Modeling Fuzzy Fidelity: Using Microsimulation to Explore Age, Period, and Cohort Effects in Secularization

This article presents a microsimulation that explores age, period, and cohort effects in the decline of religiosity in contemporary societies. The model implements a well-known and previously empirically validated theory of secularization that highlights the role of “fuzzy fidelity,” i.e., the percentage of a population whose religiosity is moderate (Voas 2009). Validation of the model involved comparing its simulation results to shifts in religiosity over 9 waves of the European Social Survey. Simulation experiments suggest that a cohort effect, based on weakened transmission of religiosity as a function of the social environment, appears to be the best explanation for secularization in the societies studied, both for the population as a whole and for the proportions of religious, fuzzy, and secular people

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast <it>Pichia pastoris</it>.</p> <p>Results</p> <p>By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.</p> <p>Conclusions</p> <p>We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant <it>P. pastoris </it>clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of <it>P. pastoris </it>clones with high expression levels of aquaporins and other classes of membrane proteins.</p

Broad-host-range <i>Rhizobium</i> species strain NGR234 secretes a family of carbamoylated, and fucosylated, nodulation signals that are <i>O</i>-acetylated or sulphated

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non‐legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod‐factors which are N‐acylated chitin pentamers carrying a variety of substituents. The terminal non‐reducing glucosamine is N‐acylated with vaccenic or palmitic acids, is N‐methylated, and carries varying numbers of carbamoyl groups. The reducing N‐acetyl‐glucosamine residue is substituted on position 6 with 2‐O‐methyl‐L‐fucose which may be acetylated or sulphated or non‐substituted. All three internal residues are N‐acetylated. At pico‐ to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non‐sulphated lipo‐oligosaccharide signals

Sphingosine Kinase-1 is Overexpressed and Correlates with Hypoxia in Osteosarcoma: Relationship with Clinicopathological Parameters

The Sphingosine kinase-1/Sphingosine 1-Phosphate (SphK1/S1P) signaling pathway is overexpressed in various cancers, and is instrumental for the adaptation to hypoxia in a number of solid tumor models, but no data are available in osteosarcoma. Here we report that SphK1 and the S1P1 receptor are involved in HIF-1α accumulation in hypoxic osteosarcoma cells. FTY720 (Fingolimod), which targets SphK1 and S1P1, prevented HIF-1α accumulation, and also inhibited cell proliferation in both normoxia and hypoxia unlike conventional chemotherapy. In human biopsies, a significant increase of SphK1 activity was observed in cancer compared with normal bones. In all sets of TMA samples (130 cases of osteosarcoma), immunohistochemical analysis showed the hypoxic marker GLUT-1, SphK1 and S1P1 were expressed in tumors. SphK1 correlated with the GLUT-1 suggesting that SphK1 is overexpressed and correlates with intratumoral hypoxia. No correlation was found between GLUT-1 or SphK1 and response to chemotherapy, but a statistical difference was found with increased S1P1 expression in patients with poor response in long bone osteosarcomas. Importantly, multivariate analyses showed that GLUT-1 was associated with an increased risk of death in flat bone, whereas SphK1 and S1P1 were associated with an increased risk of death in long bones.Anne Gomez-Brouchet, Claire Illac, Adeline Ledoux, Pierre-Yves Fortin, Sandra de Barros, Clémentine Vabre, Fabien Despas, Sophie Peries, Christelle Casaroli, Corinne Bouvier, Sébastien Aubert, Gonzague de Pinieux, Frédérique Larousserie, Louise Galmiche, Franck Talmont, Stuart Pitson, Marie-Lise Maddelein and Olivier Cuvillie

Electrotransformation of Saccharomyces cerevisiae

Intact yeast cell transformation is easily achieved by gene electrotransfer (GET). The procedure is fast and efficient in terms of transformants/mu g DNA. Yeast cells in exponential growth phase are washed, treated for a short period with dithiothreitol (DTT) and then mixed with the plasmid DNA in a buffer with a low conductivity. A single well defined electric pulsed is delivered. After a 1 h incubation in the growth medium without selection, transformants are obtained on a selective plate medium. After a short description of the present knowledge on the events affecting the yeast cell as a consequence of the pulsed electric field, a step-by-step protocol is reported for Saccharomyces cerevisiae

Production of Hydrogen from α-1,4- and β-1,4-Linked Saccharides by Marine Hyperthermophilic Archaea ▿ †

Nineteen hyperthermophilic heterotrophs from deep-sea hydrothermal vents, plus the control organism Pyrococcus furiosus, were examined for their ability to grow and produce H2 on maltose, cellobiose, and peptides and for the presence of the genes encoding proteins that hydrolyze starch and cellulose. All of the strains grew on these disaccharides and peptides and converted maltose and peptides to H2 even when elemental sulfur was present as a terminal electron acceptor. Half of the strains had at least one gene for an extracellular starch hydrolase, but only P. furiosus had a gene for an extracellular β-1,4-endoglucanase. P. furiosus was serially adapted for growth on CF11 cellulose and H2 production, which is the first reported instance of hyperthermophilic growth on cellulose, with a doubling time of 64 min. Cell-specific H2 production rates were 29 fmol, 37 fmol, and 54 fmol of H2 produced cell−1 doubling−1 on α-1,4-linked sugars, β-1,4-linked sugars, and peptides, respectively. The highest total community H2 production rate came from growth on starch (2.6 mM H2 produced h−1). Hyperthermophilic heterotrophs may serve as an important alternate source of H2 for hydrogenotrophic microorganisms in low-H2 hydrothermal environments, and some are candidates for H2 bioenergy production in bioreactors