Identification of plant semiochemicals and evaluation of their interactions with early spring insect pests of asparagus

Information is lacking on the chemical ecology of asparagus, and knowledge about the effects of its volatile emissions on its associated early season pest species is completely absent. The current study aimed to (1) evaluate whether the asparagus miner responds to asparagus volatiles, (2) identify and compare the changes in asparagus host plant volatiles from mechanical and chewing damage by the black cutworm, a temporally co-occurring species with the asparagus miner, and (3) assess how asparagus volatiles affect asparagus miner populations in the field. Results indicated that asparagus miners were significantly attracted to healthy asparagus stems when compared to clean air. Damaged asparagus headspace volatiles were quantitatively and qualitatively different from healthy plants. Volatile baits elicited a range of responses, but their effects were inconsistent between sampling years and phenology-dependent. Overall, we demonstrated that the chemical ecology of asparagus may be altered by its pest community, and volatiles identified from asparagus may impact the behavior of the asparagus miner.     

Three-Dimensional Reconstruction of Three-Way FRET Microscopy Improves Imaging of Multiple Protein-Protein Interactions

Fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for imaging the interactions between fluorescently tagged proteins in two-dimensions. For FRET microscopy to reach its full potential, it must be able to image more than one pair of interacting molecules and image degradation from out-of-focus light must be reduced. Here we extend our previous work on the application of maximum likelihood methods to the 3-dimensional reconstruction of 3-way FRET interactions within cells. We validated the new method (3D-3Way FRET) by simulation and fluorescent protein test constructs expressed in cells. In addition, we improved the computational methods to create a 2-log reduction in computation time over our previous method (3DFSR). We applied 3D-3Way FRET to image the 3D subcellular distributions of HIV Gag assembly. Gag fused to three different FPs (CFP, YFP, and RFP), assembled into viral-like particles and created punctate FRET signals that become visible on the cell surface when 3D-3Way FRET was applied to the data. Control experiments in which YFP-Gag, RFP-Gag and free CFP were expressed, demonstrated localized FRET between YFP and RFP at sites of viral assembly that were not associated with CFP. 3D-3Way FRET provides the first approach for quantifying multiple FRET interactions while improving the 3D resolution of FRET microscopy data without introducing bias into the reconstructed estimates. This method should allow improvement of widefield, confocal and superresolution FRET microscopy data.     

Living in the city: can anyone become an ‘urban exploiter'?

Aim As urban landscapes expand, shifts in biodiversity are occurring. This is leading biogeographers and ecologists to consider human‐dominated landscapes in their current work. One question that arises is: what characterizes those species that are widespread in the most highly urban environments compared with those restricted to less urbanized areas in the city? Here, we aim to identify the traits that enable species to become urban exploiters, i.e. to dominate highly urbanized surroundings. Identifying these traits may help us better predict and possibly mitigate the biotic homogenization occurring in these areas. Location Israel in general, with special focus on the city of Jerusalem. Methods Combining literature and field‐based data for birds in Israel we compared phenotypic, behavioural and life‐history traits between urban exploiters and urban adapters. The latter occur in urban landscapes, but are characteristic of the less urbanized parts of the city. We then examined the trends along a finer field‐sampled gradient of increasing urbanization from sub‐natural to downtown areas within the city of Jerusalem. Results Urban exploiters and adapters differed primarily in social structure and migratory status: exploiters were significantly more social and sedentary than urban adapters. Clear trends were also seen for dietary preferences along a gradient of increasing urbanization in Jerusalem, such that, with increasing urbanization, the proportion of granivorous species increased whereas the proportion of species feeding on invertebrates declined. In contrast, neither relative brain size nor behavioural flexibility, as measured by feeding innovations, differed significantly among urban exploiters and adapters in Israel or along the urbanization gradient in Jerusalem specifically. Main conclusions The results of our study suggest that being successful in more vs. less urbanized environments in the city is not necessarily a factor of brain size nor of how flexible and behaviourally innovative the species is; rather, it depends on a combination of traits, including diet, degree of sociality, sedentariness and preferred nesting sites.     

Comprehensive DNA signature discovery and validation

DNA signatures are nucleotide sequences that can be used to detect the presence of an organism and to distinguish that organism from all other species. Here we describe Insignia, a new, comprehensive system for the rapid identification of signatures in the genomes of bacteria and viruses. With the availability of hundreds of complete bacterial and viral genome sequences, it is now possible to use computational methods to identify signature sequences in all of these species, and to use these signatures as the basis for diagnostic assays to detect and genotype microbes in both environmental and clinical samples. The success of such assays critically depends on the methods used to identify signatures that properly differentiate between the target genomes and the sample background. We have used Insignia to compute accurate signatures for most bacterial genomes and made them available through our Web site. A sample of these signatures has been successfully tested on a set of 46 Vibrio cholerae strains, and the results indicate that the signatures are highly sensitive for detection as well as specific for discrimination between these strains and their near relatives. Our approach, whereby the entire genomic complement of organisms are compared to identify probe targets, is a promising method for diagnostic assay development, and it provides assay designers with the flexibility to choose probes from the most relevant genes or genomic regions. The Insignia system is freely accessible via a Web interface and has been released as open source software at: http://insignia.cbcb.umd.edu.     

