 7713–7719 (2013).

DNA & PROTEINS: The CD94/NKG2 “short” (hi)story

THE CD94 /NKG2 »SHORT« (HI)STORY Daniela Dukovska, National Center

for biotechnology-CSIC

Madrid, Spain

Abstract NK cells were, for a long time, thought to be regulated by the ‘missing-self’ hypothesis (Ljunggren and Karre. 1990). This theory, proposed that NK cells were constitutively active, but inhibited via inhibiting receptors and their interaction with MHC class I. However, eventually it was discovered that NK cells also express activating receptors, leading to a new paradigm of homeostasis where any given cell is controlled by a balance of both inhibiting and activating signals. The NKG2 family is one of the multiple families of immune receptors encoded in the human genome that contains members with activating and inhibitory potential. These receptors are mainly expressed on NK cells and subsets of CD8+ T cells and have been shown to play an important role in regulating immune responses against infected and tumor cells. This review looks over the current knowledge about this family of receptors, including ligand and receptor interaction as well as their roles in disease regulation, infections, and cancer. CD94/NKG2 receptors CD94/NKG2 receptors are expressed by a majority of NK cells and on subsets of CD8+ T cells. Structurally, they belong to the C-type lectin-like receptor superfamily. With the exception of the orphan NKG2F receptor and the homodimer NKG2D receptor, the other five NKG2 molecules (NKG2A, B, C, E, and H) have been shown to form disulfide-linked heterodimers with an invariant chain, CD94. The basic structure of these proteins is that of an intracellular N-terminal domain, a transmembrane domain and an extracellular carbohydrate-recognition domain, which is the ligand binding part of the molecule (Adamkiewicz et al. 1994). NKG2A and its splice variant NKG2B have been shown to possess two immunoreceptor tyrosine based inhibitory motif (ITIMs) in their cytoplasmic domains. NKG2E and -H, which are products of alternatively spliced pre-mRNA from a single gene, and NKG2C molecule lack ITIMs, but instead had a positively charged residue within the transmem-

brane region that allows association with the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor molecule DAP12 (Lanier et al. 1998). CD94/NKG2A-C receptors recognize and bind a non-classical major histocompatibility class (MHC) Ib molecule. In humans, these class Ib antigen is human leukocyte antigen (HLA)-E. HLA-E is widely distributed among various tissues, exhibit relatively low surface expression, and have limited polymorphism. Like other MHC class I molecules, HLA-E consist of three noncovalently bound components: a heavy chain, a β2 – macroglobulin, and a nanomeric peptide epitope, which usually is a peptide derived from the signal sequences of MHC class I molecules (O’Callaghan et al. 1998). HLA-E may also bind different non MHC class I derived peptide sequences. For example, HLA-E bound to a peptide derived from the ATP-binding cassette transporter multidrug resistance-associated protein 7 (MRP7) is recognized by CD94/NKG2A and inhibits NK cell activity (Wooden et al. 2005), while a signal peptide derived from heat shock protein 60 complexed with HLA-E (Michaelsson et al. 2002), or a peptide from the Epstein– Barr protein BZLF-1 (Brooks et al. 1999), are not. Finally, in addition to the recognition of HLA-E by CD94/NKG2 receptors, CD8+ T cells have been shown to recognize target cells in an HLA-E (Salerno-Goncalves et al. 2004; Hoare et al. 2006) restricted, T cell receptor dependent manner. Given the relevance of the CD94/NKG2 receptors in modulating NK and T cell responses, it is not surprising that expression of this family of receptors, and their ligands, play a role in the development of certain diseases. In the case of infectious disease like HCMV infections, NK cells play an important role in the immune control of the virus. However, the HCMV is smart virus that has reciprocally developed a series of strategies for immune evasion. One such strategy is directed toward affecting antigen presenta-

Science for you, November 2015

tion, including the reduction of levels of class I molecules on the surface of infected cells and thus avoiding CTL attack (Tortorella et al. 2000) but allowing susceptibility to NK cell recognition and killing. Consequently, HCMV has also developed an alternative way to escape NK cells by encoding a set of proteins one, UL40 giving a signal peptide that can preserve HLA-E expression at the cell surface of the infected cells in this manner protecting them from the NKG2A+ NK cell killing (Tomasec et al. 2000; Ulbrecht et al. 2000; Wang et al. 2002). Appositely, clinical studies have shown that there is a positive correlation between a HCMV infection and an increased proportion of NK and T cells expressing the CD94/NKG2C activating receptor, suggesting that HCMV infection may shape the NK cell receptor repertoire (Guma et al. 2004). In fact, co-culture of PBL from HCMV+ donors with HCMV infected fibroblasts leads to a preferential expansion of CD94/NKG2C+ NK cells by a mechanism that probably involves the interaction of the CD94/NKG2C receptor with the infected cell. Moreover, studies have shown that differentiation and expansion of the NKG2Cbright subset in response to HCMV is encompassed by profound changes in the NK-cell compartment, with potentially broader implications in immunity to other pathogens, tumors and allogeneic cells, as well as in the development of some inflammatory disorders (Muntasell et al. 2013) and (Waggoner et al. 2011). The expression of CD94/NKG2 receptors by infiltrating lymphocytes has been reported to occur in many tumors. Investigators have reported an expansion of CD94/NKG2A+ CTLs in patients with melanoma, and the lysis of melanoma cells by patient-derived CTLs is inhibited by the engagement of CD94/NKG2A (Speiser et al. 1999). In another study, cervical cancer–infiltrating T cells showed upregulated CD94/NKG2A expression compared with peripheral blood CD8+ T cells or normal cervix–infiltrating CD8+ T cells (Sheu et al. 2005). CD94 can also be expressed by lymphoma/leukemic cells. More specifically, it has been shown that the nasal extranodal NK/T cell lymphomas can express CD94 (Haedicke et al. 2000; Lin et al. 2003). The expression of CD94 transcripts by these types of lymphoma conferred a better prognosis compared with CD94 negative lymphomas (Lin et al. 2003).

In summary, during the past 10-12 years the expression and function of the CD94/NKG2A and CD94/NKG2C heterodimeric lectin-like receptors in NK and T cells, have been the subject of intensive study (Gunturi et al. 2004; Borrego et al. 2006). While a series of discoveries have explained several important functions of these receptors as well as of their cognate ligand HLAE, the first and only report of the existence of the NKG2H protein comes from the study of Teresa Bellón and her coworkers in 1999. These workers studied a αβ CD8+ T cell clone (TCC, K14B06) that could be activated using either mAbs specific for CD3 or CD94. Cell surface labelling and immunoprecipitation experiments identified a protein with molecular weight of 39-kDa that formed a disulphide-linked heterodimer with CD94, but was neither NKG2A nor NKG2C. PCR and cloning identified a novel cDNA clone, a splice variant of NKG2E that these workers named NKG2H. This cDNA was predicted to associate with CD94 and the adaptor molecule DAP12 to form an activating heterodimer. Further progress in understanding the biology and function of the NKG2H receptor (signaling, molecular interactions and ligand binding) has been seriously hampered by the lack of monoclonal antibodies specific for the NKG2H molecule. When an NKG2H specific mAb (MAB6549, R&D Systems) became commercially available, we decided to study the NKG2H receptor in more detail. We found that NKG2H was expressed at low levels on the surface of a small fraction of PBMCs derived from healthy donors. Although some expression of NKG2H was detected on NK cells and CD4+ T cells, this molecule was preferentially expressed on CD3+CD56+ and CD3+CD8+ cells implying that the signals controlling the expression of NKG2H receptor on the cell surface do not operate equally in NK and T cell subsets. Stimulation of cells via NKG2H, but not through the NKG2A or NKG2C receptors, induced apoptosis and notably reduced the activation and proliferation of the other cells in the culture. These data, together with the flow cytometry observation of preferential expression of the NKG2H receptor on CD3+ cells, implies a possible negative regulation of T cell activation mediated by NKG2H receptor signaling. Moreover, the NKG2H dependent reduction of cell culture activation did not depend on soluble factors or recognition of MHC class I molecules. Finally,

DNA & PROTEINS: The CD94/NKG2 “short” (hi)story

although it was suggested that the splice variant NKG2H, would behave identically to NKG2E and probably bind to HLA-E with similar affinity (Kaiser et al. 2005) so far, there are no data that demonstrate an interaction between HLA-E and NKG2H. In aggregate therefore, the available data suggest that it is possible that NKG2H recognizes and binds molecules that are not related, at least to conventional HLA-E-leader peptide loaded complexes. References:

Adamkiewicz, T.V., McSherry, C., Bach, F.H., Houchins, J.P., ¨Natural killer lectin-like receptors have divergent carboxy-termini, distinct from C-type lectins¨. Immunogenetics. 1994, 39: 218. Borrego, F., Masilamani, M., Marusina, A.I., Tang, X., Coligan, J.E. The CD94/NKG2 Family of receptors. Immun. Research. 2006, 35/3:263-277. Brooks AG, Borrego F, Posch PE, et al: Specific recognition of HLA-E, but not classical, HLA class I molecules by soluble CD94/NKG2A and NK cells. J Immunol 1999;162(1):305–313. Guma M, Angulo A, Vilches C, Gomez-Lozano N, Malats N, Lopez-Botet M: Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood 2004;104(12):3664–3671. Gunturi, A., Berg, R.E., Forman, J., The role of Cd94/NKG2 in innate and adaptive immunity. Immunol. Res. 2004, 30: 29-34. Haedicke W, Ho FC, Chott A, et al: Expression of CD94/NKG2A and killer immunoglobulin-like receptors in NK cells and a subset of extranodal cytotoxic T- cell lymphomas. Blood 2000;95(11):3628–3630. Hoare HL, Sullivan LC, Pietra G, et al: Structural basis for a major histocompatibility complex class Ib- restricted T cell response. Nat Immunol 2006;7(3): 256–264. Kaiser, B.K., Barahmand, F., Paulsene, W., Medley, S., Geraghty, D.E., and Strong, R.K. Interactions between NKG2x immunoreceptors and HLA-E ligands display overlapping affinities and thermodynamics. J Immunol. 2005, 174:2878– 2884. Lanier LL, Corliss B, Wu J, Phillips JH: Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity. 1998, 8:693. Lin CW, Chen YH, Chuang YC, Liu TY, Hsu SM: CD94 transcripts imply a better prognosis in nasal-type extranodal NK/T-cell lymphoma. Blood 2003;102(7): 2623–2631. Ljunggren, H.G., and Karre, K. In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol. Today. 1990, 11: 237–244. Michaelsson J, Teixeira de Matos C, Achour A, Lanier LL, Karre K, Soderstrom K: A signal peptide derived from hsp60 binds HL A-E and interferes with CD94/NKG2A recognition. J Exp Med 2002; 196(11): 1403–1414. O’Callaghan CA, Bell JI: Structure and function of the human MHC class Ib molecules HLA-E, HLA-F and HLA-G. Immunol Rev 1998. 163:129– 138. O’Callaghan CA, Tormo J, Willcox BE, et al: Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E. Mol Cell 1998. 1(4):531–541. Salerno-Goncalves R, Fernandez-Vina M, Lewinsohn DM, Sztein MB. Identification of a human HLA-E- restricted CD8+ T cell subset in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine. J Immunol 2004;173(9):5852–5862.

Sheu BC, Chiou SH, Lin HH, et al: Up-regulation of inhibitory natural killer receptors CD94/NKG2A with suppressed intracellular perforin expression of tumor- infiltrating CD8+ T lymphocytes in human cervical carcinoma. Cancer Res 2005;65(7):2921–2929. Speiser DE, Pittet MJ, Valmori D, et al: In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes. J Exp Med 1999;190(6):775–782. Tomasec, P., Braud, V.M., Rickards, C., Powell, M.B., McSharry, B.P., S. Gadola, et al. Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science, 2000, 287:1031. Tortorella, D., Gewurz, B.E., Furman, M.H., Schust, D.J., Ploegh, H.L. Viral subversion of the immune system. Annu Rev Immunol, 2000, 18:861–926. Ulbrecht, M., Martinozzi, S., Grzeschik, M., Hengel, H., Ellwart, J.W., Pla, M., et al. Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand for HLA-E and prevents NK cell-mediated lysis. J Immunol, 2000, 164: 5019–5022. Wang, E.C., McSharry, B., Retiere, C., Tomasec, P., Williams, S., Borysiewicz, L.K. et al. UL40-mediated NK evasion during productive infection with human cytomegalovirus. Proc Natl Acad Sci U S A, 2002, 99: 7570–7575. Wooden SL, Kalb SR, Cotter RJ, Soloski MJ: Cutting edge: HLA-E binds a peptide derived from the ATP- binding cassette transporter multidrug resistance-associated protein 7 and inhibits NK cell-mediated lysis. J Immunol 2005;175(3):1383–1387. Muntasell, A., Vilches, C., Angulo, A., López-Botet, M. Adaptive reconfiguration of the human NK-cell compartment in response to cytomegalovirus: a different perspective of the host–pathogen interaction. Eur J Immunol, 2013, 43:1133–1141. Waggoner, S.N., Cornberg, M., Selin, L.K., Welsh, R.M. Natural killer cells act as rheostats modulating antiviral T cell. Nature, 2011, 481 :394–398.

