BIOIMAGING.GR

Το Υποέργο “Bioimaging-GR-EIΠ”  αποτελεί μέρος της Πράξης «Ελληνική Ερευνητική Υποδομή για την Aπεικόνιση και Παρακολούθηση Θεμελιωδών Διεργασιών στη Βιολογία και την Ιατρική (BIOIMAGING–GR)»  με κωδικό ΟΠΣ (MIS 5002755) που εντάσσεται στη Δράση «Ενίσχυση Υποδομών Έρευνας και Καινοτομίας» και χρηματοδοτείται από το Επιχειρησιακό Πρόγραμμα «Ανταγωνιστικότητα, Επιχειρηματικότητα και Καινοτομία» στο πλαίσιο του ΕΣΠΑ 2014-2020, με τη συγχρηματοδότηση της Ελλάδας και της Ευρωπαϊκής Ένωσης (Ευρωπαϊκό Ταμείο Περιφερειακής Ανάπτυξης).

Το συνολικό έργο BIOIMAGING-GR αποσκοπεί στη δημιουργία μιας “Εικονικής” τεχνολογικής πλατφόρμας προηγμένης απεικόνισης, ανοιχτή στην ερευνητική κοινότητα, συνενώνοντας και δικτυώνοντας τις τεχνολογικές υποδομές, ερευνητικές δυνατότητες και υπηρεσίες λήψης και ανάλυσης εικόνας των μεγαλύτερων Μονάδων Απεικόνισης της Ελλάδας. Στόχος του συγκεκριμένου Υποέργου είναι η συνέχιση και ενίσχυση των ήδη παρεχόμενων υπηρεσιών της Μονάδας Οπτικής Μικροσκοπίας (ΜΟΜ) του Ελληνικού Ινστιτούτου Παστέρ (ΕΙΠ) προς μεγαλύτερο αριθμό χρηστών α) με τη στελέχωση της μονάδας με 3 ακόμα νέους επιστήμονες και β) την αναβάθμιση / επέκταση του υπάρχοντος εξοπλισμού της μονάδας προς την κατεύθυνση της ζωντανής απεικόνισης και γ) την ανάπτυξη νέων, προηγμένων ερευνητικών πρωτόκολλων ζωντανής απεικόνισης όπως παρατήρηση ταχέων διαδικασιών σε κύτταρα, πολυπαραμετρική ανάλυση πολλαπλών δειγμάτων και απεικόνιση δυναμικών διεργασιών σε ζωντανά πειραματόζωα. Παράλληλα στόχος του έργου αποτελεί η προσφορά υπηρεσιών απεικόνισης και ανάλυσης ψηφιακής εικόνας/video σε τεχνολογικές προσεγγίσεις αιχμής που έχουν ήδη αναπτυχθεί ή πρόκειται στα πλαίσια του έργου να αναπτυχθούν στην ΜΟΜ του ΕΙΠ, καθώς και διάχυση των ερευνητικών / τεχνολογικών εξελίξεων στην ζωντανή απεικόνιση με διοργάνωση workshops και επιστημονικών ημερίδων.

INSPIRED

INSPIRED, will offer a complete set of platforms (techniques) for the integrated Structural Biology research process comprising sample preparation, biophysical characterization, structural analysis, functional analysis & pharmacogenomics.

Joint Research Activities (JRAs) will be carried out, with the aim to deliver solutions to academic and industrial users, driven by the national/regional needs for innovative services and products.

INSPIRED consists of a dynamic infrastructure of complementary core centers of excellence.

HPI Node: devoted to research of Infectious Diseases, Immunology and Neurobiology

HPI is devoted to research of Infectious Diseases, Immunology and Neurobiology. It is also heavily involved in Public Health Services where research efforts focus mainly on the development of novel diagnostic and therapeutic strategies for human applications. In particular, HPI participates in INSPIRED with the following infrastructure:

• Protein sample preparation (Soluble expression and purification)
• Biophysical characterization
• Structural analysis
• In silico analysis and drug design

We have created the basis for synergies to exploit the expertise of the interdepartmental individual scientific groups* establishing a local scientific core capable to provide the know-how for high-level research.

The basic infrastructure is currently available. More specifically, the existing facilities are:

For protein sample preparation

• Heterologous expression of soluble/membrane proteins in bacterial, yeast (Pichia pastoris), protozoan (Leishmania tarentolae), insect & mammalian cell systems
• Phage display technology & chain-shuffling technologiesthat focuses on developing high specific monoclonal antibodies or peptides for research, diagnostic and therapeutic use (unique in Greece)
• Recombinant viruses and viral transduction systems
• Large-scale production of soluble and membrane proteins expressed in yeast and bacteria cells in the HPI fermenter unit Downstream processing of the expressed recombinant proteins, protein purification (affinity, size exclusion or ion exchange chromatography).

