Ras links cellular morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling pathways in the pathogenic fungus Candida albicans

The pathogenic fungus Candida albicans is capable of responding to a wide variety of environmental cues with a morphological transition from a budding yeast to a polarized filamentous form. We demonstrate that the Ras homologue of C. albicans, CaRas1p, is required for this morphological transition and thereby contributes to the development of pathogenicity. However, CaRas1p is not required for cellular viability. Deletion of both alleles of the CaRAS1 gene caused in vitro defects in morphological transition that were reversed by either supplementing the growth media with cAMP or overexpressing components of the filament-inducing mitogen-activated protein (MAP) kinase cascade. The induction of filament-specific secreted aspartyl proteinases encoded by the SAP4-6 genes was blocked in the mutant cells. The defects in filament formation were also observed in situ after phagocytosis of C. albicans cells in a macrophage cell culture assay and, in vivo, after infection of kidneys in a mouse model for systemic candidiasis. In the macrophage assay, the mutant cells were less resistant to phagocytosis. Moreover, the defects in filament formation were associated with reduced virulence in the mouse model. These results indicate that, in response to environmental cues, CaRas1p is required for the regulation of both a MAP kinase signalling pathway and a cAMP signalling pathway. CaRas1p-dependent activation of these pathways contributes to the pathogenicity of C. albicans cells through the induction of polarized morphogenesis. These findings elucidate a new medically relevant role for Ras in cellular morphogenesis and virulence in an important human infectious disease.     

Online optimization of surface plasmon resonance-based biosensor experiments for improved throughput and confidence

The emergence of surface plasmon resonance-based optical biosensors has facilitated the identification of kinetic parameters for various macromolecular interactions. Normally, these parameters are determined from experiments with arbitrarily chosen periods of macromolecule and buffer injections, and varying macromolecule concentrations. Since the choice of these variables is arbitrary, such experiments may not provide the required confidence in identified kinetic parameters expressed in terms of standard errors. In this work, an iterative optimization approach is used to determine the above-mentioned variables so as to reduce the experimentation time, while treating the required standard errors as constraints. It is shown using multiple experimental and simulated data that the desired confidence can be reached with much shorter experiments than those generally performed by biosensor users.     

Pyridyl and thiazolyl bisamide CSF-1R inhibitors for the treatment of cancer

The bisamide class of kinase inhibitors was identified as being active against CSF-1R. The synthesis and SAR of pyridyl and thiazolyl bisamides are reported, along with the pharmacokinetic properties and in vivo activity of selected examples.     

PGE2 reduces arachidonic acid release in murine podocytes: evidence for an autocrine feedback loop

Increased glomerularprostaglandin E₂ (PGE₂) production isassociated with the progression of diseases such as membranous nephropathy, nephrotic syndrome, and anti-Thy1 nephritis. Weinvestigated the signaling pathways that regulate the synthesis andactions of PGE₂ in glomerular podocytes. To study itsactions, we assessed the ability of PGE₂ to regulate theproduction of its own precursor, arachidonic acid (AA), in a mousepodocyte cell line. PGE₂ dose-dependently reduced phorbolester (PMA)-mediated AA release. Inhibition of PMA-stimulated AArelease by PGE₂ was found to be cAMP/PKA-dependent, becausePGE₂ significantly increased levels of this secondmessenger, whereas the inhibitory actions of PGE₂ werereversed by PKA inhibition and reproduced by the cAMP-elevating agentsforskolin and IBMX. PGE₂ synthesis in this podocyte cellline increased fourfold at 60 min in response to PMA, coinciding withupregulation of cyclooxygenase (COX)-2 but not COX-1 levels. However,PGE₂ synthesis was significantly reduced by COX-1-selectiveinhibition, yet to a lesser extent by COX-2-selective inhibition. Ourfindings suggest that PMA-stimulated PGE₂ synthesis inmouse podocytes requires both basal COX-1 activity and induced COX-2expression, and that PGE₂ reduces PMA-stimulated AA releasein a cAMP/PKA-dependent manner. Such an autocrine regulatory loop mighthave important consequences for podocyte and glomerular function in thecontext of renal diseases involving PGE₂ synthesis.

     

Study of newly synthesized proteins during bovine oocyte maturation in vitro using image analysis of two-dimensional gel electrophoresis

To investigate protein synthesis during bovine oocyte maturation in vitro, oocytes were put in culture with 35S-methionine for 4 hr periods from time zero to 28 hr. Pools of 10 oocytes were then prepared for two-dimensional gel electrophoresis (2-DE). For each time interval, three gels were obtained, digitalized, and analyzed to detect proteins. Then, the gel containing the most proteins was chosen as the reference gel and compared with the others. An averaged gel was created with proteins present in at least two gels of the three. Our results indicate that the rate of protein synthesis is higher at the beginning of maturation until the appearance of metaphase I (MI, 8-12 hr) and then it decreases and stays relatively constant. Percentages of initial proteins (0-4 hr) and remaining present during the progression decrease progressively from 100% to 53%. In contrast, when we compare proteins synthesized from the 4 to 8 hr period with proteins from the 8 to 12 or the 12 to 16 hr intervals, percentages of overall protein matching are stable with values of 81 and 79%, respectively. Comparison of proteins from 20 to 24 hr with proteins from 16 to 20 or 24 to 28 hr intervals also gives stable percentages of overall protein matching with values of 83 and 84%, respectively. Furthermore, a higher number of new proteins is observed at 4-8 hr (n=130) and 16-20 hr (n=136) of maturation. Thus, three major patterns of protein synthesis were observed during bovine oocyte maturation in vitro: one at the beginning of maturation (0-4 hr), another one in the middle (4-16 hr), and the last one after the completion of MI stage (16-28 hr).     

A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.