Conservation genetics: Linking science with practice

Conservation genetics is a well‐established scientific field. However, limited information transfer between science and practice continues to hamper successful implementation of scientific knowledge in conservation practice and management. To mitigate this challenge, we have established a conservation genetics community, which entails an international exchange‐and‐skills platform related to genetic methods and approaches in conservation management. First, it allows for scientific exchange between researchers during annual conferences. Second, personal contact between conservation professionals and scientists is fostered by organising workshops and by popularising knowledge on conservation genetics methods and approaches in professional journals in national languages. Third, basic information on conservation genetics has been made accessible by publishing an easy‐to‐read handbook on conservation genetics for practitioners. Fourth, joint projects enabled practitioners and scientists to work closely together from the start of a project in order to establish a tight link between applied questions and scientific background. Fifth, standardised workflows simplifying the implementation of genetic tools in conservation management have been developed. By establishing common language and trust between scientists and practitioners, all these measures help conservation genetics to play a more prominent role in future conservation planning and management.     

Membrane cholesterol content modulates ClC-2 gating and sensitivity to oxidative stress

ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl-beta-cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.     

A comparative study of type I and type II tryparedoxin peroxidases in Leishmania major

The genome of Leishmania major, the causative agent of cutaneous leishmaniasis, contains three almost identical genes encoding putative glutathione peroxidases, which differ only at their N- and C-termini. Because the gene homologues are essential in trypanosomes, they may also represent potential drug targets in Leishmania. Recombinant protein for the shortest of these showed negligible peroxidase activity with glutathione as the electron donor indicating that it is not a bone fide glutathione peroxidase. By contrast, high peroxidase activity was obtained with tryparedoxin, indicating that these proteins belong to a new class of monomeric tryparedoxin-dependent peroxidases (TDPX) distinct from the classical decameric 2-Cys peroxiredoxins (TryP). Mass spectrometry studies revealed that oxidation of TDPX1 with peroxides results in the formation of an intramolecular disulfide bridge between Cys35 and Cys83. Site-directed mutagenesis and kinetic studies showed that Cys35 is essential for peroxidase activity, whereas Cys83 is essential for reduction by tryparedoxin. Detailed kinetic studies comparing TDPX1 and TryP1 showed that both enzymes obey saturation ping-pong kinetics with respect to tryparedoxin and peroxide. Both enzymes show high affinity for tryparedoxin and broad substrate specificity for hydroperoxides. TDPX1 shows higher affinity towards hydrogen peroxide and cumene hydroperoxide than towards t-butyl hydroperoxide, whereas no specific substrate preference could be detected for TryP1. TDPX1 exhibits rate constants up to 8 x 10(4) m(-1).s(-1), whereas TryP1 exhibits higher rate constants approximately 10(6) m(-1).s(-1). All three TDPX proteins together constitute approximately 0.05% of the L. major promastigote protein content, whereas the TryPs are approximately 40 times more abundant. Possible specific functions of TDPXs are discussed.     

Insect pest management in the age of synthetic biology

Arthropod crop pests are responsible for 20% of global annual crop losses, a figure predicted to increase in a changing climate where the ranges of numerous species are projected to expand. At the same time, many insect species are beneficial, acting as pollinators and predators of pest species. For thousands of years, humans have used increasingly sophisticated chemical formulations to control insect pests but, as the scale of agriculture expanded to meet the needs of the global population, concerns about the negative impacts of agricultural practices on biodiversity have grown. While biological solutions, such as biological control agents and pheromones, have previously had relatively minor roles in pest management, biotechnology has opened the door to numerous new approaches for controlling insect pests. In this review, we look at how advances in synthetic biology and biotechnology are providing new options for pest control. We discuss emerging technologies for engineering resistant crops and insect populations and examine advances in biomanufacturing that are enabling the production of new products for pest control.     

‘Birth defects’ of photosystem II make it highly susceptible to photodamage during chloroplast biogenesis

High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water‐oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodeling. Employing membrane‐inlet mass spectrometry and O₂‐polarography under flashing light conditions, we demonstrate that newly synthesized PSII complexes are far more susceptible to photodamage than are mature PSII complexes. We examined these ‘PSII birth defects’ in barley seedlings and plastids (etiochloroplasts and chloroplasts) isolated at various times during de‐etiolation as chloroplast development begins and matures in synchronization with thylakoid membrane biogenesis and grana membrane formation. We show that the degree of PSII photodamage decreases simultaneously with biogenesis of the PSII turnover efficiency measured by O₂‐polarography, and with grana membrane stacking, as determined by electron microscopy. Our data from fluorescence, Q_B‐inhibitor binding, and thermoluminescence studies indicate that the decline of the high‐light susceptibility of PSII to photodamage is coincident with appearance of electron transfer capability Q_A^? → Q_B during de‐etiolation. This rate depends in turn on the downstream clearing of electrons upon buildup of the complete linear electron transfer chain and the formation of stacked grana membranes capable of longer‐range energy transfer.     