Science for you, November 2015

ASSOCIATION OF GENETIC VARIANTS IN PROMOTER REGION OF DEPTOR GENE WITH OBESITY AND INSULIN RESISTANCE DEVELOPMENT Tamara Šutuš Temovski,

DKFZ, The German Cancer Research Centre, Heidelberg Let me introduce myself briefly. My name is Tamara Šutuš Temovski and I am a goal-driven individual with multidisciplinary perspective and holistic approach. I share the idea of the legendary Steve Jobs, who said that “the only way to do a great job is to love what you do” and that’s why I always make sure to put all of my passion and enthusiasm in the project I am working on. I am currently starting my PhD project as a part of DKFZ, the German Cancer Research Centre. For the international scientific community, the DKFZ stands for top-notch research findings and ranks among foremost cancer research centers worldwide. I am honored to be part of such a great organization and to contribute to the “Research for A Life Without Cancer”. I recently joined the research group “DNA Vectors” supervised by Dr. Richard Harbottle. Our group is focused on generating novel, next-generation DNA vectors for gene therapy. My project will be focusing on investigating the epigenetic consequences of genetically modifying cells using non-integrating DNA Vectors. I studied Laboratory Biomedicine, at the Faculty of Pharmacy, University of Ljubljana, Slovenia. The research of my master’s thesis was conducted at the Unit of Special Laboratory Diagnostics, University Children’s Hospital, UKC Ljubljana, under the supervision of Dr. Katarina Trebušak Podkrajšek and Dr. Jernej Kovač. I successfully defended my master’s thesis entitled: “Association of genetic variants in promoter region of DEPTOR gene with obesity and insulin resistance development” in November 2014. Here I would like to introduce my master’s thesis research and the results I obtained.

Introduction Mammalian Target of Rapamycin (mTOR) signaling deregulation leads to obesity, increased risk of IR (insulin resistance) and type 2 diabetes. DEP domain containing mTOR interacting protein (DEPTOR) regulates mTOR signaling by binding to mTOR complex, inhibiting its activity and compromising its stability. To date, no mutation in the coding or promoter region of DEPTOR was associated with pre-diabetes and/ or obesity. Our aim was to determine gene variants in the promoter region of DEPTOR associated with development of pre-diabetes and/or obesity. 191 obese children and adolescents (91 males/100 females, mean age 13.3 ± 3.3 years, mean BMI-SDS 2.82 ± 0.66) were studied. A standard Oral Glucose Tolerance Test (OGTT) was performed for each individual. Insulin resistance was defined by HOMA-IR and WBISI according to Madsuda. Genotyping of DEPTOR promoter region was performed using High Resolution Melt (HRM) analysis and Sanger sequencing. Chi-square test was used to detect statistical significance in frequency of specific genotypes.

Results Four polymorphisms (SNP) were identified: rs7840156, rs75781905, rs117543860 and rs140142743. The SNP rs7840156 (g.120885944T>C) was associated with reduced risk of insulin resistance determined by HOMA-IR (>2.6; an upper half cut-off value for the analyzed population) and WBISI (<3). The carriers of C allele had odds ratio (OR) for insulin resistance (IR) according to HOMA-IR 0.63 (95% CI= 0.41-0.87; p=0.038) and OR for IR 0.58 (95% CI= 0.38-0.90; p=0.018) according to WBISI. Other detected SNPs were not associated with insulin resistance in the analyzed population. We were not able to associate any of the identified polymorphisms with obesity. Discussion A polymorphism in the promoter region of DEPTOR is associated with insulin resistance determined by HOMA-IR and WBISI measures. This implicates that DEPTOR is a new gene involved in the early phases of type 2 diabetes development in children and adolescents, most likely by inhibition of intracellular signaling through the insulin receptor and consequentially by altering insulin sensitivity.

DNA & PROTEINS: Pathways of deposition of H3.3 into MSC chromatin

References:

Laplante M, Horvat S, Festuccia WT, Birsoy K, Prevorsek Z, Efeyan A, et al. DEPTOR cell-autonomously promotes adipogenesis, and its expression is associated with obesity. Cell Metab.2012;16:202–12.

Blagosklonny MV. TOR-centric view on insulin resistance and diabetic complications: perspective for endocrinologists and gerontologists. Cell Death Dis. 2013; 4:e964.

Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol. 2011; 12:21–35.

Krebs M, Brunmair B, Brehm A, Artwohl M, Szendroedi J, Nowotny P, et al. The Mammalian target of rapamycin pathway regulates nutrientsensitive glucose uptake in man. Diabetes. 2007; 56:1600–7.

Xie J, Herbert TP. The role of mammalian target of rapamycin (mTOR) in the regulation of pancreatic β-cell mass: implications in the development of type-2 diabetes. Cell Mol Life Sci CMLS. 2012; 69:1289–304.

PATHWAYS OF DEPOSITION OF H3.3 INTO MSC CHROMATIN J S , ane

pirkoski

Molecular Medicine, University

Oslo

Currently, I am concluding my PhD in Philippe Collas’ group, established in the department of Molecular Medicine at the University of Oslo. The Collas lab (http://www.collaslab.com) is interested in couple of areas in the Epigenetic and Stem Cell research, such as regulation of chromatin signaling via nuclear landmarks (A-type lamins and A-kinase anchoring proteins, or AKAPs). By combining imaging, epigenomics and informatics approaches, Collas lab is examining how lamin A mutations affect chromatin organization and MSC differentiation, also characterizing novel binding partners of lamin A, whose association with the lamin is affected by laminopathy mutations. The lab, is also interested in the role of AKAP95 in tethering protein kinase and phosphatase complexes to chromatin on the regulation of cell proliferationassociated genes. Other areas of interest of the lab are the mechanistic aspects and plasticity of stem cell chromatin. Using molecular and live imaging approaches, Collas lab is investigating the pathways of deposition of newly synthesized epitope-tagged H3.3 into MSC chromatin, with a focus on the role of the various H3.3 chaperones in this process. In addition to this, combining various mathematical, statistical and bioinformatic tools, Collas group is trying to determine the 3D chromatin architecture of the stem cell genome.

Research results Histone H3 variant H3.3 incorporates into chromatin in a replication-independent manner (i.e. both during and outside S-phase) whereas the replicative H3.1 and H3.2 variants are deposited only during S phase [1]. Imaging data have shown that H3.3 accumulates mainly at sites of

transcription [1] and chromatin immunoprecipitation (ChIP) studies are consistent with these findings [2-4]. Current models suggest that histone variant H3.3 is deposited into various chromatin domains by distinct H3.3 chaperones, including death associated protein (DAXX), alpha-thalassemia/mental retardation X-linked

Figure 1. H3.3-mCherry is co-enriched at PML bodies together with H3.3 chaperones. (A) Immunostaining of H3.3-mC (24 h after H3.3mC transfection ;) with of H3.3 chaperones, co-localized in PML bodies. (B) Immunostaining of PML, HIRA, ASF1A, and DAXX at PML bodies in non-transfected cells. Scale bars, 15 μm. (C) Percentage of cells where H3.3 chaperones co-localize in PML bodies.

Science for you, November 2015

Delbarre et al. 2013. Genome Res.

syndrome protein (ATRX), histone regulator A (HIRA) and anti-silencing function 1 A (ASF1A). Furthermore, we have previously shown that a pool of neo-synthesized H3.3 transits through the promyelocytic leukaemia (PML) bodies in a DAXX-dependent manner prior to deposition into chromatin [5]. At PML bodies, epitope-tagged H3.3 in a complex with H4, interacts with DAXX, ATRX, HIRA and ASF1A [5] (Figure 1, Model). PML is a tumor suppressing protein that organizes in the nucleus to form PML nuclear bodies. PML have been shown to be involved in many cellular processes such as transcriptional regulation, chromatin remodeling, DNA damage, cellular senescence, apoptosis and angiogenesis [6]. Because of its multi-functionality, it has been suggested that PML bodies serve as scaffold for multiple protein complexes that have different functions [7, 8]. We examine a role of DAXX, ATRX, HIRA and PML on the association of endogenous and epitopetagged H3.3 with chromatin. Micrococcal nuclease (MNase) digestion reveals DAXX and PML into MNase-soluble (nucleosomal) and insoluble chromatin fractions. A pool of DAXX and PML is also Triton X-100-soluble. Thus, DAXX and PML exist both as chromatin-associated and soluble pools. DAXX knock-down does not alter PML distribution; however PML knock-down increases the detergent soluble pool of DAXX and abolishes its detection into MNase-soluble and insoluble fractions. This is corroborated in Pmlnull mouse embryonic fibroblasts (MEFs). This suggests that PML is required for targeting or maintaining DAXX in chromatin. Global chromatin enrichment in total H3 or en-

dogenous H3.3 is not affected by PML loss, based on MNase fraction profiles. However, ChIP-PCR reveals local variations in H3 density in Pmlnull MEFs. Further, newly synthesized H3.3-Flag shows in wild-type MEFs low level occupancy on promoters and coding regions (CRs) of non-expressed genes while those of expressed loci are strongly enriched. In Pml-null MEFs, enrichment in H3.3-Flag appears higher both on promoters and CRs for the genes examined. This suggests facilitated incorporation of epitope-tagged H3.3 into chromatin in the absence of PML, raising the hypothesis that PML may impose site-specificity in the deposition of H3.3. Model of (H3.3–H4) dimer recruitment to PML bodies prior to chromatin deposition. In pathway 1, DAXX recruits the (H3.3–H4) dimer to PML bodies. This process is facilitated by ASF1A (pathway 2). (H3.3–H4) loaded onto ASF1A can also be brought to additional H3.3 chaperone containing complexes (Szenker et al. 2011) (pathway 3). DAXX, ATRX, HIRA, and ASF1A are localized in a proportion of PML bodies and may speculatively be available for loading (H3.3–H4) prior to deposition into chromatin (dashed arrows).”[9] References:

1 Ahmad, K. & Henikoff, S. 2002. The Histone Variant H3.3 Marks Active Chromatin by Replication Independent Nucleosome AssemblyMol. Cell 9, 1191-1200 (2002). 2 Mito, Y., Henikoff, J.G. & Henikoff, S. 2005. Genome-scale profiling of histone H3.3 replacement patterns. Nat. Genet. 37, 1090-1097. 3 Jin, C. & Felsenfeld, G. 2007. Nucleosome stability mediated by histone variants H3.3 and H2A.Z Genes Dev. 21, 1519-1529. 4 Tamura, T. et al.2009. Inducible Deposition of the Histone Variant H3.3 in Interferon-stimulated genes J. Biol. Chem. 284, 12217-12225. 5 Delbarre, E., Ivanauskiene, K., Küntziger, T., and Collas, P. 2013. DAXXdependent supply of soluble (H3.3-H4) dimers into PML bodies pending deposition into chromatin. Genome Res. 23, 440-451. 6 Rosa Bernardi and Pier Paolo Pandolfi. 2007. Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies. Nat.Rev.Mol. Cell Biol. 8, 1006-1016. doi:10.1038/nrm2277. 7 Salomoni, P. (2013). The PML-Interacting Protein DAXX: Histone Loading Gets into the Picture. Frontiers in oncology, 3, 152-?. doi:10.3389/ fonc.2013.00152. 8 Torok, D., Ching, R. W., and Bazett-Jones, D. P. 2009. PML nuclear bodies as sites of epigenetic regulation. Front. Biosci. 14, 1325–1336. doi:10.2741/3311. 9 Szenker E, Ray-Gallet D, Almouzni G. 2011. The double face of the histone variant H3.3. Cell Res 21: 421–434.

CELLS: Transdifferentiation of canine mesenchymal stem cells into nevrons

TRANSDIFFERENTIATION OF CANINE MESENCHYMAL STEM CELLS INTO NEVRONS

Vesna Kokondoska Grgič, Center

for sharing knowledge

ScienceMIX

As a co-founder of the Center for sharing knowledge ScienceMIX in Macedonia, I am trying to fill in my free time with useful things. I based the idea for sharing knowledge into non-developed countries, especially with academic institutions from Macedonia. On the other hand, I am a biotechnologist and in accordance with my education process, I’ve got experience in animal cell cultures. My goal was to develop a novel protocol which could prove that a dog’s mesenchymal stem cells can be differentiated into neurons, which means a good opportunity for the modeling of neurodegenerative diseases. In cooperation with Prof. Dr. Gregor Majdič (Genomic center for animals, Veterinary Faculty at University of Ljubljana) and Prof. Dr. Joel Glover (Medical Faculty, University of Oslo), and Luka Mohoric, CEO of Animacel company and other coworkers, I have learned lots of new techniques and skills for characterization of transdifferentiated MSC cells into neurons. If you have any ideas for cooperation and projects don’t hesitate to contact me at vesna.kokondoska@science-mix.com.

Introduction Mesenchymal stem cells (MSCs) are self-regenerating, multipotent, adult cells of mesodermal and ectodermal origin (Ferroni et al., 2013). They are found in many tissues (adipose tissue, bone marrow, cord blood, chorionic folds of the placenta, amniotic fluid, blood, lungs, etc.), which are easily accessible and represent a potentially important source of cells for treatment. The ability of MSC differentiation into osteoblasts, chondrocytes and adipocytes in vitro has been demonstrated in many studies since the discovery of MSCs (Gronthos et al., 2001; Hauner et al., 1987). Until the year 2000, it was assumed and widely accepted that mesenchymal stem cells could differentiate only in tissues of mesodermal origin.

However, this theory was challenged by studies in the laboratory of Woodbury et al.. Follow-up studies reported mixed results and often contradictory conclusions, and, so far it was not conclusively proven that MSCs could differentiate into functional nerve cells. Most studies on transdifferentiation of MSC were carried out with human and rodent cells (Ahmadi et al. 2012; Anghileri et al. 2008; Franco Lambert et al. 2009; Zemelko et al. 2013). In veterinary medicine, the dog is increasingly used as a model of human neurological disorders, and few studies also reported the induction of canine ADSCs/MSC to neural lineages (Lim et al. 2010; Park et al. 2012; Sago et al. 2008).

