Continut:
  • Project 42-164
  • Enzimology and Applied Microbiology Laboratory
  • Project CEEX 6/2005


    • Project 42-164

    Partnership in Priority Research Area

    Molecular investigations of the acute respiratory infections brought on by non-flu viruses, assessing the involvement in the innate and child’s pathology

     

    The aim of the project is to investigate the non-flu respiratory infections (caused by the Sincitial Respiratory Virus - VRS, Human Meta Pneumo Virus - hMPV, Para flu Viruses 1, 2, 3, Adenoviruses and Corona viruses O C 43 and 229E) using molecular assays to evaluate the etiological agents and to evaluate the host reaction to them, using both clinical data and molecular laboratory tests, for the etiologic diagnostic, also to analyze the inflammatory soluble mediators (sera citokines/chemokines).

    It’s known that, if for the viral diagnostic caused by VRS and adenoviruses there are commercial diagnostic rapid tests and for the other viruses there is only the immunofluorescence assay, these tests do not have the sensibility and the specificity of RT-PCR (reverse transcription polymerase chain reaction) assay. In the same time, the acute effect, of the non-flu respiratory infections on the body, translated by the secretion of inflammatory soluble mediators, was investigated until now in a small proportion. Thus, the project main objectives are: to evaluate the infections, using both modern techniques of pathogen detection (RT-PCR/real time RT-PCR), and those assays that canalizes the host reactions using the inflammatory soluble mediators patterns (Multiplex technology - xMAP, applied on the LUMINEX 100IS platform); assessing the clinical relevance of the molecular profile, to estimate the involvement in the diagnostic strategy, management and control of respiratory infections of the child.

    The estimated results of the project are as follows:  to put together and to optimize a package of molecular diagnostic tests, enhancing the diagnostic methodology of non-flu respiratory infections (caused by the Sincitial Respiratory Virus - VRS, Human Meta Pneumo Virus - hMPV, Para flu Viruses 1, 2, 3, Adenoviruses and Corona viruses O C 43 and 229E); obtaining the secretion pattern profiles of the inflammation soluble mediators (obtainable only with the Multiplex technology), in correlation with the viral charge, clinical profile etc; to determine the virosis incidence in the innate and of the small child’s respiratory pathology that address the project’s pediatric clinics; identifying the significant parameters in evaluation of the impact on the other results.

    By the approached topic, the project can be included in the research area 4. Health, the topical priority 4.1.3 Investigational and interventional methods  based on molecular and cellular medicine, genomics and proteomics within the National research-development and innovation Plan II, Partnerships Program, because:

    1. The respiratory virosis are a major public health problem, having an important socio-economic impact
    2. The proposed study involves the use of methods and techniques specific for molecular and cellular medicine
    3. The estimated results of the research will be used to develop a new approach in the investigation and evaluation of the efficiency of strategies for the management and control of influenza infection
    4. The high complexity and interdisciplinary degree of the proposed activities call for the need to initiate a partnership involving high competence specialists from fields connected to research and medical practice (specialists in infectious diseases, laboratory physicians - virologists, specialists in molecular medicine).

    The impact of the project is major in the medical research area, both nationally and internationally ,the approached domain being a topical priority at the national level, justified by the absence of any systematic study in this field.

    The project will be carried out by the collaboration of three medical research institutitions in Romania:

    1. The initiator of the project: NIRDMI “Cantacuzino” (C.I.), with experience in basic and applied research in the field of microbiology, virology and molecular medicine
    2. Pediatric Hospital Grigore Alexandrescu-Bucuresti
    3. University of Medicine and Pharmacy Carol Davila (Pediatric Clinic IOMC Alfred Russescu, Bucuresti)
    2009
    May 16


    Enzymology and Applied Microbiology Laboratory

    Head: Nadia Bucurenci, PhD, senior researcher, nadiab@cantacuzino.ro

     

    Staff

    Adrian Onu, PhD, senior researcher, Adrian.Onu@cantacuzino.ro

    Adriana Zoe Costache, PhD student, researcher, aradulescu@cantacuzino.ro

    Ana-Maria Eftimie, MS, researcher, aeftimie@cantacuzino.ro

    Florina Toma, MS, PhD student, researcher, tflorina@cantacuzino.ro

     

    Short presentation

    Basic activities: cloning, over expression, purification of bacterial proteins (enzymes).

