MRE11 complex links RECQ5 helicase to sites of DNA damage

RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11–RAD50–NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3′→5′ exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair.     

Science-policy interfaces for biodiversity: dynamic learning environments for successful impact

To address the pressing problems associated with biodiversity loss, changes in awareness and behaviour are required from decision makers in all sectors. Science-policy interfaces (SPIs) have the potential to play an important role, and to achieve this effectively, there is a need to understand better the ways in which existing SPIs strive for effective communication, learning and behavioural change. Using a series of test cases across the world, we assess a range of features influencing the effectiveness of SPIs through communication and argumentation processes, engagement of actors and other aspects that contribute to potential success. Our results demonstrate the importance of dynamic and iterative processes of interaction to support effective SPI work. We stress the importance of seeing SPIs as dynamic learning environments and we provide recommendations for how they can enhance success in meeting their targeted outcomes. In particular, we recommend building long-term trust, creating learning environments, fostering participation and ownership of the process and building capacity to combat silo thinking. Processes to enable these changes may include, for example, inviting and integrating feedback, extended peer review and attention to contextualising knowledge for different audiences, and time and sustained effort dedicated to trust-building and developing common languages. However there are no ‘one size fits all’ solutions, and methods must be adapted to context and participants. Creating and maintaining effective dynamic learning environments will both require and encourage changes in institutional and individual behaviours: a challenging agenda, but one with potential for positive feedbacks to maintain momentum.     

Ecological scarcity 2013—new features and its application in industry and administration—54th LCA forum,Ittigen/Berne,Switzerland, December 5, 2013

The 54th LCA forum was held on December 5, 2013 to launch the fourth generation ecological scarcity method, applied to Switzerland. This conference report presents the highlights of the LCA forum. The ecological scarcity method belongs to the family of distance-to-target methods and is based on politically and legally defined environmental goals. The application of the method in industry and politics as well as its benefits, the main elements of the method and new elements such as the assessment of abiotic resources, global land use, noise and nuclear waste are presented. The losses (and not the extraction) of abiotic resources are characterised with the abiotic depletion potential. Land use impacts on flora and fauna biodiversity are quantified per land use type and for 14 different biomes. Transport noise is assessed based on the number of highly annoyed persons. Finally, nuclear waste is characterised using the radio toxicity index, a parameter commonly used in the nuclear industry. In three policy-making areas, LCA in general and the ecological scarcity method in particular are being applied: waste policy, biofuels tax exemption and Green Economy. Practical applications in administration and industry show that the eco-factors are considered useful in decision making because they cover a broad range of environmental impacts aggregated to a single score. The results of first applications and comparisons showed that the switch from third to fourth generation eco-factors hardly affects the results and conclusions although there are some significant changes in the eco-factor of individual pollutants. It was concluded that the fourth generation is a moderate evolution from the third generation published in 2008. It is considered crucial to allow for single-score methods as they allow to assess environmental impacts comprehensively and to identify environmental hot spots. The method presented thus is suited for a “true and fair” reporting on environmental information.     

The M4 muscarinic acetylcholine receptor plays a key role in the control of murine hair follicle cycling and pigmentation

Cholinergic receptors of the muscarinic class (M1-M5) are expressed in epidermal keratinocytes and melanocytes as well as in the hair follicle. Knockout (KO) mice of all five receptors have been created and resulted in different phenotypes. KO mice with a deletion of the M4 muscarinic acetylcholine receptor (M4R) present a striking hair phenotype, which we have analyzed here in greater detail by quantitative histomorphometry. Earlier studies revealed a retarded hair follicle morphogenesis in M4R KO mice, compared to age-matched wild type controls. On day 17, when mice enter the first hair growth cycle, the KO mice still showed a slightly retarded catagen phase. Subsequently, hair follicles of the KO mice stayed in a highly significantly prolonged telogen phase, while wild type mice had already far progressed in the hair cycle by entry into anagen. Most strikingly, the M4R KO mice did not engage in follicular melanogenesis and failed to produce pigmented hair shafts. The current pilot study suggests that the M4R plays a fundamental role in the control of the murine hair follicle cycling and is an essential signaling element in the control of hair follicle pigmentation.     

Testing the efficiency of three 15N-labeled nitrogen compounds for indirect labeling of grasshoppers via plants in the field

