Developing a vision for improved monitoring and reporting of riparian restoration projects

Representatives from agencies involved in natural resource management in the Murray‐Darling Basin gathered for a workshop in November 2010 to develop a vision for improved monitoring and reporting of riparian restoration projects. The resounding message from this workshop was that the effectiveness of riparian restoration depends on having sound, documented and agreed evidence on the ecological responses to restoration efforts. Improving our capacity to manage and restore riparian ecosystems is constrained by (i) a lack of ecological evidence on the effects of restoration efforts, and (ii) short‐termism in commitment to restoration efforts, in funding of monitoring and in expected time spans for ecosystem recovery. Restoration at the effective spatial scope will invariably require a long‐term commitment by researchers, funding agencies, management agencies and landholders. To address the knowledge gaps that constrain riparian restoration in the Basin, participants endorsed four major fields for future research: the importance of landscape context to restoration outcomes; spatio‐temporal scaling of restoration outcomes; functional effects of restoration efforts; and developing informative and effective indicators of restoration. To improve the monitoring and restoration of riparian zones throughout the Basin, participants advocated an integrated approach: a hierarchical adaptive management framework that incorporates long‐term ecological research.     

Intron-derived small RNAs for silencing viral RNAs in mosquito cells

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3’ UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.     

Re-establishment of Calluna vulgaris (L.) Hull in an eight-year grazing experiment on upland acid grassland

Upland heathland is an internationally important habitat but a large area in the UK has been degraded to acid grassland by intensive livestock grazing. Re-establishment of dwarf shrubs, particularly Calluna vulgaris (L.) Hull, is a key objective for restoring heathland on these sites. A replicated plot-scale experiment was set up to examine effects of disturbance and seed addition on C. vulgaris establishment in a Nardus stricta L. grassland under three grazing regimes: sheep only (1.5 ewes ha^?1 for 10 months per year); cattle only (0.5 heifers ha^?1 in summer only); and, the cattle regime combined with sheep (1.0 ewes ha^?1 for 10 months per year). Early results of the experiment have been reported previously but it was not known if these results were an indication of the longer-term restoration success. Here we evaluate the success of the restoration methods (disturbance, seeding treatments and grazing regime) eight years after the treatments began. In seeded plots, young C. vulgaris plants had greatest above-ground height, dry weight and shoot length if grazing was excluded or the cattle-only regime was applied. C. vulgaris cover was greatest, and increased most, in plots that had been disturbed, seeded and ungrazed or subjected to the cattle-only regime. The vegetation in these plots also became more similar to reference sites with 50% or more cover of C. vulgaris. The invasive Juncus effusus L. was more frequent in disturbed and grazed plots but less frequent in plots with C. vulgaris established from added seed. Previous results that showed the benefits of disturbance and seeding treatments were still valid but changes in the vegetation composition were still occurring and longer-term studies will be needed to determine when grazing regimes including sheep might be reintroduced.     

Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.     

A single-molecule Hershey-Chase experiment

Ever since Hershey and Chase used phages to establish DNA as the carrier of genetic information in 1952, the precise mechanisms of phage DNA translocation have been a mystery. Although bulk measurements have set a timescale for in vivo DNA translocation during bacteriophage infection, measurements of DNA ejection by single bacteriophages have only been made in vitro. Here, we present direct visualization of single bacteriophages infecting individual Escherichia coli cells. For bacteriophage λ, we establish a mean ejection time of roughly 5 min with significant cell-to-cell variability, including pausing events. In contrast, corresponding in vitro single-molecule ejections are more uniform and finish within 10 s. Our data reveal that when plotted against the amount of DNA ejected, the velocity of ejection for two different genome lengths collapses onto a single curve. This suggests that in vivo ejections are controlled by the amount of DNA ejected. In contrast, in vitro DNA ejections are governed by the amount of DNA left inside the capsid. This analysis provides evidence against a purely intrastrand repulsion-based mechanism and suggests that cell-internal processes dominate. This provides a picture of the early stages of phage infection and sheds light on the problem of polymer translocation.     

Human enhancement and sexual dimorphism

I argue that the existence of sexual dimorphism poses a profound challenge to those philosophers who wish to deny the moral significance of the idea of 'normal human capacities' in debates about the ethics of human enhancement. The biological sex of a child will make a much greater difference to their life prospects than many of the genetic variations that the philosophical and bioethical literature has previously been concerned with. It seems, then, that bioethicists should have something to say about the choice between a male and a female embryo. Either, 1) parents have reason to choose boys over girls; (2) parents have reason to choose girls over boys; or, (3) parents have neither reason to choose girls over boys nor reason to choose boys over girls. Embracing either of the first two alternatives has strongly counterintuitive--and arguably morally repugnant--consequences. To motivate the third option we must either make reference to the idea of 'normal human capacities' or argue that parents should consider the interests of society when thinking about what sort of children they should bring into the world - an implication that should be extremely controversial in debates about the 'new eugenics'. I conclude, then, that the idea of 'normal human capacities' is properly crucial to reasoning about the ethics of shaping future persons.     

Integrating DNA barcode data and taxonomic practice: determination, discovery, and description

DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.     

Comparative metabolism of alpha- and beta-peptides in the insect Heliothis virescens and in plant cells of black Mexican sweet maize

The tripeptide H-Val-Ala-Leu-OH and the N-Ac-tetrapeptide amide Ac-Thr-Lys-Trp-Phe-NH2, and their beta-peptidic counterparts H-beta(3)hVal-beta(3)hAla-beta(3)hLeu-OH and Ac-beta(3)hThr-(S)beta(2)hLys-beta(3)hTrp-beta(3)hPhe-NH2, respectively, have been injected into Heliothis virescens larvae and added to cell cultures of black mexican sweet maize. The body liquids of the larvae and the supernatant of the plant cell cultures were sampled 0, 2, 3, 6, 17, and/or 24 h after application and analyzed by LC/MS. While the two alpha-peptides were degraded rapidly in these environments, the concentration of the beta-peptides was found to decrease very slowly. Thus, ca. 60% of the original amount of the beta-tetrapeptide was detected in the liquids of the insect after 24 h. The plant cells did not seem to make use of the beta-peptides at all, whereas, the alpha-tripeptide completely disappeared from the supernatant after 3 h. Thus, we have demonstrated, for the first time, the high stability of beta-peptides against degradation and metabolism in an insect and a plant. Especially remarkable is the persistence of the beta-tetrapeptide with its functionalised and, thus, 'metabolisable' side chains of Thr, Lys, Trp, and Phe in the insect larvae, which are known to have a high level of activity of oxidizing enzymes. The results described here match those of ADME investigations with radioactively labeled beta-peptides in rats, where essentially complete stability has been observed, while environmental microorganisms have been found to biodegrade beta-peptides, albeit slowly. Possible implications of these findings for biomedical and pest-control applications are proposed.     

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.