Figure 1: Mesenchymal stem cells isolated and expanded from diverse sources of tissue that may be applied to various discovery and therapeutic applications (Chase et.al., 2011).

Science for you, November 2015

Figure 2: Morphological changes of ATSCs by the transdifferentiation treatment (Sago et al. 2008).

Protocols for transdifferentiation of dog adipose derived MSCs into neurons. Transdifferentiation of ADSCs refers to cell reprogramming that is characterized by the reversed differentiation of cells from a lineagespecific differentiated phenotype into a more stem cell phenotype. Most of all it is associated with pluripotency and re-entry into the cell cycle. Transdifferentiation of ADSCs can be identified by changes in cell morphology, chromatin remodeling, and gene expression (Yu et al., 2011). Adipose tissue-derived stromal cells (ADSCs) have been identified as a useful stem cell source for cellular regenerative therapy in clinical trials of degenerative diseases. International Society for Cellular Therapy (ISCT) released a consensus position paper stating the minimal criteria required for defining MSCs that is also relevant for veterinary purpose: a. Adherence to tissue culture plastic under standard culture condition. b. Cell surface marker characterization: Cells must be positive for CD73, CD90, CD105 and negative for CD11b or CD14, CD34, CD45, CD79α or CD19, HLR-DR. c. Ability to differentiate In vitro into three mesodermal linages: osteoblasts, chondrocytes and adipocytes. Neupane et al., (2008) reported for the first time the adaptation of methods to isolate human ASCs for the derivation of cells from canine adipose tissues, showing their expansion as adherent cells, expression of pluripotency genes, and differentiation into osteoblasts and adipocytes cells types. Neupane et al., (2008) group also reported that, subcutaneous adipose tissue (1 cm3) has been collected by vet-doctor from the region above the dorsal muscles. After

that, adipose sample is properly transported to laboratory after 24h from the surgical operation and professionally digested with enzymes to released the cells in medium culture. On the other side, Wu et al., (2001) were the first scientific group to report the establishment of pre-adipocytes from the canine stromal vascular fraction. The Woodbury group demonstrated that the MSCs, isolated from rat and human bone marrow, have ability to transdifferentiate into neural phenotype. Bone marrow MSCs have been exposed to 1 mmol/L β-mercaptoethanol (BME). Following this stimulation, cells expressed neuron specific enolase (NSE) and were morphologically changed to resemble mature neurons. In later experiments the same group used as the neuronal induction media DMEM, 2% dimethylsulfoxide (DMSO) and 200mM butylated hydroxyanisole (BHA) (Woodbury et al. 2000). Other research group made some modification to the Woodbury protocol (Ashjian et al. 2003; Safford et al. 2002; P A Zuk et al. 2001; Patricia A Zuk et al. 2002) using combinations of butylated hydroxyanisole, KCL, valproic acid, forskolin, hydrocortisone, insulin. In all these studies it was shown that cells express mature neuronal markers such as: glial fibrillary acid protein (GFAP), microtubule associated protein-2 (MAP2), β-III tubulin; the presence of voltage-gated calcium channels and the ability to upregulate the glutamate receptor. Sago et al., 2008 investigated in vitro differentiation of canine adipose tissuederived stromal cells into neuronal cells. ADSC were incubated with 100 μM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 125 μM isobuthylmethylxanthine (IBMX). They reported that ADSCs can differentiate into earlyto-mature neuronal cells and are candidate cells for autologous nerve regeneration therapy, al-

CELLS: Transdifferentiation of canine mesenchymal stem cells into nevrons

though additional research is needed to examine functional characteristics of differentiated ADSCs (Sago et al. 2008). Alternative methods of inducing chemical differentiation to a neural lineage involves the addition of growth factors such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), neuroblast factor (N2), B27 and retinoic acid (Anghileri et al., 2008; Zemelko et al., 2013). Neural induction is associated with the expression of several proproliferation transcription factors, downstream of FGF signaling (Foxd4l1 and Zic3 genes) or BMP inhibition (Zic1 gene)(Aruga and Mikoshiba 2011; Marchal et al. 2009). Although it is already 15 years since the first scientific evidence of the ability of human and rat MSCs to differentiate into neuronal cell linages, there is still no clear evidence that ADSC cells are capable of differentiating into functional neuronal cells in vitro. Furthermore, only few studies examined dog MSC with positive expression of selective neuronal and glial markers. Obviously new methods to induce transdifferentiation could improve the existing neuronal differentiation techniques especially usage of retinoic acid (RA) (Frey and Vogel 2011). The ability of RA to induce post-mitotic, neural phenotypes in various stem cells in vitro, served as early evidence that RA is involved in the switch between proliferation and differentiation. Genuine neuronal differentiation of adult stem cells requires full cell functionality, which may be demonstrated through electro physiology and expression of the complete profile of neuronal genes. All of this research could be applicable for neurodegenerative diseases modeling. Also, creating “disease in a dish” could be a milestone for testing new drugs and would reduce the need of animals that are involved in pre-clinical testing. References:

Ahmadi, Nafiseh, Shahnaz Razavi, Mohammad Kazemi, and Shahrbanoo Oryan. 2012. “Stability of Neural Differentiation in Human Adipose Derived Stem Cells by Two Induction Protocols.” Tissue & cell 44(2): 87–94. Anghileri, Elena et al. 2008. “Neuronal Differentiation Potential of Human Adipose-Derived Mesenchymal Stem Cells.” Stem cells and development 17(5): 909–16. Aruga, J, and K Mikoshiba. 2011. “Role of BMP, FGF, Calcium Signaling, and Zic Proteins in Vertebrate Neuroectodermal Differentiation.” Neurochemical Research 36(7): 1286–92.

Ashjian, Peter H et al. 2003. “In Vitro Differentiation of Human Processed Lipoaspirate Cells into Early Neural Progenitors.” Plastic and reconstructive surgery 111: 1922–31. Chase, Lucas C. G., Mahendra S. G. Rao, and Mohan S. Vemuri. 2011. “Mesenchymal Stem Cell Assays and Applications Methods in Molecular Biology.” 698: 534. Franco Lambert, Ana Paula et al. 2009. “Differentiation of Human Adipose-Derived Adult Stem Cells into Neuronal Tissue: Does It Work?” Differentiation; research in biological diversity 77(3): 221–28. Frey, Simone K, and Silke Vogel. 2011. “Vitamin A Metabolism and Adipose Tissue Biology.” Nutrients 3(1): 27–39. Lim, Ji-Hey et al. 2010. “Generation and Characterization of Neurospheres from Canine Adipose Tissue-Derived Stromal Cells.” Cellular reprogramming 12(4): 417–25. Marchal, Leslie, Guillaume Luxardi, Virginie Thomé, and Laurent Kodjabachian. 2009. “BMP Inhibition Initiates Neural Induction via FGF Signaling and Zic Genes - Topic1-.” Proceedings of the National Academy of Sciences of the United States of America 106(41): 17437–42. Neupane, Manish, Chia-Cheng Chang, Matti Kiupel, and Vilma Yuzbasiyan-Gurkan. 2008. “Isolation and Characterization of Canine Adipose-Derived Mesenchymal Stem Cells.” Tissue engineering. Part A 14(6): 1007–15. Park, Sung-Su et al. 2012. “Functional Recovery after Spinal Cord Injury in Dogs Treated with a Combination of Matrigel and Neural-Induced Adipose-Derived Mesenchymal Stem Cells.” Cytotherapy 14(5): 584–97. Safford, Kristine M et al. 2002. “Neurogenic Differentiation of Murine and Human Adipose-Derived Stromal Cells.” Biochemical and biophysical research communications 294(2): 371–79. Sago, Ken et al. 2008. “In Vitro Differentiation of Canine Celiac Adipose Tissue-Derived Stromal Cells into Neuronal Cells.” The Journal of veterinary medical science the Japanese Society of Veterinary Science 70(4): 353–57. Woodbury, D, E J Schwarz, D J Prockop, and I B Black. 2000. “Adult Rat and Human Bone Marrow Stromal Cells Differentiate into Neurons.” Journal of neuroscience research 61(4): 364–70. Wu, P et al. 2001. “Differentiation of Stromal-Vascular Cells Isolated from Canine Adipose Tissues in Primary Culture.” The Journal of veterinary medical science / the Japanese Society of Veterinary Science 63(1): 17–23. Yu, Ji Min, Bruce A Bunnell, and Soo-Kyung Kang. 2011. “Neural Differentiation of Human Adipose Tissue-Derived Stem Cells.” Methods in molecular biology (Clifton, N.J.) 702: 219–31. Zemelko, V. I. et al. 2013. “Neurogenic Potential of Human Mesenchymal Stem Cells Isolated from Bone Marrow, Adipose Tissue and Endometrium: A Comparative Study.” Cell and Tissue Biology 7(3): 235–44. Zuk, P A et al. 2001. “Multilineage Cells from Human Adipose Tissue: Implications for Cell-Based Therapies.” Tissue engineering 7(2): 211–28. Zuk, Patricia A et al. 2002. “Human Adipose Tissue Is a Source of Multipotent Stem Cells □.” 13(December): 4279–95.

Science for you, November 2015

STEM CELLS IN ADVANCED CELL THERAPIES AT BLOOD TRANSFUSION CENTRE OF SLOVENIA M. Jež, K. Jazbec, J.Ž. Rožman Blood Transfusion Centre

Slovenia

and

P. Rožman,

The Blood Transfusion Centre of Slovenia (BTC), is a public healthcare institution that supplies blood and blood products, renders diagnostic and therapeutic services within the transfusion and transplantation fields, manages the Registry of bone marrow donors and supplies blood-derived medicinal products within the scope of its public health service. The Centre’s primary task is to provide patients with compatible, quality and safe blood and blood products. This covers work in blood donation, selection of blood donors, blood collection, processing, storage and testing as well as the supply of blood components to hospitals. Within the scope of diagnostic services, BTC carries out immunohematological blood tests on recipients and other tests required for the transfusion of compatible blood and blood components. It also performs lab tests required for the implementation of national programs for organ and tissue transplantation, as well as advanced therapies. Furthermore, BTC provides therapeutic services, such as autotransfusion and hemapheresis. The Centre carries out research and development (R&D) in transfusion and transplantation medicine and educational activities for the general public. One of the main research directions are stem cells and advanced cell therapies. The national research program, namely Human stem cells-advanced cell therapy I (P3-0371), started in 2009 and introduced advanced therapies to Slovenia. The foundation of all three forms of advanced therapies - i.e. cell therapy, tissue engineering and gene therapy - are the human stem cells. In the current national research program, namely Human stem cells-advanced cell therapy II (P3-0371), we successfully combine and coordinate stem cell research from various Slovenian institutions and laboratories and develop protocols for collection of stem cells from various sources (umbilical cord blood, bone marrow, peripheral blood, adipose tissue, etc.), cell separation and cell sorting methods, isolation of specific subpopulations of stem cells and their long-term cryopreservation (Jež et. al. 2014; Jež et. al. 2015; Krašna et. al. 2015). One of the new cell therapies combining the knowledge from all these areas is treatment of dilated cardiomyopathy with isolated CD34+ stem and progenitor cells.

National research project: CD34+ Cell Therapy in Patients with Dilated Cardiomyopathy (J3-5508) Numerous clinical trials have shown various stem and progenitor cell populations improve cardiac function when transplanted into hearts of patients with cardiomyopathy. Several factors may contribute to the beneficial effects of stem cell therapy on cardiac function. In preclinical models, it has been shown that BMC administration can improve cardiac function through paracrine effects. These factors can attenuate apoptosis of endogenous cardiomyocytes and endothelial cells, promote angiogenesis, activate resident cardiac stem cells, or induce anti-inflammatory effects. The clinical study has been going on for more than 7 years and long-term effects of intracoronary CD34+ cell transplantation in dilated cardiomyopathy and the relationship between intramyocardial cell homing and clinical response was investigated. Intracoronary stem cell transplantation is associated with improved ventricular function, exercise tolerance, and long-term survival in patients with dilated cardiomyopa-

thy. Higher intramyocardial homing is associated with better stem cell therapy response. During the 5-year follow-up period, cell therapy was associated with a significant improvement in cardiac function and exercise capacity and a significant decrease in NT-proBNP levels. It was also found that total mortality rates were lower in patients randomized to the stem cell therapy group than in controls, with the difference being largely a consequence of reduced death rates from pump failure (Vrtovec et. al. 2013; Lezaic et. al. 2015). Despite considerable potential, cardiac cellbased therapies to date, have not been uniformly positive and have often reported only modest benefits. In order to advance the field, current and future strategies should aim to address several important issues, including what is the mechanism of action of such therapy, what are the optimal cell types, dose, route and frequency of cell administration, and how to achieve long-term engraftment of these cells at sites of injury. In order to address some of the current challenges of stem cell-based therapies, our goal is to gain further insight into the

