Engineering of benzylglucosinolate in tobacco provides proof-of-concept for dead-end trap crops genetically modified to attract Plutella xylostella (diamondback moth)

Glucosinolates are biologically active natural products characteristic of crucifers, including oilseed rape, cabbage vegetables and the model plant Arabidopsis thaliana. Crucifer‐specialist insect herbivores, like the economically important pest Plutella xylostella (diamondback moth), frequently use glucosinolates as oviposition stimuli. This suggests that the transfer of a glucosinolate biosynthetic pathway to a non‐crucifer would stimulate oviposition on an otherwise non‐attractive plant. Here, we demonstrate that stable genetic transfer of the six‐step benzylglucosinolate pathway from A. thaliana to Nicotiana tabacum (tobacco) results in the production of benzylglucosinolate without causing morphological alterations. Benzylglucosinolate‐producing tobacco plants were more attractive for oviposition by female P. xylostella moths than wild‐type tobacco plants. As newly hatched P. xylostella larvae were unable to survive on tobacco, these results represent a proof‐of‐concept strategy for rendering non‐host plants attractive for oviposition by specialist herbivores with the long‐term goal of generating efficient dead‐end trap crops for agriculturally important pests.     

Improved anticancer potency by head‐to‐tail cyclization of short cationic anticancer peptides containing a lipophilic β2,2‐amino acid

We have recently reported a series of synthetic anticancer heptapeptides (H‐KKWβ^2,2WKK‐NH₂) containing a central achiral and lipophilic β^2,2‐amino acid that display low toxicity against non‐malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising β^2,2‐amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC‐5). The results demonstrated a considerable increase in anticancer potency following head‐to‐tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High‐resolution NMR studies and molecular dynamics simulations together with an annexin‐V‐FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Genome-wide functional annotation of dual-specificity protein- and lipid-binding modules that regulate protein interactions

Emerging evidence indicates that membrane lipids regulate protein networking by directly interacting with protein-interaction domains (PIDs). As a pilot study to identify and functionally annodate lipid-binding PIDs on a genomic scale, we performed experimental and computational studies of PDZ domains. Characterization of 70 PDZ domains showed that ~40% had submicromolar membrane affinity. Using a computational model built from these data, we predicted the membrane-binding properties of 2,000 PDZ domains from 20 species. The accuracy of the prediction was experimentally validated for 26 PDZ domains. We also subdivided lipid-binding PDZ domains into three classes based on the interplay between membrane- and protein-binding sites. For different classes of PDZ domains, lipid binding regulates their protein interactions by different mechanisms. Functional studies of a PDZ domain protein, rhophilin 2, suggest that all classes of lipid-binding PDZ domains serve as genuine dual-specificity modules regulating protein interactions at the membrane under physiological conditions.     

Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods

ABSTRACT: BACKGROUND: Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. RESULTS: Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40 % depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. CONCLUSION: Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.     

Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization

An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic β-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization to form the structural equivalent of the classical hexamer. The covalently linked dimer neither bound to the insulin receptor, nor induced a metabolic response in vitro. However, it was extremely thermodynamically stable and did not form amyloid fibrils when subjected to mechanical stress, underlining the importance of oligomerization for insulin stability.     

BactMAP: An R package for integrating,analyzing and visualizing bacterial microscopy data

High-throughput analyses of single-cell microscopy data are a critical tool within the field of bacterial cell biology. Several programs have been developed to specifically segment bacterial cells from phase-contrast images. Together with spot and object detection algorithms, these programs offer powerful approaches to quantify observations from microscopy data, ranging from cell-to-cell genealogy to localization and movement of proteins. Most segmentation programs contain specific post-processing and plotting options, but these options vary between programs and possibilities to optimize or alter the outputs are often limited. Therefore, we developed BactMAP (Bacterial toolbox for Microscopy Analysis & Plotting), a command-line based R package that allows researchers to transform cell segmentation and spot detection data generated by different programs into various plots. Furthermore, BactMAP makes it possible to perform custom analyses and change the layout of the output. Because BactMAP works independently of segmentation and detection programs, inputs from different sources can be compared within the same analysis pipeline. BactMAP complies with standard practice in R which enables the use of advanced statistical analysis tools, and its graphic output is compatible with ggplot2, enabling adjustable plot graphics in every operating system. User feedback will be used to create a fully automated Graphical User Interface version of BactMAP in the future. Using BactMAP, we visualize key cell cycle parameters in Bacillus subtilis and Staphylococcus aureus, and demonstrate that the DNA replication forks in Streptococcus pneumoniae dissociate and associate before splitting of the cell, after the Z-ring is formed at the new quarter positions. BactMAP is available from https://veeninglab.com/bactmap .     

Primary Adsorption Site of Phage PBS1: the Flagellum of Bacillus subtilis

The adsorption of Bacillus subtilis phage PBS1 was studied, and it was demonstrated that the primary adsorption site for this phage is the flagellum of B. subtilis. The capacity of flagella to function for motility may be lost without the loss of their capacity to adsorb the phage and permit infection. Deoxyribonucleic acid injection by the phage is inhibited by cyanide, suggesting the requirement for cellular energy in the infection process.     

Physico-chemical environment,phytoplankton biomass and production in oligotrophic,softwater lake kalgaard,Denmark

Water chemistry of Lake Kalgaard in 1976–77 was characterized by low concentrations of total-CO₂ and inorganic nutrients. The ionic composition resembled that of precipitation (Na>Ca>Mg >K and Cl>SO₄>HCO₃). The seasonal pattern of total-CO₂ and PO₄ was regulated by internal processes and maximum concentrations as a result of decomposition processes occurred during summer stagnation. NO₃ concentrations showed the opposite pattern and were relatively high from late autumn through spring and were extremely low during summer. Total-P and PO₄ increased during summer due to release from the sediment. The phytoplankton biomass of surface water was low. The water chemistry suggested a shift from N-limitation of phytoplankton during summer to P-limitation at other seasons. Maximum algal concentrations occurred at 6 m during summer, probably due to a supply of nutrients (especially NH₄) from deeper layers. Phytoplankton productivity was often bimodal, with an upper maximum at depths of 0 or 2 m and a second maximum at 6 m.