Development of a set of plasmid vectors for genetic manipulations of the pathogenic yeast Candida parapsilosis

A system for genetic transformation of the yeast Candida parapsilosis, recently developed in our laboratory, opened a venue for investigation of this pathogenic species at the molecular level. In this study we extend the range of available experimental tools by construction of a genomic DNA library suitable for screening and isolation of genes by functional complementation of yeast mutants and a set of replicative plasmid vectors for genetic manipulation of C. parapsilosis cells. The plasmids are based on auxotrophic (CpGAL1, CpURA3, CpMET2, CpLYS4) and dominant (CaIMH3) selection markers. In addition, we constructed plasmid derivatives containing reporter genes yEGFP3 and KlLAC4 coding for enhanced version of the green fluorescent protein and Kluyveromyces lactis beta-galactosidase, respectively. The vectors facilitate propagation and expression of cloned genes in C. parapsilosis cells and allow intracellular localization of gene products and/or monitoring the activity of promoter sequences.     

Lifetime-based photoconversion of EGFP as a tool for FLIM

Background

EGFP is a fluorescent tag extensively used in biological and biomedical research. Over the years many researches have gathered collections of cell lines bearing specific EGFP-tagged proteins. Despite its popularity some photochemical properties of EGFP remain undocumented and unused. We report on so far unexplored lifetime photoconversion of EGFP usable in FLIM.

Green beards in the light of indirect genetic effects

The green‐beard effect is one proposed mechanism predicted to underpin the evolution of altruistic behavior. It relies on the recognition and the selective help of altruists to each other in order to promote and sustain altruistic behavior. However, this mechanism has often been dismissed as unlikely or uncommon, as it is assumed that both the signaling trait and altruistic trait need to be encoded by the same gene or through tightly linked genes. Here, we use models of indirect genetic effects (IGEs) to find the minimum correlation between the signaling and altruistic trait required for the evolution of the latter. We show that this correlation threshold depends on the strength of the interaction (influence of the green beard on the expression of the altruistic trait), as well as the costs and benefits of the altruistic behavior. We further show that this correlation does not necessarily have to be high and support our analytical results by simulations.     

Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order

Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.     

The meek inherit the earth: less aggressive wild mice are more successful in challenging situations

Numerous studies have shown an association between aggressiveness and several other behavioural traits. For example, more aggressive animals were bold and active explorers tending to form persistent routines whereas less aggressive animals were shy, careful but more flexible. While the former are thought to be more successful under stable conditions the latter should have advantages in more dynamic situations. These differences can apply not only to individuals but also to populations, species or groups of species with important implications to species distributions and speciation rates. Here we utilized the Morris water task (MWT) to investigate how two subspecies, Mus musculus musculus and M. m. domesticus, known to differ in aggressiveness, cope with stressful situations. We found that less aggressive musculus males performed significantly better in solving the MWT than more aggressive domesticus males. This suggests that M. m. musculus is more flexible and could be more successful under stressful and/or dynamic situations typical of dispersal bouts. It seems plausible that this difference may have had an influence on the secondary contact between musculus and domesticus populations in the past and perhaps still can affect the dynamics of the European hybrid zone between the subspecies. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 310–319.     

Effect of abiotic stress stimuli on S-nitrosoglutathione reductase in plants

S-nitrosylation of protein cysteine thiol groups has recently emerged as a widespread and important reversible post-translational protein modification, involved in redox signalling pathways of nitric oxide and reactive nitrogen species. S-nitrosoglutathione reductase (GSNOR), member of class III alcohol dehydrogenase family (EC 1.1.1.1), is considered the key enzyme in the catabolism of major low molecular S-nitrosothiol, S-nitrosoglutathione, and hence to control the level of protein S-nitrosylation. Changes of GSNOR activity after exposure to different abiotic stress conditions, including low and high temperature, continuous dark and de-etiolation, and mechanical injury, were investigated in important agricultural plants. Significantly higher GSNOR activity was found under normal conditions in leaves of Cucumis spp. genotype sensitive to biotrophic pathogen Golovinomyces cichoracearum. GSNOR activity was generally increased in all studied plants by all types of stress conditions. Strong down-regulation of GSNOR was observed in hypocotyls of etiolated pea plants, which did not recover to values of green plants even 168 h after the transfer of etiolated plants to normal light regime. These results point to important role of GSNOR during normal plant development and in plant responses to several types of abiotic stress conditions.     