CELLS: Stem cells in advanced cell therapies at Blood Transfusion Centre of Slovenia

mechanism of cardiac repair by these cells and to determine which cell subpopulations are associated with better clinical outcome. The research project is divided into 2 major subsections: clinical part and translational part. The clinical part consists of a prospective randomized double-blind, placebo-controlled trial aiming to define the safety and efficacy of CD34+ cell therapy in patients with non-ischemic dilated cardiomyopathy. Blood Transfusion Centre of Slovenia participates in the CD34+ cell mobilization, collection (immunomagnetic isolation), enumeration and finally, CD34+ cell products are sent to University Medical Centre of Ljubljana for immediate transendocardial transplantation. The remaining CD34+ cells are used for the translational part of the project A) CD34+ cell cryopreservation protocol validation, and B) CD34+ cell characterization. Our group is primarily involved in Objective B of the translational part of the study. We investigate the potential of CD34+ cells to form functional colonies, to secrete cytokines and microvesicles. Furthermore, we try to identify other cell surface markers, do the epigenetic and gene expression analyses of various factors that might be implicated in the mechanism of cardiac repair, including proangiogenic markers, markers associated with cardiomyocyte differentiation, antiapoptotic markers, pluripotent markers, telomerase and alkaline phosphatase. The project is on its way and will be completed at the end of 2016. Serial consecutive bone marrow transplantation in non-irradiated BALB/C mice Mojca Jež, Katerina Jazbec, Jasmina-Živa Rožman, Primož Rožman Recently, we have started developing some aspects of experimental hematology by studying bone marrow transplantation related phenomena in mice, aiming at the regenerative capacity of heterochronous autologous bone marrow transplantation. The first step in this process is to achieve a stabile long-term chimerism in recipient animal. Longterm chimerism after bone marrow transplantation is only possible as a result of a dynamic balance between host and donor hematopoietic

stem cells (HSC) due to bilateral tolerance (Shimoni and Nagler, 2001). Chimerism is usually induced by irradiation, antibody-mediated conditioning, chemotherapy, and/or immunosuppressive drugs, which permanently or temporarily suppress the immune system, and empty the bone marrow niches for the engraftment and proliferation of donor HSC (Duran-Struuck and Dysko, 2009). It was long believed that bone marrow transplantation can successfully lead to chimerism only with prior conditioning (Brecher et al., 1982; Rao et al., 1997; Quesenberry et al., 2005). This is still a commonly held belief, even though there are numerous mouse model studies (among them Brecher et al., 1982, Stewart et al., 1993, Nilsson et al., 1997, 1999, Rao et al., 1997, Blomberg et al., 1998, Colvin et al., 2004, 2007), in which chimerism was achieved after bone marrow transplantation in nonconditioned mice. Studies show that HSC engraftment in non-conditioned recipients is most likely a competitive engraftment, where donor stem cells compete for a place in bone marrow niches and replace a recipient’s own stem cells (Blomberg et al., 1998; Colvin et al., 2004). This competitive model shows that chimerism in a non-conditioned mouse is determined by host to donor stem cell ratios. Accordingly, most of the studies, which obtained a higher percentage of chimerism, used high cell numbers of unfractionated bone marrow for transplantation (up to 200 million cells and even more).

Non-conditioned bone marrow transplantation can offer important clinical advantages since the potentially serious complications associated with cytotoxic conditioning can be avoided. It might be of benefit in advanced cell therapies intended for tissue regeneration, and it may enable the treatment of certain clinical conditions. Autologous modified HSC can be an efficient gene delivery system, leading us into a new era of treatment and rejuvenation. Our study produced encouraging preliminary data that indicates further exploration of the subject. Conclusions Advanced cell therapies are one of the main developmental directions of biomedical research in the last decade. The final aim of our research

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program is to provide a quality medical service in the field of advanced cell therapies to all citizens. It will open the possibilities for a replacement of damaged and diseased tissues and thus high life quality. By introducing new essential technologies, our research work is also important from a scientific, technical, infrastructural and educational viewpoint. Reference Ahmadi, Nafiseh, Shahnaz Razavi, Mohammad Kazemi, and Shahrbanoo Oryan. 2012. “Stability of Neural Differentiation in Human Adipose Derived Stem Cells by Two Induction Protocols.” Tissue & cell 44(2): 87–94. Anghileri, Elena et al. 2008. “Neuronal Differentiation Potential of Human Adipose-Derived Mesenchymal Stem Cells.” Stem cells and development 17(5): 909–16. Aruga, J, and K Mikoshiba. 2011. “Role of BMP, FGF, Calcium Signaling, and Zic Proteins in Vertebrate Neuroectodermal Differentiation.” Neurochemical Research 36(7): 1286–92. Ashjian, Peter H et al. 2003. “In Vitro Differentiation of Human Processed Lipoaspirate Cells into Early Neural Progenitors.” Plastic and reconstructive surgery 111: 1922–31. Chase, Lucas C. G., Mahendra S. G. Rao, and Mohan S. Vemuri. 2011. “Mesenchymal Stem Cell Assays and Applications Methods in Molecular Biology.” 698: 534. Franco Lambert, Ana Paula et al. 2009. “Differentiation of Human Adipose-Derived Adult Stem Cells into Neuronal Tissue: Does It Work?” Differentiation; research in biological diversity 77(3): 221–28. Frey, Simone K, and Silke Vogel. 2011. “Vitamin A Metabolism and Adipose Tissue Biology.” Nutrients 3(1): 27–39. Lim, Ji-Hey et al. 2010. “Generation and Characterization of Neurospheres from Canine Adipose Tissue-Derived Stromal Cells.” Cellular reprogramming 12(4): 417–25. Marchal, Leslie, Guillaume Luxardi, Virginie Thomé, and Laurent Kodjabachian. 2009. “BMP Inhibition Initiates Neural Induction via FGF Signal-

ing and Zic Genes - Topic1-.” Proceedings of the National Academy of Sciences of the United States of America 106(41): 17437–42. Neupane, Manish, Chia-Cheng Chang, Matti Kiupel, and Vilma Yuzbasiyan-Gurkan. 2008. “Isolation and Characterization of Canine AdiposeDerived Mesenchymal Stem Cells.” Tissue engineering. Part A 14(6): 1007–15. Park, Sung-Su et al. 2012. “Functional Recovery after Spinal Cord Injury in Dogs Treated with a Combination of Matrigel and Neural-Induced Adipose-Derived Mesenchymal Stem Cells.” Cytotherapy 14(5): 584–97. Safford, Kristine M et al. 2002. “Neurogenic Differentiation of Murine and Human Adipose-Derived Stromal Cells.” Biochemical and biophysical research communications 294(2): 371–79. Sago, Ken et al. 2008. “In Vitro Differentiation of Canine Celiac Adipose Tissue-Derived Stromal Cells into Neuronal Cells.” The Journal of veterinary medical science the Japanese Society of Veterinary Science 70(4): 353–57. Woodbury, D, E J Schwarz, D J Prockop, and I B Black. 2000. “Adult Rat and Human Bone Marrow Stromal Cells Differentiate into Neurons.” Journal of neuroscience research 61(4): 364–70. Wu, P et al. 2001. “Differentiation of Stromal-Vascular Cells Isolated from Canine Adipose Tissues in Primary Culture.” The Journal of veterinary medical science / the Japanese Society of Veterinary Science 63(1): 17–23. Yu, Ji Min, Bruce A Bunnell, and Soo-Kyung Kang. 2011. “Neural Differentiation of Human Adipose Tissue-Derived Stem Cells.” Methods in molecular biology (Clifton, N.J.) 702: 219–31. Zemelko, V. I. et al. 2013. “Neurogenic Potential of Human Mesenchymal Stem Cells Isolated from Bone Marrow, Adipose Tissue and Endometrium: A Comparative Study.” Cell and Tissue Biology 7(3): 235–44. Zuk, P A et al. 2001. “Multilineage Cells from Human Adipose Tissue: Implications for Cell-Based Therapies.” Tissue engineering 7(2): 211–28. Zuk, Patricia A et al. 2002. “Human Adipose Tissue Is a Source of Multipotent Stem Cells .”: 4279–95.

CELLS: Development of an efficient feeding strategy for an industrial yeast strain

DEVELOPMENT OF AN EFFICIENT FEEDING STRATEGY FOR AN INDUSTRIAL YEAST STRAIN Jernej Horvat, Institute

Chemical Engineering, Vienna University

Technology

For the past two years, I attended master studies in Medical and Pharmaceutical Biotechnology in Krems, Austria. Six months of internship, which I performed as a part of the Bioprocess Integrated Development team, in the Biochemical Engineering research division of Institute of Chemical Engineering at Vienna University of Technology, were also part of the studies. My supervisors during my work were Dr. Christoph Herwig, Dr. Oliver Spadiut and Simona Capone, a PhD student. The project we were working on was an industrial project, during which we had a mission to develop an efficient feeding strategy for a recombinant phospholipase C producing Pichia pastoris strain. I used the project data for my master’s thesis. The mission of the Biochemical Engineering division, guided by Dr. Christoph Herwig, is the advance of methods for efficient and scientifically based bioprocess development. Cornerstone of main activities of the division are: 1) science based methods for quantitative bioprocess development; 2) connection between data and knowledge; 3) transfer from bio-refinery to biopharmaceuticals; 4) connection between process understanding and engineered strains and products, etc. Tools in use are quality by design principles, dynamics, real time sensors, data exploitation, mechanistic modelling, etc. The Integrated Bioprocess Development’s team mission, guided by Dr. Oliver Spadiut, can be described by: 1) determination of strain physiology; 2) identification of bioprocessing mode giving the highest product quantity and quality; 3) determine improvement of the production process by stress; 4) determination how do variations in the Upstream influence following unit operations; 5) determination of novel ways to purify the product and 6) identification how do critical quality attributes interrelate to previous process step.

Introduction Phospholipase C (PLC) Phospholipase C (PLC) is a class of phospholipases belonging to ubiquitous enzyme group, known for hydrolyzing a common substrate- i.e. phospholipids. Cleaving occurs just before the phosphate group, generating diacyl-glycerol, which is well-known signaling molecule in mammals (Srinivas, Rajakumari et al., 2008). In humans, it plays an important role in cell metabolism, particularly in signal transduction pathways as illustrated in Figure 1. Phosphatidylcholinehydrolyzing PLC from B. cereus is an extracellular, non-toxigenic and monomeric protein with high structural and catalytic similarity to mammalian PLC. Together with the fact that the B. cereus enzyme can mimic action of mammalian PLC (e.g. biosynthesis of prostaglandins), it can be potentially used as a model for mammalian PLCs and subject of medical research (Hough, Hansen et al., 1989). Several expressions of PLC from B. cereus shows, that production is possible in alternative hosts (e.g. Bacillus subtilis). Recombinant expressed PLC has comparable or even better stability and activity characteristics, when com-

pared to wild-type expressed PLC. Nevertheless, PLC from B. cereus is highly unstable enzyme (Kleiman and Lands, 1969), resulting in difficult production processes, which is reflected in very high price (370 € / 1 mg) of the enzyme. Therefore, application of new strategies, search for the new hosts and additional optimizations in rPLC production were needed.

Figure 1: Effect of bacterial PLC on eukaryotic cells; modified after (Titball, 1993).

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Pichia pastoris One of the candidate for the enzyme production is methylotrophic yeast P. pastoris, which has been widely used for recombinant protein production in industrial and academic area (Higgins and Cregg, 1998). The yeast offers several advantages over prokaryotic and other eukaryotic expression systems such as 1) high growth rate; 2) fermentations are possible at very high cell density; 3) high production rates; 4) endotoxin free supernatants; 5) absence of lytic viruses; 6) possible post-translational modifications including protein folding, glycosylation etc.; and 7) the ability of protein secretion. Listed advantages makes P. pastoris one of the most important tools in modern biotechnology (Weinhandl, Winkler et al., 2014). Despite many advantages, it possesses also several drawbacks. Recombinant proteins are mostly hyper-glycosylated with mannose residues, resulting in antigenic property increase, which is undesirable in therapeutic applications. However, latest optimizations in strain physiology, protein folding, secretion and glyco-engineering, brought significant improvements in recombinant protein production with P. pastoris (Damasceno, Huang et al., 2012). Heterologous protein production with P. pastoris is mainly based on strong inducible and tightly regulated promoter of methanol utilization AOX1 and AOX2 genes, where methanol is used as main inducer for protein expression. AOX system brings very high production rates of target protein, however, it also brings several disadvantages. Methanol is highly flammable and its storage in greater amount is undesirable. Methanol accumulation during fermentation can be toxic for cells and furthermore, methanol consumption causes higher heat production, increases need for oxygen and production of hydrogen peroxide during fed batch phase can be induced. Due to several drawbacks and issues of AOX promoter system, alternative promoters (e.g. de-repression promoters, GAP) were developed. rPLC production The industrial production of rPLC with P. pastoris is based on sole glycerol feed-forward regime, where PLC gene is under regulation of AOX mutated promoter. Recombinant expression of PLC is possible only in limited glycerol concentration

when promoter de-repression occurs. Industrial production of the enzyme is performed in three phases: 1) batch phase on glycerol; 2) fed batch on glycerol at high growth rate to reach high cell densities and 3) fed batch on glycerol at lower growth rate to de-repress promoter and start rPLC production. During production phase, the cells are not capable to grow further due to glycerol limitation and since production is growth rate related (Arnau, Cassa et. al, 2011 and Files, Ogawa et. al, 2001), we were wondering if rPLC production is optimal. Therefore, we developed new feeding regime as mixed feed strategy, where we introduced second substrate to possibly increase growth rate and boost rPLC production. Connection between growth rate increase and increase of target protein production was already observed and described in other experiments (Zalai, Dietzsch et al., 2012). Our developed feeding regime was under control of a specific substrate uptake rate (qs) and softsensor. Spadiut and Herwig (2014) described qs as a novel and useful physiological parameter to design and control feeding regimes in P. pastoris cultivations. A few other studies (Khatri and Hoffmann, 2006) have also described qs as very useful parameter for recombinant protein production. Online mathematical tool or soft-sensor, was implemented and used for on-line biomass estimation as described by Wechselberger and Sagmeister et al. (2013). Estimated biomass by the tool and qs parameter were integral parts of our developed feeding regime for rPLC producing P. pastoris strain. Results and discussion Prior the mixed feed experiments, we had to identify second carbon source for a mixed feed strategy and to characterize strain on glycerol, since no physiological and productivity information was available. With following two approaches (developed at Vienna University of Technology), dynamic batch with different substrate pulses and dynamic fed batch experiments, we characterized our rPLC producing strain. After dynamic batch experiment with different substrate pulses (Dietzsch et al. 2011), we identified sorbitol as the most suitable second Csource. Furthermore, with dynamic fed batch experiments (Dietzsch et al. 2011), where in two separate experiments we applied different

