The effect of biomass harvesting on greenhouse gas emissions from a rewetted temperate fen

The growing demand for bioenergy increases pressure on peatlands. The novel strategy of wet peatlands agriculture (paludiculture) may permit the production of bioenergy from biomass while avoiding large greenhouse gas emissions as occur during conventional crop cultivation on drained peat soils. Herein, we present the first greenhouse gas balances of a simulated paludiculture to assess its suitability as a biomass source from a climatic perspective. In a rewetted peatland, we performed closed‐chamber measurements of carbon dioxide, methane, and nitrous oxide exchange in stands of the potential crops Phragmites australis, Typha latifolia, and Carex acutiformis for two consecutive years. To simulate harvest, the biomass of half of the measurement spots was removed once per year. Carbon dioxide exchange was close to neutral in all tested stands. The effect of biomass harvest on the carbon dioxide exchange differed between the 2 years. During the first and second year, methane emissions were 13–63 g m^?2 a^?1 and 2–5 g m^?2 a^?1, respectively. Nitrous oxide emissions lay below our detection limit. Net greenhouse gas balances in the study plots were close to being climate neutral during both years except for the Carex stand, which was a source of greenhouse gases in the first year (in CO₂‐equivalents: 18 t ha^?1 a^?1). Fifteen years after rewetting the net greenhouse gas balance of the study site was similar to those of pristine fens. In addition, we did not find a significant short‐term effect of biomass harvest on net greenhouse gas balances. In our ecosystem, ~17 t ha^?1 a^?1 of CO₂‐equivalent emissions are saved by rewetting compared to a drained state. Applying this figure to the fen area in northern Germany, emission savings of 2.8–8.5 Mt a^?1 CO₂‐equivalents could possibly be achieved by rewetting; this excludes additional savings by fossil fuel replacement.     

Biophysical parameters of the Sec14 phospholipid exchange cycle – Effect of lipid packing in membranes

Sec14, a yeast phosphatidylinositol/phosphatidylcholine transfer protein, functions at the trans-Golgi membranes. It lacks domains involved in protein-protein or protein-lipid interactions and consists solely of the Sec14 domain; hence, the mechanism underlying Sec14 function at proper sites remains unclear. In this study, we focused on the lipid packing of membranes and evaluated its association with in vitro Sec14 lipid transfer activity. Phospholipid transfer assays using pyrene-labelled phosphatidylcholine suggested that increased membrane curvature as well as the incorporation of phosphatidylethanolamine accelerated the lipid transfer. The quantity of membrane-bound Sec14 significantly increased in these membranes, indicating that “packing defects” of the membranes promote the membrane binding and phospholipid transfer of Sec14. Increased levels of phospholipid unsaturation promoted Sec14-mediated PC transfer, but had little effect on the membrane binding of the protein. Our results demonstrate the possibility that the location and function of Sec14 are regulated by the lipid packing states produced by a translocase activity at the trans-Golgi network.     

Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution

Sec14-superfamily proteins integrate the lipid metabolome with phosphoinositide synthesis and signaling via primed presentation of phosphatidylinositol (PtdIns) to PtdIns kinases. Sec14 action as a PtdIns-presentation scaffold requires heterotypic exchange of phosphatidylcholine (PtdCho) for PtdIns, or vice versa, in a poorly understood progression of regulated conformational transitions. We identify mutations that confer Sec14-like activities to a functionally inert pseudo-Sec14 (Sfh1), which seemingly conserves all of the structural requirements for Sec14 function. Unexpectedly, the "activation" phenotype results from alteration of residues conserved between Sfh1 and Sec14. Using biochemical and biophysical, structural, and computational approaches, we find the activation mechanism reconfigures atomic interactions between amino acid side chains and internal water in an unusual hydrophilic microenvironment within the hydrophobic Sfh1 ligand-binding cavity. These altered dynamics reconstitute a functional "gating module" that propagates conformational energy from within the hydrophobic pocket to the helical unit that gates pocket access. The net effect is enhanced rates of phospholipid-cycling into and out of the Sfh1* hydrophobic pocket. Taken together, the directed evolution approach reveals an unexpectedly flexible functional engineering of a Sec14-like PtdIns transfer protein-an engineering invisible to standard bioinformatic, crystallographic, and rational mutagenesis approaches.     

Porous silicon biosensor for detection of viruses

There is a growing need for virus sensors with improved sensitivity and dynamic range, for applications including disease diagnosis, pharmaceutical research, agriculture and homeland security. We report here a new method for improving the sensitivity for detection of the bacteriophage virus MS2 using thin films of nanoporous silicon. Porous silicon is an easily fabricated material that has extremely high surface area to volume ratio, making it an ideal platform for surface based sensors. We have developed and evaluated two different methods for covalent bioconjugation of antibodies inside of porous silicon films, and we show that the pore penetration and binding efficiency depend on the wettability of the porous surface. The resulting films were used to selectively capture dye-labeled MS2 viruses from solution, and a viral concentration as low as 2 x 10(7) plaque-forming units per mL (pfu/mL) was detectable by measuring the fluorescence from the exposed porous silicon film. The system exhibits sensitivity and dynamic range similar to the Luminex liquid array-based assay while outperforming protein micro-array methods.     