For protein characterization

• Size, mass, polydispersity of purified proteins (assessed by DLS) before crystallization trials
• Functional characterization

For structural analysis

• Protein crystallization (crystallization robot) & 3D structure determination
• Modelling and simulation of biological macromolecules.

* Depts. and Labs:

• of Immunology: Lab. of Molecular Biology & Immunobiotechnology (Dr Avgi Mamalaki)
  Lab. of Immunology (Dr. K. Lazaridis)
• of Neurobiology: Lab. ofMolecular Neurobiology & Immunology (Dr Voula Zisimopoulou)
• of Microbiology: Lab. of Bacteriology (Dr Vivi Miriagou, Dr Stathis Kotsakis)
  Lab. of Molecular Virology (Dr Rania Georgopoulou)
  Lab. of Molecular Ιntracellular Parasitism (Dr Haralabia Boleti)

Selected Publications that support INSPIRED

1. Mamalaki, A., Trakas, N., Tzartos, S. Bacterial expression of a single-chain Fv fragment which efficiently protects the acetylcholine receptor against antigenic modulation caused by myasthenic antibodies. European Journal of Immunology, 23:1831845, 1993.
2. Tzartos, SJ, Tsantili, P., Papanastasiou, D., Mamalaki, A. Construction of single-chain Fv fragments of anti-MIR monoclonal antibodies. Annals of the New York Academy of Science, 841:475-7, 1998.
3. Tsantili, P., Tzartos S. and Mamalaki, A. High affinity scFv antibody fragments protecting the human nicotinic acetylcholine receptor. Journal of Neuroimmunology, 94:15-27, 1999.
4. Papanastasiou, D., Mamalaki, A., Eliopoulos, E., Poulas, K., Liolitsas, C. and Tzartos, S. Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor. Journal of Neuroimmunology, 94:182-195, 1999. (Corresponding authors: A. Mamalaki and S. Tzartos)
5. Tsouloufis, T., Mamalaki, A., Remoundos, M., Tzartos, S.J. Reconstitution of conformationally dependent epitopes of the N-terminal extracellular domain of human muscle AChR α subunit expressed in E.coli: implications for myasthenia gravis therapeutic approaches. International Immunology, 12(9):1255-1265, 2000.
6. Kontou, Μ., Leonidas, D.D., Vatzaki, E.H., Acharya, K. R., Mamalaki, A., Oikonomakos, N.G. and Tzartos, S.J. The crystal structure of a Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor. European Journal of Biochemistry, 267(8):2389-2397, 2000.
7. Kleinjung, J., Petit, M.C., Orlewski, P., Mamalaki, A., Tzartos, S., Tsikaris, V., Daitsiotis-Sakarellos, M., Sakarellos, C., Marraud, M. and Cung, M.T. The Third-Dimensional Structure of the Complex between an Fv Antibody Fragment and an Analog of the Main Immunogenic Region of the Acetylcholine Receptor: A Combined Two-Dimensional NMR, Homology and Molecular Modeling Approach. Biopolymers, 53(2):113-28, 2000.
8. Psaridi, L., Mamalaki, A., Remoundos M., and Tzartos, S. Expression of soluble ligand- and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris. Role of glycosylation in alpha -bungarotoxin binding. J Biol Chem. 277, 30, 26980-26986, 2002.
9. Mamalaki A., Gritzapis AD, Kretsovali Α., Belimezi Μ., Papamatheajis J., Perez S., Papamichail M and Baxevanis C. In vitro and in vivo antitumor activity of a mouse CTL hybridoma expressing chimeric receptors bearing the single chain Fv from HER-2/neu- specific antibody and the γ-chain from Fc(ε) RI. Cancer Immunol. Immunoth. 52(8):513-22, 2003.
10. Gritzapis A., Mamalaki A, Kretsovali Α., Papamatheajis J., Belimezi Μ, Perez S., Baxevanis C. and Papamichail M. Redirecting mouse T hybridoma against human breast and ovarian carcinomas: in vivo activity against HER-2/neu expressing cancer cells. Br J Cancer, 88(8):1292-300, 2003.
11. Psaridi-Linardaki, L., Mamalaki, A., and Tzartos, S. Future therapeutic strategies in autoimmune Myasthenia Gravis. Annals of the New York Academy of Science, 998: 539-548, 2003.
