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Protistology ■ 59
the natural fluorescence of cellular photosynthetic pigments demonstrated that the observed increase in transcription was not directly linked to their concentration in cells. Moreover, this analysis revealed heterogeneity in the pigment fluorescence among distinct cells within the model culture. Funded in part by RFBR, project 15-29-02706.
THE HIGH COMPLEXITY AND DYNAMIC EVOLUTION OF THE RAS SUPERFAMILY OF GTPASES IN NAEGLERIA Petrzelkova R.1, Herman E.K.2, Dacks J.B.2, Elias M.1
1 - University of Ostrava, Faculty ofScience, Department ofBiology and Ecology, Ostrava, Czech Republic
2 - Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada [email protected]
Ras superfamily GTPases constitute a vast group of proteins involved in many eukaryote-specific processes. The last eukaryotic common ancestor appears to have possessed at least several tens of Ras superfamily genes, but gene duplications and losses in different eukaryotic lineages have modified this ancestral set such that substantially different gene complements may be present in different eukaryote groups. One extreme are taxa harbouring an extensively expanded Ras superfamily complement, as is the case of a free-living amoeboflagellate Nae-gleria gruberi (Heterolobosea). Recently, genome sequences of three strains of Naegleria fowleri, a causative agent of primary amebic meningoencephalitis (PAM), became available for analysis. In order to assess the differences between the two species and the three strains, we identified and annotated the Ras superfamily genes in the newly sequenced N. fowleri genomes and reannotated the respective gene complement in the previously published N. gruberi genome. The sets of Ras superfamily genes turned out to differ substantially between the two species, as N. gruberi harbours over 350 genes, whereas N. fowleri exhibits a much less expanded set with "only" over 200 genes. In contrast, little, if any, differences were found for the three N. fowleri strains. Phylogenetic analyses revealed both species-specific duplications and losses as the factors responsible for the different gene numbers in the two species. The evolution of the Ras superfamily in the genus Naegleria is thus surprisingly dynamic and points to a hidden level of differentiation in cellular physiology of different Naegleria species.
EXPRESSION AND PURIFICATION OF A PNEUMOCYSTIS JIROVECII SYNTHETIC RECOMBINANT ANTIGEN AND APPLICATION IN THE DEVELOPMENT OF A SEROLOGICAL RAPID DIAGNOSTIC TEST Pinto Mafalda12, Cardoso Fernando2, Gomes Ines1,3, Pereira Eulália4, Peixoto Miguel4, Franco Ricardo1, Matos Olga2
1 - UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa (UNL), Portugal
2 - Medical Parasitology Unit, Group ofOpportunistic Protozoa/HIV and Other Protozoa, GHTM, Instituto de Higiene e Medicina Tropical, UNL, Portugal
3 - Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal
4 - UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciencias, Universidade do Porto, Portugal
Pneumocystis pneumonia (PcP) is an infectious disease caused by Pneumocystis jirovecii, an atypical fungus. PcP remains a major cause of respiratory illness among immunosuppressed patients. Current PcP diagnosis is based on the detection of P. jirovecii in respiratory specimens, obtained by invasive methods such as bronchoalveolar lavage, followed by cytochemical staining, immunofluorescent staining with monoclonal antibodies (IF/Mab) or PCR. Therefore, the possibility of an early diagnostic method allowing the use of biological specimens obtained non-invasively, is highly desirable. Rapid diagnostic tests (RDTs) using gold nanoparticles (AuNPs) allow a more sensitive, fast and cheap diagnosis, to be used in developing countries. The goal of this work is to develop an immunochromatographic RDT for the detection of P. jirovecii in non-invasive specimens like serum. In this test, spherical AuNPs are conjugated with a multi-epitope synthetic recombinant antigen (msr) which will allow the detection of circulating anti-P. jirovecii antibodies in sera. In order to obtain the highest amount of pure antigen, the expression vector pLATE 31, which contains the coding sequence for the MSG g antigen, was isolated and cloned in E. coli XJb (DE3). Extraction and purification through affinity chromatography with immobilized metallic ions, ELISA, SDS-PAGE and Western-Blot, were performed with the objective to obtain the maximum quantity of antigen and determine its purity. The antigen was then used to form bionanoconjugates with AuNPs, previously