    Major scientific interests:

    •  
      • bacterial nucleoside monophosphate kinase family as new therapeutic targets for antibacterial agents
      • new diagnostic methods using mono- or polyclonal antibodies or coupled enzymatic reactions (toxin sub-units, bacterial antigens etc.)
      • enzymatic regeneration of ATP and NADH in industrial enzymatic syntheses

     

    Research report

    Enzymatic regeneration of ATP in industrial enzymatic syntheses

    Phosphorylation is often used in industrial enzymatic syntheses. ATP (phosphate donor) needs to be regenerated during the synthesis in order to have an economically convenient procedure.

    Syntheses: phosphorylation of adenosine monophosphate to adenosine diphosphate, phosphorylation of guanosine to guanosine monophosphate,  phosphorylation of glucose to glucose-6-phosphate

    ATP regeneration systems: creatine kinase / creatine phosphate; acetate kinase (AcK) / acetyl phosphate (AcP)

    AcP: Na salt, Li salt, generated in situ

    Generating AcP in situ: we cloned and used pyruvate oxidase, which catalyses the synthesis of AcP from pyruvate, phosphate and oxygen; to avoid damages caused by hydrogen peroxide (the other reaction product) we had to add several coupled reactions including a NADH regenerating system. The overall reaction involves AcK, pyruvate oxidase, catalase, alcohol dehydrogenase, glucose dehydrogenase and glucose mutarotase.

    Results: The best yield (87%) obtained for the phosphorylation of glucose to glucose-6-phosphate, using acetyl phosphate generated in situ, proved that ATP was regenerated and the whole system was functional. 

    Phosphorylation of Gua to GMP is still under study because the yield obtained was not satisfactory probably due to enzyme inhibition by reaction products or intermediary.

     

    Development of a new reagent for tuberculosis (TB) skin testing using specific recombinant antigens

    The traditional tuberculin skin test lacks specificity for TB. Cross-reactions are due to vaccination with BCG or to infection with other mycobacteria.

    Development of new TB-specific skin test requires identification of M.tuberculosis (M.tb)-specific antigens that elicit DTH responses in TB patients but not in BCG vaccinated persons. Since culture filtrate antigens are usually potent in DTH-based immunoassays we chose proteins secreted by M.tb during its early and active growth phase.

    Four such proteins (ESAT6, CFP10, MPT64, PPE68), already identified as being unique for M.tb, were found in M.tb H37Rv genome and the corresponding genes were cloned. The CFP10 protein was over expressed, purified and tested for DTH response in guinea pigs, with promising results.

     

    Implementation and optimization of a technological process for obtaining therapeutic sera with F(ab’)2 fragments against highly pathogenic bacterial and viral agents

    The major objective of the project is the development of laboratory scale methods for obtaining therapeutic sera with divalent F(ab’)2 fragments as active components.

    The project has a distinct impact on the national health system because it offers the premises for autonomy at national level concerning the production of therapeutic sera used for the treatment of some severe acute diseases with bacterial or viral etiology .

    Innovative technological solutions :

    • harvesting of hyper-immune plasma by plasmapheresis
    • fibrin/fibrinogen removing from plasma by cryoprecipitation
    • controlled pepsin enzymatic digestion of plasma in acidic medium
    • processing, purification and concentration methods with single-use systems
    • flexible technological process adjustable for different antigen types

    Antigens produced and characterized: tetanus anatoxin from Clostridium tetani TD21 (Lister Institute - London), anthrax antigen from Bacillus anthracis 34 F2 (Sterne), viral antigens from H5N1 – NIBRG-14 strain (reassortant virus prepared by reverse-genetics from A/Vietnam/1194/04 and A/PR/8/34 – H1N1 strains).

    Hyper-immune plasma (serum) obtained : horse serum and plasma against tetanus anatoxin, sheep serum against NIBRG – 14 H5N1 virus, sheep serum against PR/8/34 virus, horse serum and plasma against Bacillus anthracis 34 F2.