12. Avramopoulou, A. Mamalaki and S. Tzartos. Soluble oligomeric and ligand-binding extracellular domain of human neuronal alpha7 acetylcholine receptor expressed in the yeast Pichia pastoris: Replacement of the hydrophobic Cys-loop by the hydrophilic loop of ACh-binding protein enhances protein solubility. J. Biol. Chemistry 279(37):38287-38293, 2004.
13. Psaridi-Linardaki, A. Mamalaki, N. Trakas and S. J. Tzartos. Specific immunoadsorption of the autoantibodies from myasthenic patients using the extracellular domain of the human muscle acetylcholine receptor alpha-subunit. Development of an antigen-specific therapeutic strategy. Journal of Neuroimmunology 159(1-2):183-91, 2005.
14. Ε. Fostieri, Socrates J. Tzartos, Sonia Berrih-Aknin, David Beeson, and Avgi Mamalaki. Isolation of potent human Fab fragments against a novel highly immunogenic region on human muscle acetylcholine receptor which protect the receptor from myasthenic autoantibodies. Eur J Immunol. 35(2):632-43, 2005.
15. Evdokia Protopapadakis, Anna Kokla, Socrates J. Tzartos and Avgi Mamalaki.  Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci. Eur J Immunol. 35(6):1960-8, 2005.
16. Belimezi, D. Papanastassiou, E. Merkouri, C. Baxevanis and A. Mamalaki Growth inhibition of breast cancer cell lines overexpressing Her2/neu by a novel internalized fully human Fab antibody fragment. Cancer Immunol Immunother. 55: 1091-1099, 2006.
17. Tsitoura, U. Georgopoulou and P. Mavromara (2006). HSV-1 based amplicon vectors as an alternative system for the expression of functional HCV proteins Curr Gene Ther  6(3): 393-398 (REVIEW).
18. Kostelidou K, Trakas N, Zouridakis M, Bitzopoulou K, Sotiriadis A, Gavra I & Tzartos SJ (2006)Expression and characterisation of soluble forms of the extracellular domains of the β, γ and ε subunits of the human muscle acetylcholine receptor. FEBS Journal 273, 3557-3568
19. Zouridakis M, Kostelidou K, Sotiriadis A, Stergiou C, Eliopoulos E, Poulas K & Tzartos SJ (2007)Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide aninsight into their structure. International Journal of Biological Macromolecules 41(4):423-9
20. Zouridakis M, Zisimopoulou P, Eliopoulos E, Jacobson L, Poulas K & Tzartos SJ (2007)Recombinant extracellular domains of human neuronal nicotinic receptors. Preliminary studies on mutant forms for the improvement of solubility. Neurophysiology 39 (4/5) 302-306
21. Tsitoura, U. Georgopoulou, S. Petres, A. Varaklioti, A. Karafoulidou, D. Vagena, C. Politis and P. Mavromara (2007). Evidence for cellular uptake of recombinant hepatitis C virus non-enveloped capsid-like particles FEBS Lett 581(21): 4049-4057.
22. Kouvatsis, R. Argnani, E. Tsitoura, M. Arsenakis, U. Georgopoulou, P. Mavromara and R.Manservigi (2007). Characterization of herpes simplex virus type 1 recombinants that express and incorporate high levels of HCV E2-gC chimeric proteins Virus Res.123(1):40-49.
23. Nianiou, K. Kalantidis, P. Madesis, U Georgopoulou, P.Mavromara and A. Tsaftaris (2008). Expression of an HCV core antigen coding gene in tobacco (N. tabacum L.) Prep Biochem Biotechnol 38(4): 411-421.
24. Katsarou, E. Serti, P. Tsitoura ,A. Lavdas ,A. Varaklioti ,A. Pickl-Herk ,D. Blaas ,D. Oz-Arslan, R. Zhu, P.Hinterdoefer, P. Mavromara, U. Georgopoulou (2009). Green-Fluorescent protein-tagged HCV non-enveloped capsid like particles: Development of a new tool for tracking HCV core uptake. Biochimie 91(7):903-915.
25. Madesis, P.; Osathanunkul, M.; Georgopoulou, U.; Gisby, MF; Mudd, EA; Nianiou, I; Tsitoura, P; Mavromara, P; Tsaftaris, A; Day, A (2010). A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera. Journal of Biotechnology, 145 (4), 377-386.
