Cost-effectiveness of managing Natura 2000 sites: an exploratory study for Finland,Germany, the Netherlands and Poland

Natura 2000 sites are expected to assure the long-term survival of Europe’s most valuable and threatened species and habitats. It follows that successful management of the sites is of great importance. Next to goal attainment, cost-effectiveness is increasingly recognised as a key requirement for gaining social and political acceptance for costly conservation measures. We identify and qualitatively examine issues of cost-effectiveness related to the design and implementation of management measures in Natura 2000 sites in Finland, Germany, the Netherlands and Poland. Given the wide variety of management design and implementation options within the four countries, our study is purely of an exploratory nature. We derive recommendations for improving the cost-effectiveness of management in Natura 2000 sites and for future research. Examples of policy recommendations include guaranteeing the availability of funds for longer periods, and ensuring the appropriate allocation of funds between the different tasks of designing and implementing management plans. Further research should examine the cost-effectiveness of controversial suggestions such as, for example, more tailored payment schemes for conservation measures that result in higher ecological outputs but are costly to administer. Moreover, more research is needed to better understand how rules for administrations, as well as rules and governance structures for tasks within administrations, should be designed.     

WNK1 activates ERK5 by an MEKK2/3-dependent mechanism

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.     

Stable-isotope probing and metagenomics reveal predation by protozoa drives E. coli removal in slow sand filters

Stable-isotope probing and metagenomics were applied to study samples taken from laboratory-scale slow sand filters 0.5, 1, 2, 3 and 4 h after challenging with ¹³C-labelled Escherichia coli to determine the mechanisms and organisms responsible for coliform removal. Before spiking, the filters had been continuously operated for 7 weeks using water from the River Kelvin, Glasgow as their influent source. Direct counts and quantitative PCR assays revealed a clear predator–prey response between protozoa and E. coli. The importance of top-down trophic-interactions was confirmed by metagenomic analysis, identifying several protozoan and viral species connected to E. coli attrition, with protozoan grazing responsible for the majority of the removal. In addition to top-down mechanisms, indirect mechanisms, such as algal reactive oxygen species-induced lysis, and mutualistic interactions between algae and fungi, were also associated with coliform removal. The findings significantly further our understanding of the processes and trophic interactions underpinning E. coli removal. This study provides an example for similar studies, and the opportunity to better understand, manage and enhance E. coli removal by allowing the creation of more complex trophic interaction models.     

MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC₅₀s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.     

Role of DptE and DptF in the lipidation reaction of daptomycin

Daptomycin and A21987C antibiotics are branched, cyclic, nonribosomally assembled acidic lipodepsipeptides produced by Streptomyces roseosporus. The antibacterial activity of daptomycin against gram-positive bacteria strongly depends on the nature of the N-terminal fatty acid moiety. Two genes, dptE and dptF, localized upstream of the daptomycin nonribosomal peptide synthetase genes, are thought to be involved in the lipidation of daptomycin. Here we describe the cloning, heterologous expression, purification and biochemical characterization of the enzymes encoded by these genes. DptE was proven to preferentially activate branched mid- to long-chain fatty acids under ATP consumption, and these fatty acids are subsequently transferred onto DptF, the cognate acyl carrier protein. Additionally, we demonstrate that lipidation of DptF by DptE in trans is based on specific protein-protein interactions, as DptF is favored over other acyl carrier proteins. Study of DptE and DptF may provide useful insights into the lipidation mechanism, and these enzymes may be used to generate novel daptomycin derivatives with altered fatty acids.     

The anti‐apoptotic Bcl‐2 protein regulates hair follicle stem cell function

Maintaining the architecture, size and composition of an intact stem cell (SC) compartment is crucial for tissue homeostasis and regeneration throughout life. In mammalian skin, elevated expression of the anti‐apoptotic Bcl‐2 protein has been reported in hair follicle (HF) bulge SCs (BSCs), but its impact on SC function is unknown. Here, we show that systemic exposure of mice to the Bcl‐2 antagonist ABT‐199/venetoclax leads to the selective loss of suprabasal BSCs (sbBSCs), thereby disrupting cyclic HF regeneration. RNAseq analysis shows that the pro‐apoptotic BH3‐only proteins BIM and Bmf are upregulated in sbBSCs, explaining their addiction to Bcl‐2 and the marked susceptibility to Bcl‐2 antagonism. In line with these observations, conditional knockout of Bcl‐2 in mouse epidermis elevates apoptosis in BSCs. In contrast, ectopic Bcl‐2 expression blocks apoptosis during HF regression, resulting in the accumulation of quiescent SCs and delaying HF growth in mice. Strikingly, Bcl‐2‐induced changes in size and composition of the HF bulge accelerate tumour formation. Our study identifies a niche‐instructive mechanism of Bcl‐2‐regulated apoptosis response that is required for SC homeostasis and tissue regeneration, and may suppress carcinogenesis.     

The dynamics of surface acoustic wave‐driven scaffold cell seeding

Flow visualization using fluorescent microparticles and cell viability investigations are carried out to examine the mechanisms by which cells are seeded into scaffolds driven by surface acoustic waves. The former consists of observing both the external flow prior to the entry of the suspension into the scaffold and the internal flow within the scaffold pores. The latter involves micro‐CT (computed tomography) scans of the particle distributions within the seeded scaffolds and visual and quantitative methods to examine the morphology and proliferation ability of the irradiated cells. The results of these investigations elucidate the mechanisms by which particles are seeded, and hence provide valuable information that form the basis for optimizing this recently discovered method for rapid, efficient, and uniform scaffold cell seeding. Yeast cells are observed to maintain their size and morphology as well as their proliferation ability over 14 days after they are irradiated. The mammalian primary osteoblast cells tested also show little difference in their viability when exposed to the surface acoustic wave irradiation compared to a control set. Together, these provide initial feasibility results that demonstrate the surface acoustic wave technology as a viable seeding method without risk of denaturing the cells. Biotechnol. Bioeng. 2009;103: 387–401. © 2009 Wiley Periodicals, Inc.     

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.