Kinetic analysis of IgG antibodies to beta-amyloid oligomers with surface plasmon resonance

Surface plasmon resonance was used to investigate the kinetics, affinity, and specificity of binding between anti-Aβ (beta-amyloid) IgG antibodies and oligomeric Aβ. Two factors were needed to accurately characterize the IgG binding kinetics. First, a bivalent model was necessary to properly fit the kinetic association and dissociation sensograms. Second, a high concentration of IgG was necessary to overcome a significant mass transport limitation that existed regardless of oligomer density on the sensor surface. Using high IgG concentrations and bivalent fits, consistent kinetic parameters were found at varying sensor surface ligand densities. A comparison of binding specificity, affinity, and kinetic flux between monoclonal and natural human anti-Aβ IgG antibodies revealed the following findings. First, monoclonal antibodies 6E10 and 4G8 single-site binding affinity is similar between Aβ oligomers and monomers. Second, natural human anti-Aβ IgG binding readily binds Aβ oligomers but does not bind monomers. Third, natural human anti-Aβ IgG binds Aβ oligomers with a higher affinity and kinetic flux than 6E10 and 4G8. Both the current analytical methodology and antibody binding profiles are important for advances in antibody drug development and kinetic biomarker applications for Alzheimer’s disease.     

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.     

A restatement of recent advances in the natural science evidence base concerning neonicotinoid insecticides and insect pollinators

A summary is provided of recent advances in the natural science evidence base concerning the effects of neonicotinoid insecticides on insect pollinators in a format (a ‘restatement'') intended to be accessible to informed but not expert policymakers and stakeholders. Important new studies have been published since our recent review of this field (Godfray et al. 2014 Proc. R. Soc. B 281, 20140558. (doi:10.1098/rspb.2014.0558)) and the subject continues to be an area of very active research and high policy relevance.     

Plantagora: modeling whole genome sequencing and assembly of plant genomes

Background

Genomics studies are being revolutionized by the next generation sequencing technologies, which have made whole genome sequencing much more accessible to the average researcher. Whole genome sequencing with the new technologies is a developing art that, despite the large volumes of data that can be produced, may still fail to provide a clear and thorough map of a genome. The Plantagora project was conceived to address specifically the gap between having the technical tools for genome sequencing and knowing precisely the best way to use them.

Methodology/Principal Findings

For Plantagora, a platform was created for generating simulated reads from several different plant genomes of different sizes. The resulting read files mimicked either 454 or Illumina reads, with varying paired end spacing. Thousands of datasets of reads were created, most derived from our primary model genome, rice chromosome one. All reads were assembled with different software assemblers, including Newbler, Abyss, and SOAPdenovo, and the resulting assemblies were evaluated by an extensive battery of metrics chosen for these studies. The metrics included both statistics of the assembly sequences and fidelity-related measures derived by alignment of the assemblies to the original genome source for the reads. The results were presented in a website, which includes a data graphing tool, all created to help the user compare rapidly the feasibility and effectiveness of different sequencing and assembly strategies prior to testing an approach in the lab. Some of our own conclusions regarding the different strategies were also recorded on the website.

Conclusions/Significance

Plantagora provides a substantial body of information for comparing different approaches to sequencing a plant genome, and some conclusions regarding some of the specific approaches. Plantagora also provides a platform of metrics and tools for studying the process of sequencing and assembly further.     

Use of Electronic Loggers to Measure Changes in the Rates of Hand Washing with Soap in Low-Income Urban Households in India

We evaluated the utility of electronic loggers to measure the effects of a simple intervention designed to influence the rates of hand washing with soap within enclosed toilets and bathrooms in low-income urban households in Kerala, India. 58 households were given three items with embedded electronic loggers for a period of 2-5 days. Two logged soaps tracked hand and body washing in the bathroom. The third logged item was a water vessel used for flushing the toilet and for post-defecation anal cleansing; this served as a marker of toilet use. In addition, 28 households in a Soap by toilet arm were given an additional logged soap, to be kept by the toilet, and used for hand washing. Compared with the Soap in bathroom arm, the loggers in the Soap by toilet households recorded 73% greater daily use of soaps designated for hand washing (t(36)=2.92, p<0.01) and 172% greater use within 2 minutes of the use of the water vessel (t(36)=3.51, p = 0.001). We conclude that the loggers were capable of detecting changes in the rates of hand washing with soap and changes in hand washing with soap after use of the toilet. Further adoption of logger technologies would enable more insightful studies of hand washing within urban environments.     

Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat

Renin-angiotensin-aldosterone system (RAAS) activation mediates increases in reactive oxygen species (ROS) and impaired insulin signaling. The transgenic Ren2 rat manifests increased tissue renin-angiotensin system activity, elevated serum aldosterone, hypertension, and insulin resistance. To explore the role of aldosterone in the pathogenesis of insulin resistance, we investigated the impact of in vivo treatment with a mineralocorticoid receptor (MR) antagonist on insulin sensitivity in Ren2 and aged-matched Sprague-Dawley (SD) control rats. Both groups (age 6-8 wk) were implanted with subcutaneous time-release pellets containing spironolactone (0.24 mg/day) or placebo over 21 days. Systolic blood pressure (SBP) and intraperitoneal glucose tolerance test were determined. Soleus muscle insulin receptor substrate-1 (IRS-1), tyrosine phosphorylated IRS-1, protein kinase B (Akt) phosphorylation, GLUT4 levels, and insulin-stimulated 2-deoxyglucose uptake were evaluated in relation to NADPH subunit expression/oxidase activity and ROS production (chemiluminescence and 4-hydroxy-2-nonenal immunostaining). Along with increased soleus muscle NADPH oxidase activity and ROS, there was systemic insulin resistance and reduced muscle IRS-1 tyrosine phosphorylation, Akt phosphorylation/activation, and GLUT4 expression in the Ren2 group (each P < 0.05). Despite not decreasing blood pressure, low-dose spironolactone treatment improved soleus muscle insulin signaling parameters and systemic insulin sensitivity in concert with reductions in NADPH oxidase subunit expression/activity and ROS production (each P < 0.05). Our findings suggest that aldosterone contributes to insulin resistance in the transgenic Ren2, in part, by increasing NADPH oxidase activity in skeletal muscle tissue.