CELLS: Development of an efficient feeding strategy for an industrial yeast strain

feed rates (qs control parameter) of glycerol and sorbitol over time, we determined optimal glycerol and sorbitol qs to design a mixed feed strategy. After several mixed feed experiments, we designed our final mixed feed strategy in the following order: 1) batch on glycerol; 2) fed batch on glycerol at high qs of glycerol to get higher cell densities; 3) decrease in glycerol feed to de-repression qs and; 4) start of sorbitol feed at pre-determined qs in parallel with glycerol feed. Main differences between industrial feeding regime and our regime was introduction of sorbitol at glycerol de-repression phase and use of qs and soft-sensor for feed control instead of growth rate. Comparison of industrial feeding regime with developed feeding regime To have reference values to our developed mixed feed strategy, we mimicked industrial feeding regime with fed batch at sole glycerol. When we compared values of both experiments, we observed 5.2-fold increase in growth rate, 2.7-fold increase in volumetric productivity and 2.3-fold increase in specific productivity during mixed feed strategy. These values brought us to a conclusion, that our developed mixed feed strategy brings significant increase not only in growth rate but also in rPLC production, when compared to industrial feeding regime at sole glycerol. Furthermore, we determined that rPLC production also depends on a specific ratio between glycerol and sorbitol qs.

References:

Arnau C., Casas C., Calero F. (2011). The effect of glycerol mixed substrate on the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system. Biochemical Engineering Journal - BIOCHEM ENG J, 01/2011; 57:30-37. Damasceno, L. M., et al. (2012). Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol, 93(1): 31-39. Higgins, D. R. and J. M. Cregg (1998). Introduction to Pichia pastoris. Methods Mol Biol, 103: 1-15. Hough, E., et al. (1989). High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus. Nature, 338(6213): 357-360. Khatri, N. K. and F. Hoffmann (2006). Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris. Appl Microbiol Biotechnol, 72(3): 492498. Kleiman, J. H. and W. E. Lands (1969). Purification of a phospholipase C from Bacillus cereus. Biochim Biophys Acta, 187(4): 477-485. Spadiut, O. and C. Herwig (2014). Dynamics in bioprocess development for Pichia pastoris. Bioengineered, 5(6). Srinivas, M., et al. (2008). Functional characterization of the phospholipase C activity of Rv3487c and its localization on the cell wall of Mycobacterium tuberculosis. J Biosci, 33(2): 221-230. Titball, R. W. (1993). Bacterial phospholipases C. Microbiol Rev, 57(2): 347-366. Wechselberger, P., et al. (2013). Real-time estimation of biomass and specific growth rate in physiologically variable recombinant fed-batch processes. Bioprocess Biosyst Eng, 36(9): 1205-1218. Weinhandl, K., et al. (2014). Carbon source dependent promoters in yeasts. Microb Cell Fact 13: 5. Zalai, D., et al. (2012). A dynamic fed batch strategy for a Pichia pastoris mixed feed system to increase process understanding. Biotechnol Prog, 28(3): 878-886.

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DETECTION AND IMAGE ANALYSIS OF CLINICAL FUNGAL BIOFILMS Marina Pekmezović, Institute

Microbiology

and Immunology,

Faculty

Medicine, University

Belgrade

National Reference Laboratory for Medical Mycology (NRL-MM; Faculty of Medicine, University of Belgrade) was named in 2008 in order to deal with problems caused by microscopic fungi in medicine, taking into account high prevalence of superficial fungal infections (SFI) and the high mortality of invasive fungal infections (IFI) in high-risk patients worldwide. SFI are the fourth most common disease in humans, fungi that cause them are highly contagious, diseases are chronic and require long-term course of treatment that may fail without laboratory confirmation. On the other hand, IFIs are severe, often fatal diseases occurring primarily in neonates, immunocompromised persons, as well as people with hemato-oncological malignancies and transplanted organs/tissues. Mortality is very high (40-100%) because IFI often remain unrecognized, undiagnosed, untreated and unregistered. Therefore, IFI are in the focus of research, because it is estimated to cause mortality as much as malaria or tuberculosis. NRL-MM is the only laboratory in Serbia which performs expert diagnosis of both SFIs and IFI, based on novel immunological and molecular techniques. Additionally, in NRL-MM many research activities are performed based on improvement of prevention, diagnosis and therapy of SFI and IFI.

Introduction Hospitalized high risk patients, especially neonates, patients with hemato-oncological malignancies, transplantation or any other condition of severe and prolonged immunosuppression are under risk for developing invasive fungal infections (IFI) (Rüping et al., 2008). The widespread use of antibiotics, frequent use of indwelling medical devices and a trend toward increased patient immunosuppression lead to opportunity for clinically important fungi, especially Candida spp., to develop adherent biofilm structures on different surfaces (Odds, 1988; Ramage and Williams, 2013) from which fungal cells can detach and cause an IFI associated with high mortality that makes fungal biofilms increasingly important clinical problem (Ramage and Williams, 2013). Biofilms are associated with drastically enhanced resistance against most antimicrobial agents, leading to the persistence of infection despite antifungal therapy (Douglas, 2003; Mukherjee and Chandra, 2004). Optimal diagnostic approach is therefore based on detection of biofilm-forming fungi and its susceptibility testing. However, simple methods for its analysis are lacking, so the aim of this study was to prospectively collect clinical Candida strains from blood in one-year period and test them for biofilm-testing capability. Additionally, we performed quantitative analysis of biofilms in vitro by digital analysis of biofilm photomicrographs

and developed of kinetic model for description and prediction of the fungal biofilm dynamics. Results A total of 32 Candida isolates were collected: C. albicans (18/32; 56.3%), C. parapsilosis (11/32; 34.4%) and C. tropicalis (3/32; 9.3%). The ability of biofilm formation was present in 84.4% (27/32) of tested strains: 88.8% of tested C. albicans (16/18), 72.7% of C. parapsilosis (8/11) and all C. tropicalis strains (3/3, 100%). In order to analyze structure and dynamics, biofilms were developed in vitro on plastic cover slips and photographed in different time points. The image analysis was done in software “Image J” and two biofilm parameters were quantitatively evaluated: the size by measuring the area (A) and the complexity of biofilm by fractal analysis of image (D). Logistic kinetic model was used to describe the increase of the two parameters of 2D biofilm image during its growth based on A and D values obtained during time. The experimental data successfully fitted exponential equation. The agreement between the predicted and experimental values was observed which proved that the kinetic model was appropriate for the description and prediction of A and D parameters. Kinetic model provided rate constants for both parameters (αsize and αcomp for A and D, respectively) where αsize had a higher value than αcomp (5.968±0.062 and 0.409±0.024, respectively). A positive linear correlation was observed between A and D (r=0.9, p<0.05).

CELLS: Detection and image analysis of clinical fungal biofilms

Discussion Candida species are the most common etiologic agent of fungal-related biofilm infection (Wisplinghoff et al., 2004; Ramage and Williams, 2013). Patients with candidemia caused by Candida biofilm-forming isolates have higher hospital mortality, longer hospital stay and higher costs of antifungal therapy comparing to those infected by Candida non-biofilm-forming isolates (Tumbarello et al, 2012). The majority of strains in our study were able to form biofilm (84.4%) so the next step in this study is to correlate biofilm formation with clinical patient data on risk factors, therapy and outcome. Providing the data on biofilm capability of isolates to clinicians can be useful for optimal therapy approach for their patients. The biofilm structure consists of complex features and it is impossible to analyze the morphology of biofilm without using some analysis of image. Fractal analysis offers a quantitative description of two-dimensional pattern of biofilm, which objectively evaluates its geometrical complexity (Yang et al. 2000). We calculated two parameters biofilm features: the size of the biofilm (A) and its complexity (D). Similar analyses have been performed previously, but these were aimed to quantifying biofilm structure in an arbitrary division into 2 developmental periods (Yang et al. 2000) or attempted to quantify development of fungal biomass in solid-state fermentation (Yingyi et al., 2012). Our results suggest that changes of the A and D of the biofilm during its growth can be presented with a logistic kinetic model which enables prediction of biological process. The differences in constants for these two parameters indicated that biofilms tend to first enlarge their size and then to increase complexity, which explains why antifungal agents are less effective in mature biofilms. Prediction and better understanding of biofilm dynamics can provide more insight useful for local eradication of formed biofilms on different structures such as human skin/mucosa and medical device surfaces. This can reduce the risk for entering of Candida in the patient bloodstream and prevent of Candida-related IFI. Biofilm images used for modeling were obtained using novel, cost-effective and simple approach which can provide detection and prediction of

the biofilm dynamics. This can be useful for further studies on kinetics of fungal biofilm and contribute to better clinical management of local biofilms and prevention of IFI. References:

1. Douglas, L.J. Candida biofilms and their role in infection. Trends Microbiol. 2003; 11: 30–36. 2. Mukherjee PK, Chandra J. Candida biofilm resistance. Drug Resist Updat. 2004; 7(4-5):301-9. 3. Odds, F. Candida and candidosis (2nd ed.). London 1988: Bailliere Tindall. 4. Ramage G and Williams C. The clinical importance of fungal biofilms. Advances in Applied Microbiology. 2013; 84: 27-83. 5. Rüping MJ, Vehreschild JJ, Cornely OA. Patients at high risk of invasive fungal infections: when and how to treat. Drugs. 2008; 68(14):1941-62. 6. Tumbarello M, Fiori B, Trecarichi EM, Posteraro P, Losito AR, De Luca A, Sanguinetti M, Fadda G, Cauda R, Posteraro B. Risk factors and outcomes of candidemia caused by biofilm-forming isolates in a tertiary care hospital. PLoS One. 2012; 7(3):e33705. 7. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis. 2004; 39(3): 309-17. 8. Yang XM, Beyenal H, Harkin G, Lewandowski Z. Quantifying biofilm structure using image analysis. J Microbiol Methods 2000; 39:109-19. 9. Duana Y, Wanga L, Chen H. Digital image analysis and fractal-based kinetic modelling for fungal biomass determination in solid-state fermentation. Biochemical Engineering Journal 2012; 67(15):60–7.

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PRESENTATION OF WORK AT THE CENTRE FOR PLANT BIOTECHNOLOGY Štajner N., Jakše J., Bohanec B., Luthar Z., Murovec J., Javornik B. University

Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia

The main mission of the Centre for Plant Biotechnology and Breeding is education at graduate and postgraduate levels, training pedagogic, research and professional personnel and presenting the research to the wider public. Members of the group have been teaching at agricultural and biotechnology study programs courses on genetics, plant breeding, plant biotechnology and related subjects. The research of the Centre includes basic and applied research in the field of food and agriculture. The group has developed contemporary biotechnological methods for application in plant breeding and applied numerous molecular approaches in studies of the genetic mechanisms underlying various crop properties. The current group at the Centre has provided a strong research core for a number of years. The group has developed several methods in the field of plant breeding and introduced a series of molecular marker systems. The group has set up a genomic (PCR cyclers, real-time PCR, DNA sequencer, bioinformatics facilities) and proteomic (2-D system, bioinformatics facilities) lab and has experience in maintenance and management of genetic resources (gene banks) in hop, buckwheat, olive and grapevine. The research group has also introduced novel methods for plant pathogen diagnostics and has great expertise in the analysis of plant-pathogen interactions and molecular variability for distinguishing and identifying varieties (and intra-varietal variability). The most advanced facility just recently acquired by the Centre is NGS facility (Ion Proton sequencer), which opens new possibilities of research and development. The research program of our group consists of different modules, which are presented in the text below:

Development of biotechnological methods in plant breeding Procedures for the induction of haploid plants have been developed on a long term basis with two aims: to accelerate breeding goals and for use in genetic studies. Such studies have been completed in onions (Jakše et al., 2010). A haploid induction method was handed over to a Slovenian seed company, by whom it is applied. Haploid induction in Mimulus aurantiacus was published as the first such protocol (Murovec and Bohanec, 2013), enabling further studies in this ornamental species, which is often used as a model species in evolutionary studies. We have also developed a protocol for adventitious organogenesis in this species (Murovec et al., 2010), and an efficient genetic transformation protocol (Susič et al., 2014). Study of oil seed pumpkins and zucchini has been directed at protocol development and cultivar release (Košmrlj et al., 2013, 2014, 2015). A haploid induction method was developed some years ago in white cabbage and other vegetable brassicas (Rudolf et al. 1999) and is now applied in breeding hybrid cultivars. We planned a study of gynogenesis of olive trees in our program but, due to recalcitrance, this research was redirected into other biotechnological procedures for application in olive breeding. We carried out several studies, two of which were particularly successful: the use of induced

mutations in in vitro culture systems (Oražem et al., 2013) and identification of endogenous pathogenic fungi (Oražem et al. in press). We have performed some minor studies of in vitro production of complex secondary metabolites, in particular of mandragora and St. John’s wort. In the latter, we established in vitro protocols (cell suspension cultures) and discovered major genetic diversity, which is reflected both on various levels of ploidy and in the production of active ingredients. Studies of genetic variability Genetic variability and diversity has been studied with the tools of molecular markers and statistical approaches and the obtained results have assisted in various studies: assessment of the origins of Allium ampeloprasum horticultural groups and their molecular phylogeny (Hirschegger et al., 2006, 2010); genetic structures and the origin of germplasm of sage (Radosavljević et al. 2011, 2012); specialisation and identification of plant pathogens within fungi species of Verticillium (Radišek et al. 2006), Monilinia (Gril et al. 2008, 2010) and some hop viroids (Jakše et al. 2014); evaluation of accessions in gene banks and assessment of relationships among varieties of hop (Štajner et al. 2008; Howard et al. 2011, Čerenak et al. 2012), grapevines (Štajner et al. 2008, 2009, 2011, 2013, 2015; Tomić et al. 2012, 2013) and olives (Bandelj et