In field studies of plant–insect herbivore interactions it is often difficult to establish which herbivore has fed on a particular plant. We investigated the suitability of three different ¹⁵N‐labeled nitrogen compounds (ammonium, nitrate, and glycine) for indirect marking of three grasshopper species [Omocestus viridulus (L.), Chorthippus parallelus (Zett.), and Chorthippus biguttulus (L.) (Orthoptera: Acrididae)] through labeling their food plants in the field. In two short‐term experiments grassland plots of 1 m² were separately labeled with either one of the different nitrogen compounds. Grasshoppers were caged on three food‐plant species [Dactylis glomerata L., Holcus lanatus L. (Poaceae), and Trifolium repens L. (Fabaceae)] present in these plots for 72 h. Significantly enriched δ¹⁵N values in grasshoppers were found in all plant/grasshopper combinations. Enrichment in grasshoppers was positively correlated with the enrichment of plants and labeling with nitrate resulted in highest ¹⁵N enrichment. In a long‐term experiment, individuals of C. biguttulus were placed in a cage covering an area of 1 m² for 37 days, with sampling of grasshoppers at regular intervals. δ¹⁵N values of the grasshopper and a common food plant, D. glomerata, increased steadily over time, up to 40‐fold by the end of the experiment. Our results demonstrate that ¹⁵N‐labeling of plants is an appropriate tool for the investigation of insect–plant interactions under natural conditions.     

Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a

The oxidative folding, particularly the arrangement of disulfide bonds of recombinant extracellular N-terminal domains of the corticotropin-releasing factor receptor type 2a bearing five cysteines (C2 to C6), was investigated. Depending on the position of a His-tag, two types of disulfide patterns were found. In the case of an N-terminal His-tag, the disulfide bonds C2-C3 and C4-C6 were found, leaving C5 free, whereas the C-terminal position of the His-tag led to the disulfide pattern C2-C5 and C4-C6, and leaving C3 free. The latter pattern is consistent with the disulfide arrangement of the extracellular N-terminal domain of the corticotropin-releasing factor (CRF) receptor type 1, which has six cysteines (C1 to C6) and in which C1 is paired with C3. However, binding data of the two differently disulfide-bridged domains show no significant differences in binding affinities to selected ligands, indicating the importance of the C-terminal portion of the N-terminal receptor domains, particularly the disulfide bond C4-C6 for ligand binding.     

Four terpene synthases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpa

After herbivore damage, many plants increase their emission of volatile compounds, with terpenes usually comprising the major group of induced volatiles. Populus trichocarpa is the first woody species with a fully sequenced genome, enabling rapid molecular approaches towards characterization of volatile terpene biosynthesis in this and other poplar species. We identified and characterized four terpene synthases (PtTPS1-4) from P. trichocarpa which form major terpene compounds of the volatile blend induced by gypsy moth (Lymantria dispar) feeding. The enzymes were heterologously expressed and assayed with potential prenyl diphosphate substrates. PtTPS1 and PtTPS2 accepted only farnesyl diphosphate and produced (−)-germacrene D and (E,E)-α-farnesene as their major products, respectively. In contrast, PtTPS3 and PtTPS4 showed both mono- and sesquiterpene synthase activity. They produce the acyclic terpene alcohols linalool and nerolidol but exhibited opposite stereospecificity. qRT-PCR analysis revealed that the expression of the respective terpene synthase genes was induced after feeding of gypsy moth caterpillars. The TPS enzyme products may play important roles in indirect defense of poplar to herbivores and in mediating intra- and inter-plant signaling.     

Inhibition of DNA replication by fish oil-treated cytoplasm is counteracted by fish oil-treated nuclear extract

We have recentlynoted that cells treated with fish oil and n-3-fatty acids showslower DNA replication rates than cells treated with a control emulsionor corn oil only. However, it is not clearly understood howsuch an effect is induced. Fish oil and its metabolites are known tohave several modulating effects on signal transduction pathways.Alternatively, they may influence DNA replication by interactingdirectly with nuclear components. To investigate this problem ingreater detail, we have studied the kinetics of DNA synthesis in acell-free system derived from HeLa cells. Nuclei and cytosolic extractwere isolated from cells synchronized in early S phase after treatmentwith control emulsion, corn oil, or fish oil, respectively. The nucleiwere reconstituted with cytosolic extract and a reaction mixturecontaining bromodeoxyuridine (BrdU) triphosphate to label newlysynthesized DNA. The rate of DNA synthesis was measured by bivariateDNA/BrdU analysis and flow cytometry. We show that fish oil-treatedcytosol inhibits the elongation of newly synthesized DNA by ~80% incontrol nuclei. However, nuclei treated with fish oil escape thisinhibitory effect. We also show that addition of nuclear extract fromfish oil-treated cells reverses the inhibitory effect seen in thereconstitution system of control nuclei and fish oil-treated cytosol.These results indicate that polyunsaturated fatty acids can modulateDNA synthesis through cytosolic as well as soluble nuclear factors.

     

Characterization of conserved properties of hemagglutinin of H5N1 and human influenza viruses: possible consequences for therapy and infection control

Background  

Epidemics caused by highly pathogenic avian influenza virus (HPAIV) are a continuing threat to human health and to the world's economy. The development of approaches, which help to understand the significance of structural changes resulting from the alarming mutational propensity for human-to-human transmission of HPAIV, is of particularly interest. Here we compare informational and structural properties of the hemagglutinin (HA) of H5N1 virus and human influenza virus subtypes, which are important for the receptor/virus interaction.