     

    Our research was supported by national (The Romanian Agency for Research) grants

     

    Publications (2000 - 2008):

    • Genetic and Biochemical Characterization of Salmonella enterica Serovar Typhi Deoxyribokinase - L.Tourneux, N.Bucurenci, C.Saveanu, P.A.Kaminski, M.Bouzon, E.Pistotnik, A.Namane, P.Marliere, O.Barzu, I.L.de la Sierra, J.Neuhard, A.M.Gilles, 2000, J.Bact., 182(4), 869 - 873
    • Sugar specificity of bacterial CMP kinases revealed by crystal structures and mutagenesis of Escherichia coli enzyme - T.Bertrand, P.Briozzo, L.Assairi, A.Ofiteru, N.Bucurenci, H.Munier-Lehmann, B.Golinelli-Pimpaneau, O.Barzu, A-M.Gilles, 2002, J.Mol.Biol., 315, 1099-1110
    • Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase - G.Labesse, N.Bucurenci, D.Douguet, H.Sakamoto, S.Landais, C.Gagyi, A-M.Gilles, O.Barzu, 2002, Biochem.Biophys.Res.Commun., 294, 173-179
    • UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity - Cristina Gagyi, Nadia Bucurenci, Ovidiu Sirbu, Gilles Labesse, Mihaela Ionescu, Augustin Ofiteru,Liliane Assairi, Stephanie Landais, Antoine Danchin, Octavian Barzu and Anne-Marie Gilles, 2003, Eur.J.Biochem., 270, 3196–3204
    • Factors associated with virulence and survival in environmental and clinical isolates of Vibrio cholerae 01 and non 01 in Romania - Anca Israil, Carmen Balotescu, Nadia Bucurenci, Nadia Nacescu, Claudia Cedru, Cornelia Popa, C.Ciufecu, 2003, Roum. Arch. Microbiol. Immunol., 62(3-4), 155-177
    • Comparative study of different methods for detection of toxic and other enzymatic factors in Vibrio cholerae strains - Anca Israil, Carmen Balotescu, Maria Damian, Cristina Dinu and Nadia Bucurenci, 2004, Roum. Arch. Microbiol. Immunol., 63 (1-2), 63-77
    • Characterization of Vibrio cholerae strains isolated in the Republic of Moldavia between 1995-1999 - Anca Israil, Carmen Balotescu, Ionela Alexandru, Radu Cojocaru, Nadia Bucurenci, Valeriu Chicu, Ludmila Mutoi, 2005, Roum. Biotechnol. Lett., 10 (6), 2441-2445
    • Calorimetric and crystallographic analysis of the oligomeric structure of Escherichia coli GMP kinase - Guillaume Hible, Louis Renault, Francis Schaeffer, Petya Christova, Adriana Zoe Radulescu, Cecile Evrin, Anne-Marie Gilles, Jacqueline Cherfils, 2005, J.Mol.Biol., 352, 1044-1059
    • Structural and functional consequences of single amino acid substitutions in the pyrimidine base binding pocket of Escherichia coli CMP kinase - Augustin Ofiteru, Nadia Bucurenci, Emil Alexov, Thomas Bertrand, Pierre Briozzo, Hélène Munier-Lehmann, Anne-Marie Gilles, 2007, FEBS Journal, 274(13) , 3363–3373
    • Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria - Evrin C, Straut M, Slavova-Azmanova N, Bucurenci N, Onu A, Assairi L,Ionescu M, Palibroda N, Barzu O, Gilles AM.,  2007, J.Biol. Chem, 282(10), 7242-7253
    • Fluorescent Study of the Interaction between Staphylococcus aureus UMP kinase and UTP - C.Chilom, D. Gazdaru, N. Bucurenci, M. Straut, A. Popescu, 2007, J.of Optoelectronics and Advanced Materials, 9(8), 2585-2588
    • Characterization of Guanylate Kinase from gram positive and gram negative microorganisms; preliminary results – Ana-Maria Ruxandra Eftimie, Florina Toma, Adriana-Zoe Costache and Nadia Bucurenci, 2007, Roum. Arch. Microbiol. Immunol.,  66(1-2), 22-25
    • Stereoselective synthesis of L-[15N] amino acids with glucose dehydrogenase and galactose mutarotase as NADH regenerating system - Maria Chiriac, Iulia Lupan, N. Bucurenci, O. Popescu, N. Palibroda, Journal of Labelled Compounds and Radiopharmaceuticals , 2008, 51 (4) , 171-174