Impact of phagostimulants on effectiveness of OMRI‐listed insecticides used for control of spotted‐wing drosophila (Drosophila suzukii Matsumura)

Spotted‐wing drosophila, Drosophila suzukii Matsumura, is an invasive pest in the United States that causes considerable damage to fruit crops. It is responsible for many millions of dollars of revenue loss. The female D. suzukii has a heavily sclerotized ovipositor and can lay eggs in ripening or ripe fruit. The arrival of this invasive species has disrupted existing integrated pest management programmes, and growers rely on repeated insecticide applications to protect fruit. Organic growers have few chemical control options, and their reliance on spinosad increases the risk of developing insecticide resistance. We hypothesized that combining phagostimulants with insecticides would increase insecticide efficacy by prompting flies to spend more time in contact with residues. Therefore, the objective of this study was to evaluate the effectiveness of sucrose and the yeast Saccharomyces cerevisiae as phagostimulants in combination with organic biopesticides against D. suzukii in blueberries. Adding sucrose with or without yeast did not improve insecticide efficacy in terms of adult fly mortality or fruit infestation. Spinosad was very effective in all experiments, and for this product, there is little room for improvement. The phagostimulants had no effect on residual activity of any insecticide. The addition of sucrose with or without yeast did not improve the effectiveness of organic insecticides for D. suzukii. Concentrations of these phagostimulants in our experiments (0.36%) may have been too low to elicit a response. Further research is recommended to test different types and concentrations of phagostimulants.     

Cortisol metabolism in the domestic cat and implications for non-invasive monitoring of adrenocortical function in endangered felids

Three domestic cats were given i.m. injections of ³H-cortisol to determine the time course and relative proportion of excreted ³H-cortisol metabolites into urine and feces. Most urinary radioactivity was detected in the first sample collected at 3.9 ± 2.5 hr postinjection and accounted for 13.9 ± 2.1% of the total radioactivity recovered. High performance liquid chromatography (HPLC) detected four urinary metabolites, one of which (13.7% urinary radioactivity) eluted with the ³H-cortisol reference tracer and was quantifiable using a commercial cortisol radioimmunoassay (RIA). The majority of cortisol metabolites in feces (85.9 ± 2.1%) was excreted at 22.3 ± 6.2 hr. HPLC analysis detected several fecal metabolites consisting primarily of nonhydolyzable water-soluble forms, none of which eluted with ³H-cortisol or ³H-corticosterone reference tracers. No immunoreactivity was detected in HPLC-separated fecal eluates using the cortisol RIA; however, two of the more polar metabolites were quantifiable using a commerical cortisosterone RIA. The physiological relevance of the immunoreactive fecal metabolites was determined in four domestic cats given an adrenocorticotropin (ACTH) challenge. Increased serum cortisol concentrations were detected within 30 min of ACTH injection, which was maintained for at least 6 hr. A corresponding increase in fecal cortisol metabolite concentrations (ranging from 238% to 826% over individual baseline values) was observed 24–48 hr later. These data indicate that adrenocortical activity can be monitored nonivasively in the cat by measuring cortisol metabolites excreted in feces. This procedure is a potentially valuable tool for endangered felid management to help evaluate responses to physiological and psychological stressors associated with environmental conditions and husbandry practices. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Ring‐opening of azetidiniums by nucleophiles. Synthesis of polysubstituted linear amines

This microreview focuses on the nucleophilic ring‐opening of azetidiniums presenting various substitution patterns at C2, C3, and C4. In most cases, the nucleophilic ring‐opening occurred in a stereoselective and regioselective fashion producing functionalized linear amines. Experimental selectivities associated with Density Functional Theory (DFT) calculations have allowed a better understanding of the parameters governing the regioselectivities.     

PEtab—Interoperable specification of parameter estimation problems in systems biology

Reproducibility and reusability of the results of data-based modeling studies are essential. Yet, there has been—so far—no broadly supported format for the specification of parameter estimation problems in systems biology. Here, we introduce PEtab, a format which facilitates the specification of parameter estimation problems using Systems Biology Markup Language (SBML) models and a set of tab-separated value files describing the observation model and experimental data as well as parameters to be estimated. We already implemented PEtab support into eight well-established model simulation and parameter estimation toolboxes with hundreds of users in total. We provide a Python library for validation and modification of a PEtab problem and currently 20 example parameter estimation problems based on recent studies.     

Rearrangements within human spliceosomes captured after exon ligation

In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3′ exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3′ exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex). In comparison to C complex, which is blocked at a point following first-step chemistry, we detect specific differences in RNA substrate interactions near the splice sites. These differences include extended protection across the exon junction and changes in protein crosslinks to specific sites in the 5′ and 3′ exons. Using selective reaction monitoring (SRM) mass spectrometry, we quantitatively compared P and C complex proteins and observed enrichment of SF3b components and loss of the putative RNA-dependent ATPase DHX35. Electron microscopy revealed similar structural features for both complexes. Notably, additional density is present when complexes are chemically fixed, which reconciles our results with previously reported C complex structures. Our ability to compare human spliceosomes before and after second-step chemistry has opened a new window to rearrangements near the active site of spliceosomes, which may play roles in exon ligation and mRNA release.