Microbiota within the perennial ice cover of Lake Vida, Antarctica

Lake Vida, located in the McMurdo Dry Valleys, Antarctica, is an 'ice-sealed' lake with approximately 19 m of ice covering a highly saline water column (approximately 245 ppt). The lower portions of the ice cover and the lake beneath have been isolated from the atmosphere and land for circa 2800 years. Analysis of microbial assemblages within the perennial ice cover of the lake revealed a diverse array of bacteria and eukarya. Bacterial and eukaryal denaturing gradient gel electrophoresis phylotype profile similarities were low (<59%) between all of the depths compared (five depths spanning 11 m of the ice cover), with the greatest differences occurring between surface and deep ice. The majority of bacterial 16S rRNA gene sequences in the surface ice were related to Actinobacteria (42%) while Gammaproteobacteria (52%) dominated the deep ice community. Comparisons of assemblage composition suggest differences in ice habitability and organismal origin in the upper and lower portions of ice cover. Specifically, the upper ice cover microbiota likely reflect the modern day transport and colonization of biota from the terrestrial landscape, whereas assemblages in the deeper ice are more likely to be persistent remnant biota that originated from the ancient liquid water column of the lake that froze.     

A fast and accessible methodology for micro-patterning cells on standard culture substrates using Parafilm™ inserts

Micropatterning techniques provide direct control over the spatial organization of cells at the sub-mm scale. Regulation of these spatial parameters is important for controlling cell fate and cell function. While micropatterning has proved a powerful technique for understanding the impact of cell organization on cell behaviour, current methods for micropatterning cells require complex, specialized equipment that is not readily accessible in most biological and bioengineering laboratories. In addition, currently available methods require significant protocol optimization to ensure reliable and reproducible patterning. The inaccessibility of current methods has severely limited the widespread use of micropatterning as a tool in both biology and tissue engineering laboratories. Here we present a simple, cheap, and fast method to micropattern mammalian cells into stripes and circular patterns using Parafilm?, a common material found in most biology and bioengineering laboratories. Our method does not require any specialized equipment and does not require significant method optimization to ensure reproducible patterning. Although our method is limited to simple patterns, these geometries are sufficient for addressing a wide range of biological problems. Specifically, we demonstrate i) that using our Parafilm? insert method we can pattern and co-pattern ARPE-19 and MDCK epithelial cells into circular and stripe micropatterns in tissue culture polystyrene (TCPS) wells and on glass slides, ii) that we can contain cells in the desired patterns for more than one month and iii) that upon removal of the Parafilm? insert we can release the cells from the containment pattern and allow cell migration outward from the original pattern. We also demonstrate that we can exploit this confinement release feature to conduct an epithelial cell wound healing assay. This novel micropatterning method provides a reliable and accessible tool with the flexibility to address a wide range of biological and engineering problems that require control over the spatial and temporal organization of cells.     

The hows and whys of aerobic H2 metabolism

The bacterial [NiFe]-hydrogenases have been classified as either 'standard' or 'O2-tolerant' based on their ability to function in the presence of O2. Typically, these enzymes contain four redox-active metal centers: a Ni-Fe-CO-2CN- active site and three electron-transferring Fe-S clusters. Recent research suggests that, rather than differences at the catalytic active site, it is a novel Fe-S cluster electron transfer (ET) relay that controls how [NiFe]-hydrogenases recover from O2 attack. In light of recent structural data and mutagenic studies this article reviews the molecular mechanism of O2-tolerance in [NiFe]-hydrogenases and discusses the biosynthesis of the unique Fe-S relay.     

Moths complement bumblebee pollination of red clover: a case for day-and-night insect surveillance

Recent decades have seen a surge in awareness about insect pollinator declines. Social bees receive the most attention, but most flower-visiting species are lesser known, non-bee insects. Nocturnal flower visitors, e.g. moths, are especially difficult to observe and largely ignored in pollination studies. Clearly, achieving balanced monitoring of all pollinator taxa represents a major scientific challenge. Here, we use time-lapse cameras for season-wide, day-and-night pollinator surveillance of Trifolium pratense (L.; red clover) in an alpine grassland. We reveal the first evidence to suggest that moths, mainly Noctua pronuba (L.; large yellow underwing), pollinate this important wildflower and forage crop, providing 34% of visits (bumblebees: 61%). This is a remarkable finding; moths have received no recognition throughout a century of T. pratense pollinator research. We conclude that despite a non-negligible frequency and duration of nocturnal flower visits, nocturnal pollinators of T. pratense have been systematically overlooked. We further show how the relationship between visitation and seed set may only become clear after accounting for moth visits. As such, population trends in moths, as well as bees, could profoundly affect T. pratense seed yield. Ultimately, camera surveillance gives fair representation to non-bee pollinators and lays a foundation for automated monitoring of species interactions in future.     

Effects of Cortisol and Dexamethasone on Insulin Signalling Pathways in Skeletal Muscle of the Ovine Fetus during Late Gestation

Before birth, glucocorticoids retard growth, although the extent to which this is mediated by changes in insulin signalling pathways in the skeletal muscle of the fetus is unknown. The current study determined the effects of endogenous and synthetic glucocorticoid exposure on insulin signalling proteins in skeletal muscle of fetal sheep during late gestation. Experimental manipulation of fetal plasma glucocorticoid concentration was achieved by fetal cortisol infusion and maternal dexamethasone treatment. Cortisol infusion significantly increased muscle protein levels of Akt2 and phosphorylated Akt at Ser473, and decreased protein levels of phosphorylated forms of mTOR at Ser2448 and S6K at Thr389. Muscle GLUT4 protein expression was significantly higher in fetuses whose mothers were treated with dexamethasone compared to those treated with saline. There were no significant effects of glucocorticoid exposure on muscle protein abundance of IR-β, IGF-1R, PKCζ, Akt1, calpastatin or muscle glycogen content. The present study demonstrated that components of the insulin signalling pathway in skeletal muscle of the ovine fetus are influenced differentially by naturally occurring and synthetic glucocorticoids. These findings may provide a mechanism by which elevated concentrations of endogenous glucocorticoids retard fetal growth.     

The novel ruthenium—γ‐linolenic complex [Ru2(aGLA)4Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential,increased reactive oxygen species generation and apoptosis in vitro

The present study reports the synthesis of a novel compound with the formula [Ru₂(aGLA)4Cl] according to elemental analyses data, referred to as Ru₂GLA. The electronic spectra of Ru₂GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru₂GLA was synthesized with the aim of combining and possibly improving the anti‐tumour properties of the two active components ruthenium and γ‐linolenic acid (GLA). The properties of Ru₂GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru₂GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru₂GLA enters the cells and ICP‐AES elemental analysis found an increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub‐G1 apoptotic cell population was increased by Ru₂GLA (22 ± 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru₂GLA (44 ± 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 ± 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru₂GLA exposed cells. The EC₅₀ for Ru₂GLA decreased with increasing time of exposure from 285 µM at 24 h, 211 µM at 48 h to 81 µM at 72 h. In conclusion, Ru₂GLA is a novel drug with antiproliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas. Copyright © 2009 John Wiley & Sons, Ltd.