PLANT BIOTECHNOLOGY: Presentation of work at the Centre for Plant Biotechnology

al. 2004; Bandelj and Javornik 2007; Jakše et al. 2013; Arbeiter et al. 2014) as well as in a search for new candidate genes in olives (Rešetič et al. 2013) and QTLs for different traits of hop (Čerenak et al. 2009, McAdam 2013). We have also developed a large number of genomic and genic microsatellite markers in hops (Štajner et al. 2005; Jakše et al. 2008, 2011) and in common sage (Radosavljević et al. 2011) and RGA resistance gene analogous (Kozjak et al. 2009) markers and EST-RGA markers in hops (Majer et al. 2014). Studies of biotic stress Our research group has great expertize in studying the phytopathogenic fungus Verticillium albo-atrum, which causes lethal damage to hop (Humulus lupulus L.), since the only effective way of combating the disease is host resistance. Our efforts are therefore aimed at increasing the efficiency of hop resistance breeding based on knowledge of the mechanisms of hop resistance to Verticillium wilt and of the pathogenicity of V. albo-atrum. We have constructed a linkage map using all available markers on the mapping population segregating for Verticillium wilt (Jakše et al. 2013). By means of QTL analysis, we were able to identify significant QTL for wilt resistance in one linkage group on both parental maps, accounting for 24.2 to 26.0% of the phenotypic variance. This is the first identified QTL for Verticillium wilt resistance in hop flanked by closely linked markers, which should be further tested for their use in marker assisted selection. Hop-Verticillium interactions have been studied on proteome (Mandelc et al. 2009, 2013; Mandelc and Javornik 2015) and transcriptome levels (Štajner et al. 2013; Cregeen et al. 2015) following changes in susceptible and resistant hops after infection with V. albo-atrum. On the proteome level, specific changes in the susceptible cultivar but no infection-specific changes were shown in the incompatible interaction, although some identified lectin isoforms may be implicated in the resistance reaction. Transcriptional analysis has so far identified differentially express genes implicated in resistance, which were verified by qPCR. On the fungus side, we have undertaken a whole genome sequencing approach with the aim of finding the virulence factor in highly aggressive

strains to explain the pathogenicity of V.alboatrum. In parallel, we have also established deletion mutation methods and a virulence assay to be able to test candidate virulence genes for their function. Studies of sex determination in plants In studies of sex determination in hops, a large number of hermaphrodite hop plants from various control crosses were phenotypically evaluated in the field and analysed by microsatellite markers and by means of flow cytometry (Jakše et al. 2008, Škof et al 2012). We showed that triploids can occur spontaneously in crosses and that they originate from unreduced gametes. Additionally, we found four new DaRT markers with known sequences tightly linked to the male sex locus and successfully converted them into SCAR markers. Plant genetic resources at the Biotechnical Faculty of the University of Ljubljana In the framework of the National Program of Slovenian Plant Gene Bank, there is also an important gene bank at the Department of Agronomy at the Biotechnical Faculty of University in Ljubljana. It stores genetic resources of agricultural plants: buckwheat, wheat (to a minor extent barley, rye, oats and millet), maize, forage species (perennial and Italian ryegrasses, cocksfoot, red and white clover, woolly honey grass, alfalfa, birdsfoot trefoil), fruit plants (apple trees and pears), as well medicinal and aromatic plants, especially those which are most endangered (Gentiana lutea, Rhodiola rosea, Arnica montana, Carum carvi, Salvia officinalis, Thymus spp., Satureja montana, Origanum vulgare). The main aim of our gene bank is to preserve genetic variability in interesting populations, and wild plants that have never been raised and cultivated. The primary reasons for collecting and maintaining gene bank samples are the abandonment of cultivation of some varieties and populations, the possibility that varieties and populations could outcross and the preservation of genetic variability within varieties and populations, which can be used in various genetic studies and provide a valuable source of genes for breeding new varieties (Bohanec 2012, Luthar et al. 2012; Luthar 2012; Luthar et al. 2010). Collected samples in gene banks are kept, multiplied and evaluated according to

Science for you, November 2015

agreed international standards for plant gene banks.

highly aggressive pathogen on hop. Plant Pathology, ISSN 0032-0862, 2014.

References:

JAKŠE, Jernej, ŠTAJNER, Nataša, KOZJAK, Petra, ČERENAK, Andreja, JAVORNIK, Branka. Trinucleotide microsatellite repeat is tightly linked to male sex in hop (Humulus lupulus L.). Molecular breeding, ISSN 13803743. [Tiskana izd.], 2008, vol. 21, no. 2, str. 139-148.

BANDELJ MAVSAR, Dunja, JAKŠE, Jernej, JAVORNIK, Branka. Assessment of genetic variability of olive varieties by microsatellite and AFLP markers. Euphytica, ISSN 0014-2336. [Print ed.], 2004, vol. 136, str. 93-102. BANDELJ MAVSAR, Dunja, JAVORNIK, Branka. Microsatellites as a powerful tool for identification of olive (Olea europaea L.) planting material in nurseries. Annales, Series historia naturalis, ISSN 1408-533X, 2007, letn. 17, št. 1, str. 133-138.

JAKŠE, Jernej, ŠTAJNER, Nataša, LUTHAR, Zlata, JELTSCH, Jean-Marc, JAVORNIK, Branka. Development of transcript-associated microsatellite markers for diversity and linkage mapping studies in hop (Humulus lupulus L.). Molecular breeding, ISSN 1380-3743. [Tiskana izd.], 2011, vol. 28, no. 2, str. 227-239.

BOHANEC B. 2012. Genebanks as cultural heritage of mankind. Acta agriculturae Slovenica, 99, 3: 289¬-293.

JAKŠE, Jernej, ŠTAJNER, Nataša, TOMIĆ, Lidija, JAVORNIK, Branka. Application of microsatellite markers in grapevine and olives. V: POLJUHA, Danijela (ur.), SLADONJA, Barbara (ur.). The Mediteranean genetic code - grapevine and olive. Rijeka: InTech, 2013, str. 25-50.

ČERENAK, Andreja, JAKŠE, Jernej, ŠTAJNER, Nataša, JAVORNIK, Branka. Vrednotenje genskih virov hmelja z molekulskimi markerji = Evaluation of hop genetic resources with molecular markers. Acta agriculturae Slovenica, ISSN 1581-9175. [Tiskana izd.], 2012, letn. 99, št. 3, str. 355-361.

JAKŠE, Marijana, HIRSCHEGGER, Pablo, BOHANEC, Borut, HAVEY, Michael J. Evaluation of gynogenic responsiveness and pollen viability of selfed doubled haploid onion lines and chromosome doubling via somatic regeneration. Journal of the American Society for Horticultural Science, ISSN 0003-1062. [Print ed.], 2010, vol. 135, no. 1, str. 67-73.

ČERENAK, Andreja, ŠATOVIĆ, Zlatko, JAKŠE, Jernej, LUTHAR, Zlata, CAROVIĆ-STANKO, Klaudija, JAVORNIK, Branka. Identification of QTLs for alpha acid content and yield in hop (Humulus lupulus L.). Euphytica, ISSN 0014-2336. [Print ed.], 2009, vol. 170, no. 1-2, str. 141-154.

KOŠMRLJ, Kristina, KASTELEC, Damijana, BOHANEC, Borut. Styrian oil pumpkin pollen germinability at higher irradiation doses : optimization of the in vitro germination protocol and irradiation procedure. Turkish journal of biology, ISSN 1300-0152, 2014, vol. 38, iss. 4, str. 516-522.

CREGEEN, Sara, RADIŠEK, Sebastjan, MANDELC, Stanislav, TURK, Boris, ŠTAJNER, Nataša, JAKŠE, Jernej, JAVORNIK, Branka. Different gene expressions of resistant and susceptible hop cultivars in response to Infection with a highly aggressive strain of Verticillium albo-atrum. Plant molecular biology reporter, ISSN 0735-9640, 2015, vol. 33, iss. 3, str. 689-704.

KOŠMRLJ, Kristina, KLADNIK, Aleš, BOHANEC, Borut. Adventitious regeneration in styrian oil pumpkin in relation to the endoreduplication pattern and induced tetraploidy on fusaric acid-supplemented media. Plant growth regulation, ISSN 0167-6903, 2015, vol. 75, iss. 3, str. 587594.

GRIL, Tjaša, CELAR, Franci Aco, JAVORNIK, Branka, JAKŠE, Jernej. Fluorescent AFLP fingerprinting of Monilinia fructicola = FluoreszenzAFLP-Fingerprinting von Monilinia fructicola. Journal of plant diseases and protection, ISSN 1861-3829. [Print ed.], 2010, vol. 117, no. 4, str. 168-172. GRIL, Tjaša, CELAR, Franci Aco, MUNDA, Alenka, JAVORNIK, Branka, JAKŠE, Jernej. AFLP analysis of intraspecific variation between Monilinia laxa isolates from different hosts. Plant disease, ISSN 0191-2917, dec. 2008, vol. 92, no. 12, str. 1616-1624. HIRSCHEGGER, Pablo, GALMARINI, Claudio, BOHANEC, Borut. Characterization of a novel form of fertile great headed garlic (Allium sp.). Plant breeding, ISSN 0179-9541. [Print ed.], 2006, vol. 125, str. 635-637. HIRSCHEGGER, Pablo, JAKŠE, Jernej, TRONTELJ, Peter, BOHANEC, Borut. Origins of Allium ampeloprasum horticultural groups and a molecular phylogeny of the section Allium (Allium: Alliacea). Molecular phylogenetics and evolution, ISSN 1055-7903, 2010, vol. 54, no. 2, str. 488-497. HOWARD, E. L., WHITTOCK, S., JAKŠE, Jernej, CARLING, J., MATTHEWS, P., PROBASCO, G., HENNING, J. A., DARBY, Peter E., ČERENAK, Andreja, JAVORNIK, Branka, KILIAN, A., KOUTOULIS, A. High-throughput genotyping of hop (Humulus lupulus L.) utilising diversity arrays technology (DArT). Theoretical and Applied Genetics, ISSN 0040-5752. [Print ed.], 2011, vol. 122, no. 7, str. 1265-1280. JAKŠE, Jernej, ČERENAK, Andreja, RADIŠEK, Sebastjan, SATOVIĆ, Zlatko, LUTHAR, Zlata, JAVORNIK, Branka. Identification of quantitative trait loci for resistance to Verticillium wilt and yield parameters in hop (Humulus lupulus L.). Theoretical and Applied Genetics, ISSN 0040-5752. [Print ed.], 2013, vol. 126, no. 6, str. 1431-1443. JAKŠE, Jernej, LUTHAR, Zlata, JAVORNIK, Branka. New polymorphic dinucleotide and trinucleotide microsatellite loci for hop Humulus lupulus L. Molecular ecology resources, ISSN 1755-098X, 2008, vol. 8, no. 4, str. 769-772. JAKŠE, Jernej, RADIŠEK, Sebastjan, POKORN, Tine, MATOUŠEK, Jaroslav, JAVORNIK, Branka. Deep-sequencing revealed a CBCVd viroid as a

KOŠMRLJ, Kristina, MUROVEC, Jana, BOHANEC, Borut. Haploid induction in hull-less seed pumpkin through parthenogenesis induced by Xray-irradiated pollen. Journal of the American Society for Horticultural Science, ISSN 0003-1062. [Print ed.], 2013, vol. 138, no. 4, str. 310-316. KOZJAK, Petra, JAKŠE, Jernej, JAVORNIK, Branka. Isolation and sequence analysis of NBS-LRR disease resistance gene analogues from hop Humulus lupulus L. Plant science, ISSN 0168-9452. [Print ed.], 2009, vol. 176, issue 6, str. 775-782. LUTHAR et al. 2012. Genebank of the Agronomy department of the Biotechnical faculty in Ljubljana. Acta agriculturae Slovenica, 99, 3: 301¬306. LUTHAR Z. 2012. Buckwheat genebank ¬ a source of Slovenian genetic variability. Acta agriculturae Slovenica, 99, 3: 307-¬316. LUTHAR, Zlata, ROZMAN, Ludvik, OSTERC, Gregor, ČOP, Jure. Genska banka Oddelka za agronomijo, Biotehniške fakultete, Univerze v Ljubljani. V: MEGLIČ, Vladimir (ur.). Slovenska rastlinska genska banka v mednarodnem letu biodiverzitete : knjiga povzetkov, [Ljubljana, 19. oktobra 2010]. Ljubljana: Kmetijski inštitut Slovenije, 2010, str. 12. MAJER, Aljaž, JAVORNIK, Branka, ČERENAK, Andreja, JAKŠE, Jernej. Development of novel EST-derived resistance gene markers in hop (Humulus lupulus L.). Molecular breeding, ISSN 1380-3743. [Tiskana izd.], 2014, vol. 33, issue 1, str. 61-74. MANDELC, Stanislav, JAVORNIK, Branka. The secretome of vascular wilt pathogen Verticillium albo-atrum in simulated xylem fluid. Proteomics, ISSN 1615-9853. [Print ed.], 2015, v tisku. MANDELC, Stanislav, RADIŠEK, Sebastjan, JAMNIK, Polona, JAVORNIK, Branka. Comparison of mycelial proteomes of two Verticillium alboatrum pathotypes from hop. European journal of plant pathology, ISSN 0929-1873, 2009, vol. 125, no. 1, str. 159-171. MANDELC, Stanislav, TIMPERMAN, Isaak, RADIŠEK, Sebastjan, DEVREESE, Bart, SAMYN, Bart, JAVORNIK, Branka. Comparative proteomic profiling in compatible and incompatible interactions between hop roots and Verticillium albo-atrum. Plant physiology and biochemistry, ISSN 0981-9428. [Print ed.], 2013, vol. 68, str. 23-31.