     

    2009
    May 14


     

    Project CEEX 6/2005

    Advanced biotechnologies for preparation of diagnosis and therapeutic products of human use with integration of optimized methods for quality monitoring and control (in accordance with EU guidelines and standards

    (acronym: BIOMEDGMP)

    Abstract

    The concept of GMP (Good Manufacturing Practice) has been talked about for the first time more than 100 years ago, at the same time with Food and Drug Administration (FDA) foundation; later on it has been implemented in other countries. This concept proved its usefulness after some unfortunate experiences regarding the uncautious usage of drugs such as sulphanilamide or thalidomide. GMP was created by FDA as a set of clearly defined regulations in 1962, but it was adjusted on international level only since 1990.

    This set of regulations is relatively new as compared with the development of production technologies and is based upon the infrastructure of several drug control institutions; therefore it consists of several basic rules and control methods. GMP usage limited the extent of manufacturing area development, constraining to remodeling of current technologies. New concepts such as “process analytical tools” (PAT) are currently expanding to allow development of real-time control and consequently flexibility of production.

    The aim of the present project is therefore to improve the biotechnologies for preparation of diagnosis and therapeutic products of human use, to develop processes of production for new bioproducts, to design and realize the premises and equipment framework for a fully closed operating system in accordance with EU GMP (good manufacturing practices for biopharmaceutical products) standards in “Cantacuzino” Institute.

    The first step of the project involved the design of a global model for preparation biotechnologies optimization regarding therapeutic products of human use. The starting point was the necessity to enforce GMP standards in manufacturing of such products, with particular utilization in biotechnological processes.

    Based on the aforementioned standards and regulations the project proposes a model of flexible development which implies the existence of modular units in the manufacturing framework. The model employs the concepts of “process analytical tool” and “comparability protocol”, promoting the remodeling of current biotechnological processes.

    The model was applied to a particular biotechnological process currently in use for the production of a bacterial immunomodulator but it might be conveniently applied to other therapeutic or diagnostic products (currently or would-be manufactured). Analyzing the current process (including the kinetic model of bacterial growth within the fermentor) there has been devised a theoretical model of technological flow-chart in a closed system, GMP regulations being observed. The result was a model of flexible development using modular units within the manufacturing framework - flexible and sustainable solution for improvement (background for production efficiency and resilience). Each ongoing step can be easily evaluated and costs can be assessed using projective economic analysis (break-even analysis).

    Practical validation was applied to several steps considered to be of crucial importance. The first one was the optimization of bacterial growth and gathering of necessary information towards culture medium optimization. Beside the classical monitoring methods (optical density, amino nitrogen, bacterial mass), new methods for amino acid analysis, such as high performance liquid chromatography (HPLC), have been employed. The extraction using organic solvents (critical step due to necessity of output improvement but also of solvent volume reduction to comply with current environmental regulations) has been evaluated as a model for analysis of physical and chemical properties of the final bioproducts and their intermediary compounds. The model focused mainly on improvement of methods related to extraction output optimization.

    To the aim of expanding the methodology of PAT’s there has also been assessed the bidimensional electrophoresis (2D electrophoresis), which is considered to be the most sensitive method of protein isolation and identification (be it normal or abnormal proteins). The protein patterns thus obtained correlated with every time point in the bacterial growth. Several advanced analysis methods pertaining to physics, chemistry, biochemistry, immunology and molecular biology have also been evaluated; interesting results were thus gathered using differential scanning calorimetry. Genomics was employed in bacterial strain characterization for the bacteria used in bioprocesses. Furthermore, the bioproducts were assessed in terms of biochemical and immunological proprieties using in vivo and in vitro tests.

    Model validation and remodeling of bacterial growth conditions was accomplished using new generation (single-use) fermentors. The closed system concept was tested in several versions using methods developed within the project. 

    These results fully prove the proficiency of alternative manufacturing systems and contribute to define the background for the development of improved manufacturing areas.

    2009
    May 14