26. Koliaraki, M. Marinou, M. Samiotaki, G. Panayotou, K. Pantopoulos, A. Mamalaki Iron regulatory and bactericidal properties of human recombinant hepcidin expressed in Pichia pastoris. Biochimie 90(5):726-35, 2008.
27. Koliaraki V, Marinou M, Vassilakopoulos TP, Vavourakis E,Tsochatzis E, Pangalis GA, Papatheodoridis G, Stamoulakatou A, Swinkels D, PapanikolaouG, Mamalaki A. A novel immunological assay for hepcidin quantification in human serum. PLoS One. 2009;4(2):e4581.
28. Alexios Dimitriadis, Chrysanthi Gontinou, Constantin N. Baxevanis and Avgi Mamalaki The mannosylated extracellular domain of Her2/neu produced in P. pastoris induces protective antitumor immunity..BMC cancer, 2009 Oct 30;9:386
29. Zouridakis M, Zisimopoulou P, Eliopoulos E, Poulas K & Tzartos SJ (2009) Design and expression of human α7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties Biochim Biophys Acta1794(2):355-66 doi: 10.1016/j.bbapap.2008.11.002
30. Kotsakis SD, Papagiannitsis CC, Tzelepi E, Tzouvelekis LS, Miriagou V. Extended-spectrum properties of CMY-30, a Val211Gly mutant of CMY-2 cephalosporinase. Antimicrobial Agents and Chemotherapy 2009, 53:3520-3.
31. Kotsakis SD, Miriagou V, Tzelepi E, Tzouvelekis LS. Comparative biochemical and computational study of the role of naturally occurring mutations at Ambler’s positions 104 and 170 in GES β-lactamases. Antimicrobial Agents and Chemotherapy 2010, 54:4864-71
32. Stefan Gattenlohner, Hannah Jörißen, Michael Huhn, Angela C. Vincent, David Beeson, S. Tzartos, Avgi Mamalaki, Benjamin Etschmann, Hans-Konrad Müller-Hermelink, Eva Koscielniak, Stefan Barth and Alexander Marx. A Human Recombinant Autoantibody-based Immunotoxin Specific for the Fetal Acetylcholine Receptor Inhibits Rhabdomyosarcoma Growth in vitro and in a Murine Transplantation Model. Journal of Biomedicine and Biotechnology, 2010:187621
33. Sidera K, El Hamidieh A, Mamalaki A, Patsavoudi E. The 4C5 cell-impermeable anti-HSP90 antibody with anti-cancer activity, is composed of a single light chain dimer. PLoS One. 2011;6(9):e23906.
34. Stergiou, Ch., Zisimopoulou, P., and Tzartos, S.J. (2011).Expression of water-soluble, ligand-binding concatameric extracellular domains of the human neuronal nicotinic receptor alpha4 and beta2 subunits in the yeast pichia pastoris. J. Biol. Chem. 286: 8884-92.
35. Papagiannitsis CC, Kotsakis SD, Petinaki E, Vatopoulos AC, Tzelepi E, Miriagou V, Tzouvelekis LS. Characterization of metallo-β-lactamase VIM-27, an A57S mutant of VIM-1 associated with Klebsiella pneumoniae ST147. Antimicrobial Agents and Chemotherapy 2011,55:3570-2.
36. Kotsakis SD, Tzouvelekis LS, Petinaki E, Tzelepi E, Miriagou V. Effects of the Val211Gly substitution on molecular dynamics of CMY-2 cephalosporinase: implications on hydrolysis of expanded-spectrum cephalosporins. Proteins 2011, 79:3180-92.
37. Kotsakis SD, Tzouvelekis LS, Zerva L, Liakopoulos A, Petinaki E. Staphylococcus lugdunensis strain with a modified PBP1A/1B expressing resistance to β-lactams. Eur J Clin Microbiol Infect Dis 2012 31(2):169-172.
38. Niarchos A, Zouridakis M, Douris V, Georgostathi A, Kalamida D, Sotiriadis A, Poulas K, Iatrou K, Tzartos SJ(2013) Expression of a highly antigenic and native-like folded extracellular domain of the human α1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis. PLoS One 2013 Dec 20;8(12) doi: 10.1371/journal.pone.0084791
39. Kotsakis SD, Caselli E, Tzouvelekis LS, Petinaki E, Prati F, Miriagou V. (2013) Interactions of oximino-substituted boronic acids and β-lactams with the CMY-2-derived extended-spectrum cephalosporinases CMY-30 and CMY-42. Antimicrobial Agents &Chemotherapy; 57(2): 968-76.