Activation of HGF/c-Met pathway contributes to the reactive oxygen species generation and motility of small cell lung cancer cells

Small cell lung cancer (SCLC) is a difficult disease to treat and sometimes has overexpression or mutation of c-Met receptor tyrosine kinase. The effects of c-Met/hepatocyte growth factor (c-Met/HGF, ligand for c-Met) on activation of reactive oxygen species (ROS) was determined. HGF stimulation of c-Met-overexpressing H69 SCLC cells (40 ng/ml, 15 min) resulted in an increase of ROS, measured with fluorescent probe 2'-7'-dichlorofluorescein diacetate (DCFH-DA) or dihydroethidine (DHE) but not in c-Met-null H446 cells. ROS was increased in juxtamembrane (JM)-mutated variants (R988C and T1010I) of c-Met compared with wild-type c-Met-expressing cells. ROS was significantly inhibited by preincubation of SCLC cells with pyrrolidine dithiocarbamate (PDTC, 100 microM) and/or SU11274 (small molecule c-Met tyrosine kinase inhibitor, 2 microM) for 3 h. PDTC and SU11274 also abrogated the HGF proliferative signal and cell motility in a cooperative fashion. H(2)O(2) treatment of SCLC cells (over 15 min) led to phosphorylation of c-Met receptor tyrosine kinase and further upregulated downstream phosphorylation of phospho-AKT, ERK1/2, and paxillin in a dose-dependent manner (125 microM to 500 microM). c-Met is an important target in lung cancer, and the pathways responsible for ROS generation together may provide novel therapeutic intervention.     

Pericytes Regulate Vascular Basement Membrane Remodeling and Govern Neutrophil Extravasation during Inflammation

During inflammation polymorphonuclear neutrophils (PMNs) traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs) of matrix proteins in the vascular basement membrane (BM) for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discovered that direct PMN-pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation.     

Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein alpha subunit at the endosome

In the yeast Saccharomyces cerevisiae, the G protein beta gamma subunits are essential for pheromone signaling. The Galpha subunit Gpa1 can also promote signaling, but the effectors in this pathway are not well characterized. To identify candidate Gpa1 effectors, we expressed the constitutively active Gpa1(Q323L) mutant in each of nearly 5000 gene-deletion strains and measured mating-specific responses. Our analysis reveals a requirement for both the catalytic (Vps34) and regulatory (Vps15) subunits of the sole phosphatidylinositol 3-kinase in yeast. We demonstrate that Gpa1 is present at endosomes, where it interacts directly with both Vps34 and Vps15 and stimulates increased production of phosphatidylinositol 3-phosphate. Notably, Vps15 binds to GDP-bound Gpa1 and is predicted to have a seven-WD repeat structure similar to that of known G protein beta subunits. These findings reveal two new components of the pheromone signaling pathway. More remarkably, these proteins appear to comprise a preformed effector-G beta subunit assembly and function at the endosome rather than at the plasma membrane.     

Trans-Golgi network and endosome dynamics connect ceramide homeostasis with regulation of the unfolded protein response and TOR signaling in yeast

Synthetic genetic array analyses identify powerful genetic interactions between a thermosensitive allele (sec14-1^ts) of the structural gene for the major yeast phosphatidylinositol transfer protein (SEC14) and a structural gene deletion allele (tlg2Δ) for the Tlg2 target membrane-soluble N-ethylmaleimide-sensitive factor attachment protein receptor. The data further demonstrate Sec14 is required for proper trans-Golgi network (TGN)/endosomal dynamics in yeast. Paradoxically, combinatorial depletion of Sec14 and Tlg2 activities elicits trafficking defects from the endoplasmic reticulum, and these defects are accompanied by compromise of the unfolded protein response (UPR). UPR failure occurs downstream of Hac1 mRNA splicing, and it is further accompanied by defects in TOR signaling. The data link TGN/endosomal dynamics with ceramide homeostasis, UPR activity, and TOR signaling in yeast, and they identify the Sit4 protein phosphatase as a primary conduit through which ceramides link to the UPR. We suggest combinatorial Sec14/Tlg2 dysfunction evokes inappropriate turnover of complex sphingolipids in endosomes. One result of this turnover is potentiation of ceramide-activated phosphatase-mediated down-regulation of the UPR. These results provide new insight into Sec14 function, and they emphasize the TGN/endosomal system as a central hub for homeostatic regulation in eukaryotes.     

Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein

SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.