Temporal and Spatial Distribution of the Microbial Community of Winogradsky Columns

Winogradsky columns are model microbial ecosystems prepared by adding pond sediment to a clear cylinder with additional supplements and incubated with light. Environmental gradients develop within the column creating diverse niches that allow enrichment of specific bacteria. The enrichment culture can be used to study soil and sediment microbial community structure and function. In this study we used a 16S rRNA gene survey to characterize the microbial community dynamics during Winogradsky column development to determine the rate and extent of change from the source sediment community. Over a period of 60 days, the microbial community changed from the founding pond sediment population: Cyanobacteria, Chloroflexi, Nitrospirae, and Planctomycetes increased in relative abundance over time, while most Proteobacteria decreased in relative abundance. A unique, light-dependent surface biofilm community formed by 60 days that was less diverse and dominated by a few highly abundant bacteria. 67–72% of the surface community was comprised of highly enriched taxa that were rare in the source pond sediment, including the Cyanobacteria Anabaena, a member of the Gemmatimonadetes phylum, and a member of the Chloroflexi class Anaerolinea. This indicates that rare taxa can become abundant under appropriate environmental conditions and supports the hypothesis that rare taxa serve as a microbial seed bank. We also present preliminary findings that suggest that bacteriophages may be active in the Winogradsky community. The dynamics of certain taxa, most notably the Cyanobacteria, showed a bloom-and-decline pattern, consistent with bacteriophage predation as predicted in the kill-the-winner hypothesis. Time-lapse photography also supported the possibility of bacteriophage activity, revealing a pattern of colony clearance similar to formation of viral plaques. The Winogradsky column, a technique developed early in the history of microbial ecology to enrich soil microbes, may therefore be a useful model system to investigate both microbial and viral ecology.     

Cellular processes underlying maturation of P19 neurons: Changes in protein folding regimen and cytoskeleton organization

Embryonal carcinoma P19 cells provide an ideal model to study molecular programs along differentiation. Upon induction by retinoic acid (RA), the cells undergo a program of differentiation that generates functioning neurons within 60 h. RA induced cells that were plated as sparse (1000 cells/mm(2)) or dense (4000 cells/mm(2)) cultures showed a marked difference in the culture morphology with the dense cultures exhibiting rapid maturation and accelerated neurite outgrowth. The protein expression levels of the sparse and dense cultures were compared 48 h following RA. Cell extracts were separated by 1-DE and 2-DE and differential expression (>four-fold) proteins were identified by MS. Here, we focus on 20 proteins associated with cytoskeletal regulation and stress-dependent protein refolding. The first group includes drebrin, cofilin, alpha-internexin, vimentin, and nestin. Among the proteins in the second group are subunits of the TCP-1, and several chaperones of the Hsp70 and Hsp90 families. We show that coordinated remodeling of the cytoskeleton and modulations in chaperone activity underlie the change in neurite extension rate. Furthermore, a proteomics-based analysis applied on P19 neurons demonstrated pathways underlying neuronal outgrowth, suggesting that a malfunction of such pathways leads to neuropathological conditions.     

PROCEED: A proteomic method for analysing plasma membrane proteins in living mammalian cells.

Elucidating the profile of extracellular integral membrane proteins on live cells is vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates. Exploring the realm of these proteins has proved to be an intricate task, mainly due to their hydrophobic nature and low abundance. Furthermore, the level of purity achieved by classical methods of purification and cell fractionation is insufficient. These restrictions pose major limitations for gel electrophoresis or chromatography-based separation techniques as the preferred methodologies for high-throughput analysis. Mass spectrometry has alleviated most of the difficulties in the identification of proteins in general; however, the Achilles' heel is still the isolation and separation of membrane proteins. In order to circumvent these limitations, a high-throughput platform has been devised, whereby proteases are applied to whole intact living cells. The resulting peptide fragments are then analysed by liquid chromatology followed by tandem MS (LC-MS/MS) technology to provide a detailed profile of proteins exposed on the surface of the plasma membrane. This kind of protein trimming offers the advantages that no prior manipulation or fractionation of the cell is required, contaminating proteins are remarkably reduced and the procedure is adequate for high-throughput purposes. This method, referred to as PROCEED (PROteome of Cell Exposed Extracellular Domains) is compatible with isotope labelling techniques which facilitate comparative protein expression studies. The methodology is extendable to all cell types including yeast and bacteria. Finally, the advantages and the limitations of PROCEED are discussed in view of other current technologies.     

Coat Polymorphism in Eurasian Lynx: Adaptation to Environment or Phylogeographic Legacy?

Journal of Mammalian Evolution - We studied the relationship between the variability and contemporary distribution of pelage phenotypes in one of most widely distributed felid species and an array...