PLANT BIOTECHNOLOGY: Presentation of work at the Centre for Plant Biotechnology

MCADAM, Erin L., FREEMAN, Jules S., WHITTOCK, Simon P., BUCK, Emily J., JAKŠE, Jernej, ČERENAK, Andreja, JAVORNIK, Branka, LILIAN, Andrzej, WANG, Cai-Hong, ANDERSEN, Dave, VAILLANCOURT, René E., CARLING, Jason, BEATSON, Ron, GRAHAM, Lawrence, GRAHAM, Donna, DARBY, Peter E., KOUTOULIS, Anthony. Quantitative trait loci in hop (Humulus lupulus L.) reveal complex genetic architecture underlying variation in sex, yield and cone chemistry. BMC genomics, ISSN 14712164, 2013, vol. 14, art. no. 360, str. 1-27. MUROVEC, Jana, BOHANEC, Borut. Haploid induction in Mimulus aurantiacus Curtis obtained by pollination with gamma irradiated pollen. Scientia horticulturae, ISSN 0304-4238. [Print ed.], 2013, vol. 162, str. 218-225 MUROVEC, Jana, ELER, Klemen, BOHANEC, Borut. Adventitious shoot regeneration from leaf and internodal explants of Mimulus aurantiacus Curtis. Propagation of ornamental plants, ISSN 1311-9109. [Tiskana izd.], 2010, vol. 10, no. 1, str. 18-23 ORAŽEM, Petra, ŠTAJNER, Nataša, BOHANEC, Borut. Effect of X-ray irradiation on olive shoot culture evaluated by morphological measurements, nuclear DNA content and SSR and AFLP markers. Trees, ISSN 0931-1890, 2013, vol. 27, issue 6, str. 1587-1595 RADIŠEK, Sebastjan, JAKŠE, Jernej, JAVORNIK, Branka. Genetic variability and virulence among Verticilium albo-atrum isolates from hop. European journal of plant pathology, ISSN 0929-1873, 2006, 116, str. 301-314. RADOSAVLJEVIĆ, Ivan, JAKŠE, Jernej, JAVORNIK, Branka, SATOVIĆ, Zlatko, LIBER, Zlatko. New microsatellite markers for Salvia officinalis (Lamiaceae) and cross-amplification in closely related species. American journal of botany, ISSN 0002-9122, 2011, vol. 98, no. 11, str. 316-318. RADOSAVLJEVIĆ, Ivan, SATOVIĆ, Zlatko, JAKŠE, Jernej, JAVORNIK, Branka, GREGURAŠ, Danijela, JUG-DUJAKOVIĆ, Marija, LIBER, Zlatko. Development of new microsatellite markers for Salvia officinalis L. and its potential use in conservation-genetic studies of narrow endemic Salvia brachyodon Vandas.International journal of molecular sciences, ISSN 1661-6596, 2012, vol. 13, issue 9, str. 12082-12093. REŠETIČ, Tjaša, ŠTAJNER, Nataša, BANDELJ MAVSAR, Dunja, JAVORNIK, Branka, JAKŠE, Jernej. Validation of candidate reference genes in RT-qPCR studies of developing olive fruit and expression analysis of four genes involved in fatty acids metabolism. Molecular breeding, ISSN 1380-3743. [Tiskana izd.], 2013, vol. 32, issue 1, str. 211-222. RUDOLF PILIH, Katarina, BOHANEC, Borut, HANSEN, Magnor. Microspore culture of white cabbage, Brassica oleracea var. capitata L. : Genetic improvement of non-responsive cultivars and effect of genome doubling agents. Plant breeding, ISSN 0179-9541. [Print ed.], 1999, 118, str. 237-241

ŠKOF, Suzana, ČERENAK, Andreja, JAKŠE, Jernej, BOHANEC, Borut, JAVORNIK, Branka. Ploidy and sex expression in monoecious hop (Humulus lupulus). Botany, ISSN 1916-2790. [Tiskana izd.], 2012, vol. 90, no. 7, str. 617-626. ŠTAJNER, Nataša, ANGELOVA, Elizabeta, BOZINOVIĆ, Zvonimir, PETKOV, Mihail, JAVORNIK, Branka. Microsatelite marker analysis of Macedonian grapevines (Vitis vinifera L.) compared to Bulgarian and Greek cultivars. Journal international des sciences de la vigne et du vin, ISSN 1151-0285, 2009, vol. 43, no. 1, str. 29-34. ŠTAJNER, Nataša, JAKŠE, Jernej, KOZJAK, Petra, JAVORNIK, Branka. The isolation and characterisation of microsatellites in hop (Humulus lupulus L.). Plant science, ISSN 0168-9452. [Print ed.], 2005, vol. 168, no. 1, str. 213-221. ŠTAJNER, Nataša, ŠATOVIĆ, Zlatko, ČERENAK, Andreja, JAVORNIK, Branka. Genetic structure and differentiation in hop (Humulus lupulus L.) as inferred from microsatellites. Euphytica, ISSN 0014-2336. [Print ed.], 2008, vol. 161, no. 1-2, str. 301-311. ŠTAJNER, Nataša, KOROŠEC-KORUZA, Zora, RUSJAN, Denis, JAVORNIK, Branka. Microsatellite genotyping of old Slovenian grapevine varieties (Vitis viniferaL.) of the Primorje (coastal) winegrowing region. Vitis, ISSN 0042-7500, 2008, vol. 47, no. 4, str. 201-204. ŠTAJNER, Nataša, RUSJAN, Denis, KOROŠEC-KORUZA, Zora, JAVORNIK, Branka. Genetic characterization of old Slovenian grapevine varieties of Vitis vinifera L. by microsatellite genotyping. American journal of enology and viticulture, ISSN 0002-9254. [Print ed.], 2011, vol. 62, issue 2, str. 250-255. ŠTAJNER, Nataša, TOMIĆ, Lidija, BACILIERI, Roberto, PROGAR, Vasja, POKORN, Tine, LACOMBE, Thierry, LAUCOU, Valérie, BOURSIQUOT, Jean-Michel, JAVORNIK, Branka. Genetic clustering and parentage analysis of Western Balkan grapevines (Vitis vinifera L.). Vitis 54, 2015 (Special issue), ?–? (accepted). ŠTAJNER, Nataša, TOMIĆ, Lidija, IVANIŠEVIĆ, Dragoslav, KORAĆ, Nada, JOVANOVIĆ CVETKOVIĆ, Tatjana, BELESKI, Klime, ANGELOVA, Elizabeta, MARAŠ, Vesna, JAVORNIK, Branka. Microsatellite inferred genetic diversity and structure of Western Balkan grapevines (Vitis vinifera L.). Tree genetics & genomes, ISSN 1614-2942, 2013, vol. 10, iss. 1, str. 127-140. TOMIĆ, Lidija, ŠTAJNER, Nataša, JAVORNIK, Branka. Characterization of grapevines by the use of genetic markers. V: POLJUHA, Danijela (ur.), SLADONJA, Barbara (ur.). The Mediteranean genetic code - grapevine and olive. Rijeka: InTech, 2013, str. 3-23. TOMIĆ, Lidija, ŠTAJNER, Nataša, JOVANOVIĆ-CVETKOVIĆ, Tatjana, CVETKOVIĆ, Miljan, JAVORNIK, Branka. Identity and genetic relatedness of Bosnia and Herzegovina grapevine germplasm. Scientia horticulturae, ISSN 0304-4238. [Print ed.], 2012, vol. 143, str. 122-126.

Science for you, November 2015

BIOINFORMATICS – BEGINNERS’ GUIDE Luka Ausec, Genialis d.o.o.

Bioinformatics may seem like a holy grail of life sciences – the long awaited magic wand that will help to push our understanding of the biological reality onto whole new levels. Bioinformaticists everywhere are among the most desired colleagues and often whole departments cling on the spare moments of those busy men and women. Let us try to distil the characteristics of these half-gods and list the skills needed to approach divinity. Introductory articles describing bioinformatics often begin by explaining the unprecedented accumulation of molecular data that has now exceeded the Moore’s law. There are claims (with some restrictions) that resequencing the organism’s genome is now cheaper that longterm storage of the raw data used to obtain the sequence in the first place. Everyone is aware of the big data, so this hardly needs mentioning. Or does it?

Working in the fields of bioinformatics in academia as well as in a private company, we are often surprised about the extent to which big data is not taken seriously. Individual research groups and even small institutes dream of investing several thousand euros into a new desktop computer and a brand new PhD student that will somehow solve the problems produced by their newly acquired sequencing machine. The heads of the departments, often blissfully unaware of the amount of computational power as well as computer skills needed to analyze this data, are quite content to leave the worries to their unassuming young employees. Even if the computational infrastructure is provided, it is often just expected that the required skills will be picked up by the student and immediately put into practice.

In reality, the transition from a wet-lab expert into a fairly confident bioinformatician takes years of dedicated work with a lot of side-tracking and dead-ends and general frustration in the late night hours when the rest of the world seems to be resting. What is more, the supervisors often fail to provide any guidance as to what skills are necessary and in what order. This short essay aims help the young life-scientists as well their bosses in identifying the important areas that probably require immediate focus.

Linux: horror of the black screen UNIX based systems such as Linux are of outmost importance in bioinformatics. The plain command line may seem outdated in the era of polished graphical user interfaces, but it stood the test of time and it is here to stay. Modern Linux distributions (e.g. Ubuntu and its derivatives) are increasingly user friendly and not only support most features expected by a life-scientist but often exceeds the expectations. The powerful and versatile command line is often at the very top of the list of pleasant surprises. Admittedly, it takes a few hours to learn how to navigate the file system and perform simple everyday actions, but you do not need to become a Linux guru to achieve surprising results and in some cases eliminate the need for custom computer programs. With the abundant online resources as well as free Linux distributions, learning the command line is mostly an exercise in determination. Clever manipulation of files, advanced search and replace with regular expressions and ability to build pipelines are beyond doubt well worth your time.

YOUTH & ENTREPRENEURSHIP: Bioinformatics - Beginners’ guide

Super computers: when 4 cores are not enough The amount of data in real-life NGS experiments soon exceeds the capabilities of normal desktop computers not only in terms of processing power for the more greedy steps of the analysis (e.g. assembly of short reads into contigs), but also in terms of pure storage and data management. It is not trivial to keep track of the files, their origin or purpose, when the files are tens of GB in size and produced by slight alterations of existing pipelines. Desktop computers usually cannot satisfy these needs and cloud computing has recently emerged as a compelling solution providing virtually unlimited computing power and storage capacity for a reasonable price. When resorting to online providers, one has to pay special attention to the details, such as the pricing model, access to common databases such as GenBank, availability of required tools (such as BLAST) and possibilities to add them. These services are often accessed using the command line, some providers also have graphical interfaces enabling detailed annotation of each file thus managing different projects as well as different computational approaches within each project.

Programming: scripting vs. pipetting Most researchers realize sooner or later that a particular software or functionality within the available software does not exist in spite of the (for them) obvious and universal need for it. Programming enables researchers to tackle those genuinely interesting questions that nobody has asked before. Moreover, the liberation and empowerment that comes from automating repetitive tasks is inspiring and gives more time to actual science. Computer programming languages often seem daunting and life-scientists are often put off by their high personal goals of mastering the language. In reality, one usually doesn’t start programming once every little nuisance of the language is understood, but rather learns as he or she goes along. Solving particular problems

from the start provides the best motivation and actually delivers results, while limiting yourself to perfection before you actually start programming is just daydreaming. Different languages are suitable for different tasks. Novices are advised to tackle high-level scripting languages. Python is now among the most popular languages in life sciences. It is easy to learn, has a clean syntax and, most importantly, has dedicated packages of functions for different disciplines, e.g. for bioinformatics. Biopython thus lets you interact easily with online databases and automate tasks such as blasting sequences on NCBI. Another good choice is R. While its syntax is somewhat more cumbersome, it has almost unparalleled power in statistics, data exploration and visualisation. Perl used to be the standard language for bioinformatics for many years, but is now largely replaced by Python.