40. Papadimitropoulou A, Mamalaki A. The glycosylated IgII extracellular domain of EMMPRIN is implicated in the induction of MMP-2. Mol Cell Biochem. 2013 Jul;379 (1-2):107-13.
41. Lazaridis, K., Zisimopoulou, P., Giastas, P., Bitzopoulou, K., Evangelakou, P., Sideri, A., Tzartos, S.J. Expression of human AChR extracellular domain mutants with improved characteristics (2014) International Journal of Biological Macromolecules, 63, pp. 210-217. DOI: 10.1016/j.ijbiomac.2013.11.003
42. Zouridakis M, Giastas P, Zarkadas E, Chroni-Tzartou D, Bregestovski P & Tzartos SJ (2014) Crystal structures of free and antagonist-bound states of human α9 nicotinic receptor extracellular domain. Nature Struct Mol Biol. doi: 10.1038/nsmb.2900
43. Kouvatsos N, Niarchos A, Zisimopoulou P, Eliopoulos E, Poulas K, and Tzartos(2014) Purification and functional characterization of a truncated human α4β2
44. Kotsakis SD, Miriagou V, Vetouli EE, Bozavoutoglou E, Lebessi E, Tzelepi E, Tzouvelekis LS. (2015) Increased hydrolysis 1 of oximino-β-lactams by CMY-107, aTyr199Cys mutant of CMY-2 produced by Escherichia coli. Antimicrobial Agents & Chemotherapy; 59(12): 7894-8.
45. Azam L, Papakyriakou A, Zouridakis M, Giastas P, Tzartos SJ & McIntosh JM (2015) Molecular interaction of α-conotoxin RgIA with the rat α9 nAChR. Mol Pharm doi: 10.1124/mol.114.096511
46. Arnaouteli, S., Giastas, P., Andreou, A., Tzanodaskalaki, M., Aldridge, C., Tzartos, S.J., Vollmer, W., Eliopoulos, E., Bouriotis, V. Two putative polysaccharide deacetylases are required for osmotic stability and cell shape maintenance in Bacillus anthracis (2015) Journal of Biological Chemistry, 290 (21), pp. 13465-13478. DOI: 10.1074/jbc.M115.640029
47. Lykhmus O, Koval L, Pastuhova D, Zouridakis M,Tzartos S, Komisarenko S, Skok M (2016) The role of carbohydrate component of recombinant α7 nicotinic acetylcholine receptor extracellular domain in its immunogenicity and functional effects of resulting antibodies. Immunobiology doi: 10.1016/j.imbio.2016.07.012
48. Kouvatsos, N., Giastas, P., Chroni-Tzartou, D., Poulopoulou, C., Tzartos, S.J. Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer (2016) Proc.  Natl. Acad. Sci., USA, 113 (34), pp. 9635-9640. DOI: 10.1073/pnas.1602619113
49. Giastas P, Zouridakis M& Tzartos SJ¶ (2017) Understanding structure-function relationships of the human neuronal acetylcholine receptor: insights from the first crystal structures of neuronal subunits. Br J Pharmacol. doi: 10.1111/bph.13838
50. Ivanova II, Mihaylova NM, Manoylov IK, Makatsori D, Lolov S, Nikolova MH, Mamalaki A, Prechl J, Tchorbanov AI. Targeting of Influenza Viral Epitopes to Antigen-Presenting Cells by Genetically Engineered Chimeric Molecules in a Humanized NOD SCID Gamma Transfer Model. Hum Gene Ther. 2018 Sep;29(9):1056-1070.
51. Andreou, A., Giastas, P., Christoforides, E., Eliopoulos, E.E. Structural and evolutionary insights within the polysaccharide deacetylase gene family of bacillus anthracis and bacillus cereus (2018) Genes, 9 (8), art. no. 386, DOI: 10.3390/genes9080386
52. Giastas, P., Andreou, A., Papakyriakou, A., Koutsioulis, D., Balomenou, S., Tzartos, S.J., Bouriotis, V., Eliopoulos, E.E. Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors (2018) Biochemistry, 57 (5), pp. 753-763 DOI:10.1021/acs.biochem.7b00919
53. Kryukova, E.V., Ivanov, I.A., Lebedev, D.S., Spirova, E.N., Egorova, N.S., Zouridakis, M.,et al. (2018). Orthosteric and/or Allosteric Binding of alpha-Conotoxins to Nicotinic Acetylcholine Receptors and Their Models. Mar Drugs 16(12). doi: 10.3390/md16120460.
54. Doukas A., Karena E, Papakostas K, Papadaki A., Tziouvara O. Xingi E., Frillingos S., Boleti H.“Heterologous expression of the mammalian sodium-nucleobase transporter rSNBT1 in Leishmania tarentolae (under revision).