Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch.

Campylobacter jejuni is a prevalent cause of food-borne diarrhoeal illness in humans. Understanding of the physiological and metabolic capabilities of the organism is limited. We report a detailed analysis of the C. jejuni growth cycle in batch culture. Combined transcriptomic, phenotypic and metabolic analysis demonstrates a highly dynamic 'stationary phase', characterized by a peak in motility, numerous gene expression changes and substrate switching, despite transcript changes that indicate a metabolic downshift upon the onset of stationary phase. Video tracking of bacterial motility identifies peak activity during stationary phase. Amino acid analysis of culture supernatants shows a preferential order of amino acid utilization. Proton NMR (1H-NMR) highlights an acetate switch mechanism whereby bacteria change from acetate excretion to acetate uptake, most probably in response to depletion of other substrates. Acetate production requires pta (Cj0688) and ackA (Cj0689), although the acs homologue (Cj1537c) is not required. Insertion mutants in Cj0688 and Cj0689 maintain viability less well during the stationary and decline phases of the growth cycle than wild-type C. jejuni, suggesting that these genes, and the acetate pathway, are important for survival.

Treatment of Phaeodactylum tricornutum cells with papain facilitates lipid extraction.

Triacylglycerols (TAGs) from microalgae have the potential to be used for biodiesel, but several technical and economic hurdles have to be overcome. A major challenge is efficient extraction of intracellular TAGs from algae. Here we investigate the use of enzymes to deconstruct algal cell walls/membranes. We describe a rapid and simple assay that can assess the efficacy of different enzyme treatments on TAG-containing algae. By this means crude papain and bromelain were found to be effective in releasing TAGs from the diatom Phaeodactylum tricornutum, most likely because of their cysteine protease activity. Pre-treating algal biomass with crude papain enabled complete extraction of TAGs using heptane/isopropyl alcohol. Heptane as a single solvent was also effective, although complete recovery of TAG was not obtained. Economic implications of these findings are discussed, with the aim to reduce the complexity of, and energy needed in, TAG extraction.

Treatment of Phaeodactylum tricornutum cells with papain facilitates lipid extraction

Triacylglycerols (TAGs) from microalgae have the potential to be used for biodiesel, but several technical and economic hurdles have to be overcome. A major challenge is efficient extraction of intracellular TAGs from algae. Here we investigate the use of enzymes to deconstruct algal cell walls/membranes. We describe a rapid and simple assay that can assess the efficacy of different enzyme treatments on TAG-containing algae. By this means crude papain and bromelain were found to be effective in releasing TAGs from the diatom Phaeodactylum tricornutum, most likely because of their cysteine protease activity. Pre-treating algal biomass with crude papain enabled complete extraction of TAGs using heptane/isopropyl alcohol. Heptane as a single solvent was also effective, although complete recovery of TAG was not obtained. Economic implications of these findings are discussed, with the aim to reduce the complexity of, and energy needed in, TAG extraction. © 2012 Elsevier B.V.

Rapid patterning of 1-D collagenous topography as an ECM protein fibril platform for image cytometry.

Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells' response to ROCK inhibition, because of ROCK's key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among potential applications as culture platforms for drug screening.