Gastrointestinal Parasites of Savanna Chimpanzees (Pan troglodytes schweinfurthii) in Ugalla,Tanzania

Understanding variability in patterns of parasite infections requires studies of multiple populations inhabiting a variety of habitats. Gastrointestinal parasites of chimpanzees (Pan troglodytes) have been studied extensively at several forested sites, but the parasite fauna of chimpanzees living in dry, open habitats is less well known. We studied the parasites of savanna chimpanzees (Pan troglodytes schweinfurthii) living in the Issa Valley, Ugalla (Tanzania). We examined 119 fresh fecal samples using standard coproscopical methods. We detected protozoans including Blastocystis sp., Entamoeba coli, E. histolytica/dispar, Iodamoeba buetschlii, Troglodytella abrassarti, and Troglocorys cava, but only two types of spirurid nematodes among the helminths. The parasites of the Ugalla chimpanzees differ from those of forest chimpanzees in the absence of Strongyloides sp. and strongylid nematodes and a high prevalence of spirurids. Strongylids and Strongyloides sp. have thin-shelled eggs and larvae, which develop in the external environment; thus they may not be able to survive for prolonged periods in the extreme environment of Ugalla. The Ugalla chimpanzees also live at a lower population density and exhibit a larger home range than forest chimpanzees, factors that may lead to lower exposure to infective nematode larvae. Spirurid eggs, however, have thick shells and a life cycle dependent on intermediary hosts, making their survival and transmission in such extreme conditions more feasible. These differences between parasite fauna of closed and open forest chimpanzees contribute to our understanding of the ecology of infectious disease, and have the potential to contribute to conservation policies and practices.     

Analysis of metabolites in the terpenoid pathway of Catharanthus roseus cell suspensions

In Catharanthus roseus cell cultures, the monoterpenoid pathway has been shown to be a limiting factor in terpenoid indole alkaloid (TIA) production. This could be due to competition at the level of isopentenyl diphosphate::dimethylallyl diphosphate (C5) which leads to the biosynthesis of different terpenoid groups. For future engineering of the terpenoid pathway, chemical characterization of C. roseus cell cultures is a necessity. Therefore, in this study nine C. roseus cell suspension lines were characterized by analyzing the levels of the major terpenoids derived from different biosynthetic pathways which may compete for the same precursors; TIA (monoterpenoid, C10), carotenoids (tetraterpenoid, C40), and sterols (triterpenoid, C30). Among the cell lines, CRPP (S) was the most promising TIA-producing cell line which provided more TIA [24 μmol g^?1 dry weight (DW)] than carotenoids (15 μmol g^?1 DW) and sterols (2 μmol g^?1 DW). However, when considering the distribution of the isopentenyl-precursor (C5), the carotenoids which assemble from 8× C5 represent twofold more C5-units (122 μmol g^?1 DW) than the TIA in this cell line. In the CRPP (G), A12A2 (G), and A12A2 (S) cell lines, the C5 distribution was predominant toward carotenoid biosynthesis as well, resulting in a relatively high accumulation of carotenoids. The geranylgeranyl diphosphate (C20) pathway toward carotenoid production is therefore considered competitive toward TIA biosynthesis. For channeling more precursors to the TIA, the branch point for C10 and C20 seems an interesting target for metabolic engineering. Using principal component analysis of the chromatographic data, we characterized the cell lines chemically based on their metabolite levels. The information on the metabolic composition of C. roseus cell cultures is useful for developing strategies to engineer the metabolic pathways and for selection of cell lines for future studies.     

Radio-sensitization of human leukaemic MOLT-4 cells by DNA-dependent protein kinase inhibitor,NU7441

We studied the effect of pre-incubation with NU7441, a specific inhibitor of DNA-dependent protein kinase (DNA-PK), on molecular mechanisms triggered by ionizing radiation (IR). The experimental design involved four groups of human T-lymphocyte leukaemic MOLT-4 cells: control, NU7441-treated (1 μM), IR-treated (1 Gy), and combination of NU7441 and IR. We used flow cytometry for apoptosis assessment, Western blotting and ELISA for detection of proteins involved in DNA repair signalling and epifluorescence microscopy for detection of IR-induced phosphorylation of histone H2A.X. We did not observe any major changes in the amount of DNA-PK subunits Ku70/80 caused by the combination of NU7441 and radiation. Their combination led to an increased phosphorylation of H2A.X, a hallmark of DNA damage. However, it did not prevent up-regulation of neither p53 (and its phosphorylation at Ser 15 and 392) nor p21. We observed a decrease in the levels of anti-apoptotic Mcl-1, cdc25A phosphatase, cleavage of PARP and a significant increase in apoptosis in the group treated with combination. In conclusion, the combination of NU7441 with IR caused increased phosphorylation of H2A.X early after irradiation and subsequent induction of apoptosis. It was efficient in MOLT-4 cells in 10× lower concentration than the inhibitor NU7026. NU7441 proved as a potent radio-sensitizing agent, and it might provide a platform for development of new radio-sensitizers in radiotherapy.