(Not)obvious: reproducible science Like too often in real life, considerations regarding reproducibility come last in our short review of basic bioinformatic tools. While many life-scientist have become really detailed with their description of wet-lab protocols, the description of the data analysis is often reduced to “statistics was done in MS Excel.” Unlimited copy-pasting data from table to table justifiably raises doubts about reproducibility and even validity of conclusions. Packaging the analyses procedures into executable code (scripts) relieves this problem and most often helps the original author to modify and refine the used approaches. Moreover, version control software such as Git help you track the development of your research and maintain order in the chaos of creativity. Conclusions Developing of these skills take time. However, we would argue that mastery of these skills saves even more time. And they open the doors to new possibilities especially in the careers of young scientists.

Science for you, November 2015

SCIENCE-MIX Non-profit

organisation

It is an international life-science organization for everyone who loves science and would like to make new friends from all around the world. Taking into consideration the importance of biotechnology for the development of modern science and also it’s influence on, biology, chemistry, pharmacy and medicine, an idea about this project was born spontaneously in 2009 year and realized in the year of 2010.

Figure 1: Infographis - Science mix developement and working vision

After five years of hard work, our team aims at achieving worldwide cooperation with students and experts from different countries thus we decided “Biotechnology days in Macedonia” to be renamed into “ScienceMIX”. Furthermore, we also embrace cooperation with all life-sciences scopes where biotechnology has an impact.

Figure 2: 5th Biotecnology conference in year 2014 in Stip, Macedoinia

The initial aim was to connect Macedonian and Slovenian students and to create informative environment where students could exchange and broaden their ideas about biotechnology field. Initially this project was created exclusively for undergraduate students, but since 2012 we spread our public group also for PhD students, as well as for junior and senior scientists with experience in biotechnology, medical and pharmaceutical field, which gave more significance to our ScienceMIX event.

We strongly believe that motivated students could change the future. No matter where are you in your professional career, you always need to study and feel like a student. Please contact as for any ideas for cooperation at info@science-mix.com or visit our web page www.science-mix.com. We inspire everyone, because we take care about the future of young scientist profiles.

YOUTH & ENTREPRENEURSHIP: Non-formal science

NON-FORMAL SCIENCE Larisa Lara Tomsic, A Biotechnology

student,

University

Ljubljana, Notranjska Regional Park

TV shows, museums, podcasts, radio-shows and channels prove that the science is becoming more and more popular, easy accessible and its importance is being recognised. Most of the children have first contact with an expression »science« in their schools. It could be discussed about a lack of inspirational science in schools but in contrary the next paragraphs will focus on the good examples of non-formal and informal education that focus on promotion, education and inspiration of children for science. Introduction Formal learning is always organised and structured, and it has learning objectives. Learner’s objective is always to gain knowledge, skills or competences. On the other hand, informal learning is never organised, has no set objective in terms of learning outcomes and is never intentional from the learner’s standpoint. Often it is referred to as learning by experience or just as experience. The idea is that the simple fact of existing constantly exposes the individual to learning situations, at work, at home or during leisure time for instance. A combination of both is non-formal learning. It is rather organised and can have learning objectives. Non-formal learning therefore gives some flexibility between formal and informal learning, which must be strictly defined as operational therefor being mutually exclusive, and avoid overlap. (OECD, 2015) In the article about importance of non-formal education the authors stated they found life, informal learning, and work experiences, non-formal learning, are relatively more important for gaining self-reported skills than formal training/study. These forms of learning have a great advantage of dynamic interrelatedness. (Cameron & Harrison, 2012) The other article took into consideration non-formal and informal science learning in schools. The conclusion was that the teachers consider the relationship among formal, non-formal and informal science complementary. Most teachers utilize non-formal and informal learning in ways that attract students’ interest. Children choose the form of learning, and therefore they are actively involved and attracted to the educational process. In formal education process should be implemented more non-formal and informal teaching material. (Sevdalis & Skoumios, 2014) Science holidays Different organisations provide activities for children based on a non-formal learning, learning by doing. This summer Notranjska Regional Park, nature and heritage protecting, preserving and exploring organisation, organised science holidays for children. The main aim was to show children what surrounds them in a compelling way with a by-product, teaching them. The activities combined experiments, field trips, games and the most important, learning about different science prospective. There were three projects within six months with 50 participants all together. The groups were formed by 7-15 children and two ‘teachers’ each. The interdisciplinary approach was found to be very successful. Five year olds isolated DNA and they understood what it is and why have we used some the ingredients. In conventional school the themes are divided by children’s age. This is a rough approximation of what suits the population the most. For example,

younger children are able to understand photosynthesis concepts and what mixes with what. But if the knowledge wants to be provided surficial the method used should be chosen carefully. Children love stories therefore I made up many. From how the trees grow to why is important for us and animals. There is a lack of connection between observations and learning materials in formal education. The cause may be its changing dynamic due to group specifics or just a lack of time to implement new methods even though most of the topics could be explained directly form nature or/and with experiments. Results The evaluation showed purpuses children applied to the program were: their wish, a lack of local knowledge and to put time to good advantage. Children liked the program because they have learned a lot of new things, they have spent time with their friends, and they have done experiments with plants, rocks, DNA, they have learned how to catch fish, extract invasive plants and so on. Nevertheless when they proudly explained to their parents what they have learned and explored showed us that we were doing something good. In addition, when we meet after few months, they could still remember the important things we have done and this is priceless. References:

Sevdalis C. and Skoumios M. (2014) Non-formal and Informal Science Learning: Teachers’ Conceptions. The International Journal of Science in Society, vol. 5, no. 4, pp. 13-25. Cameron R. and Harrison JL. (2012) The Interrelatedness of Formal, Non-Formal and Informal Learning: Evidence from Labour Market Program Participants. Australian Journal of Adult Learning, vol. 52, no. 2, pp. 277-309. http://www.oecd.org/edu/skills-beyond-school/recognitionofnon-formalandinformallearning-home.html (October 2015)

Figure 1: Experiments are children’s favourite activity (Source: NRP)

Science for you, November 2015

A YOUNG SCIENTIST’S JOURNEY Sabrina Lakotta, Institute

Microbiology

and Immunology,

Faculty

Medicine, University

Munich

I am a biological technical assistant (BTA) or lab technician for almost 7 years now. I got my first job at the Ludwig Maximillian University of Munich, at the Clinical Center in Großhadern ( or like a German would say : Klinikum Großhadern der LMU ). There, I was part of the neurosurgical research team and due to the positive working environment and changing challenges, I stayed there for over 6 years. During the years, I have worked on several projects concerning the understanding of the interaction of cell subpopulations within a brain tumor (mainly WHO°IV) and investigating, in general, the biology of primary brain tumors and the physiological role of stem cells, immune cells and the vasculature of the brain tumors. Moreover, exploring new strategies for the diagnosis and treatment of primary brain tumors (http://www.klinikum.uni-muenchen.de/Neurochirurgische-Klinik-und-Poliklinik/en/Research/index.html). Firstly, I got employed for an European project with cooperation partners in Munich, Berlin and Slovenia. Consequently, in the period from 2008 – 2012, I was part of the Systher project. “It was established to investigate and improve solid tumor treatment based on systems biology approaches. Identification of target genes and proteins, and subsequent mathematical modeling of candidate pathways, intracellular networks and interaction dynamics of immunocompetent, tumor as well as stem cell, aim at elucidating mechanisms of tumor immunomodulation and the role of tumor stem cells in solid, non-metastasizing tumors, improving thus therapeutic strategies. Serum antibody profiling and gene expression analysis of tumor and stem cells shall further lead to the understanding of immunomodulation mechanisms.” You can read all about this online (https://www.biologie.hu-berlin.de/de/gruppenseiten/systher) and since many data are not published yet or are in ongoing processes, I am not allowed to talk much about results. But what does it mean from the technical point of view? It was investigated that there are mesenchymal stem cells (MSC) existing in malignant brain tumors. Our lab isolated these cell types from GBM tissue provided from our operation room. By simply homogenizing the tissue and digesting it with collagenases, and then running the cell suspension over a Ficoll gradient, you can get the cells of interest. So, the protocol is almost the same as isolating MSC from bone marrow. After characterizing the MSC via differentiation assays (adipogenesis and osteogenesis) and flow cytometry profile (yes, I know that there still is a big discussion about the right markers), we tested them for different markers, which may bring value to diagnosis. Furthermore, we performed experiments observing the interaction of MSC with known gbm cell lines (U87, U373) in direct and indirect co-culturing. Also, there were experiments done under hypoxia conditions to get a closer picture if a hypoxic environment - as it is in some areas of a tumor - has an impact on the migration and proliferation behavior of the cells. As I said, I cannot go to detail. Just this much: tumor cells and MSC ARE attracted to each other! (what a surprise…) Over which factors is still an ongoing project. One of the theories is that it are factors transported in exosomes. My former group is right now working on that. Another part of the research processed the topic angiogenesis, especially the investigation of VEGFR3, -R2 and its antagonists. http://edoc.ub.uni-muenchen.de/17450/ Since 2011, our lab ran under the new lead of Prof. Dr. rer. nat. Rainer Glaß, PhD. The new main focus was now the investigation of neuronal progenitor cells (NPC) but also investigating already obtained effects not just in vitro but in vivo. Therefore, former experiments concerning angiogenesis and others were now strengthened by animal experiments. We implanted tumors in the mouse brain. The method was well established and performed with different cell types. As a known model we would use cell lines as well as experiments with self-isolated primary cells, which were of interest, concerning their behavior in tumor growth and expression of several markers. Additionally, we tested the effect of potential drugs against tumor treatment by implanting osmotic pumps which provided a constant delivery of the drug directly inside the tumor. Analyzes were done by isolating the brains after treatment or incubation time of the tumor (of course all animals were sacrificed to EU laws of handling laboratory animals), fixing them and then either measuring the tumor volume or doing immunochemical stainings. Moreover, our lab had an own breeding of mice, which enabled us to isolate NPCs of the genotype of interest. By isolating the subventricular zone out of the brain of young mice, we extracted viable NCPs for further experiments. At the end of February 2015, I have started with new challenges in Slovenia. Nowadays, I live in Ljubljana, Slovenia and I am doing an internship at EDUCELL d.o.o., which includes a cooperation project with the Blood Transfusion Center of Ljubljana (Zavod Republike Slovenije za transfuzijsko medicine). I am still curious of new experiences and knowledge and I think the field of science is the best to live this excitement about new things and learn to understand. “Any fool can know. The point is to understand.” ― Albert Einstein If anyone has questions about my article, researches I have done or my person, please do not hesitate to contact me. E-mail: sabrina.lakotta@gmail.com

Science for you, November 2015

THIS PUBLICATION WAS FOUNDED BY: The Student Organisation of the University of Ljubljana - ŠOU Ljubljana organises weekly meetings for all incoming students where students can pick up all sorts of information about sports activities, pub-crawls and weekend trips around Slovenia that will soon get them acquainted with Slovenia, its people and culture. Activities of ŠOU Ljubljana In order to realise the objectives for which it was founded, ŠOU Ljubljana performs the following functions and activities: - it represents student interests and works for their realisation, - it organises and implements activities for students and supports student activities in the fields of culture, sport, education, technology, tourism, international cooperation, public media, civil initiatives and other interest areas of students of the University of Ljubljana. The permanent activities of ŠOU Ljubljana are carried out by: the Student Advice Service, which offers social and legal advice and a free room exchange (m2), Student Food, which sells vouchers for student meals, KISS (the ŠOU Ljubljana communications and information centre), which offers students free access to the internet and an electronic mailbox, the Ljubljana Secondary School Pupils’ Association, and the Information Office, which keeps students informed about ŠOU Ljubljana’s projects and activities. ŠOU Ljubljana is one of the most representative university student organisations in Europe.

SOBF or the Student Organisation of the Biotechnical Faculty is an organisation that is primarily intended for organising extracurricular activities for students of the BF. At the same time, it also strives with the aid of SCBF and SCU in Ljubljana, to which SOBF actually belongs, for the rights of students on the level of the faculty, university and even state. All students enrolled in the faculty are members of the SOBF, and it is headed by an administrative council consisting of representatives of all departments and inter-departmental studies, and the president, vice-president and representative of the student council.

Genialis is offering bioinformatics services and support, where a team of data scientists with both computer and life science background analyze your data and help you with the interpretation. Most of our previous projects covered different kind of basic data analysis, biostatistics and data mining tasks, however we also provide custom solutions for NGS and microarray data management, analysis automation and real-time interactive visualizations. We also share our in-house knowledge by organising bioinformatics hands-on workshops called BioBash. They are ideal for life scientists who are starting to implement bioinformatics into their research. www.genialis.com | www.biobash.com

Sciencemix is an international life-science organization for everyone who loves science and would like to make new friends from all around the world. Taking into consideration the importance of biotechnology for the development of modern science and also itâ&#x20AC;&#x2122;s influence on, biology, chemistry, pharmacy and medicine, an idea about this project was born spontaneously in 2009 year and realized in the year of 2010.

The fundamental mission of the Biotechnical Faculty is to provide university level, advanced professional, and postgraduate education, as well as to carry out scientific research and technical and consulting work concerning the sciences of living nature (biology, microbiology) as well as agriculture, forestry and fisheries (forestry, animal husbandry, agronomy) and the related production technologies (wood technology, food technology, biotechnology). The common denominator of all academic and scientific disciplines at the Biotechnical Faculty is natural resources (soil, physical space, flora, fauna, and water).

BĹ D or Biotechnology student club/association is non-profit student organization located in Ljubljana. Our mission is to connect all biotechnology student from Biotechnical faculty with other colleagues abroad. In 10 years, lot of projects were done and we made some impact for our members and representing our international cooperation.