Comparative binding of Cry1Ab and Cry1F Bacillus thuringiensis toxins to brush border membrane proteins from Ostrinia nubilalis, Ostrinia furnacalis and Diatraea saccharalis (Lepidoptera: Crambidae) midgut tissue

The European (Ostrinia nubilalis Hübner) and Asian corn borers (Ostrinia furnacalis Guenée) are closely related and display similar sensitivity to Cry1 toxins. In this study, we compared the binding patterns of Cry1Ab and Cry1F toxins between both Ostrinia spp., as well as the expression of putative cadherin- and aminopeptidase-N (APN)-like protein receptors. Additionally, cDNA sequences of these putative toxin receptors from both Ostrinia species were compared. Ligand blots for both species indicated a similar binding pattern for Cry1Ab with the strongest immunoreactive band at 260 kDa in both species. In addition, similar expression of the putative cadherin- and APN-like protein receptors were observed at 260 and 135 kDa, respectively. A high degree of similarity (98% amino acid sequence identity) of cDNA sequences for both putative receptor sequences was observed. The Cry1F ligand blot revealed that O. furnacalis and O. nubilalis BBMV exhibited slightly different binding patterns, with strong binding to putative proteins at 150 and 140 kDa, respectively. Both proteins appeared to also bind Cry1Ab, although the signal intensity was much reduced with Cry1Ab. O. furnacalis showed an additional but weaker band at 210 kDa relative to the 150 kDa band. Diatraea saccharalis (Fabricius), which was used as an outgroup species, exhibited different binding patterns than either Ostrinia species, with both Cry1Ab and Cry1F toxins binding to a 210 kDa protein. These results support the previous experiments indicating that O. nubilalis and O. furnacalis share similar patterns of susceptibility to Cry toxins.     

Unusual phenolic compounds contribute to ecophysiological performance in the purple‐colored green alga Zygogonium ericetorum (Zygnematophyceae,Streptophyta) from a high‐alpine habitat

The filamentous green alga Zygogonium ericetorum (Zygnematophyceae, Streptophyta) was collected in a high‐alpine rivulet in Tyrol, Austria. Two different morphotypes of this alga were found: a purple morph with a visible purple vacuolar content and a green morph lacking this coloration. These morphotypes were compared with respect to their secondary metabolites, ultrastructure, and ecophysiological properties. Colorimetric tests with aqueous extracts of the purple morph indicated the presence of soluble compounds such as phenolics and hydrolyzable tannins. High‐performance liquid chromatography‐screening showed that Z. ericetorum contained several large phenolic peaks with absorption maxima at ~280 nm and sometimes with minor maxima at ~380 nm. Such compounds are uncommon for freshwater green microalgae, and could contribute to protect the organism against increased UV and visible (VIS) irradiation. The purple Z. ericetorum contained larger amounts (per dry weight) of the putative phenolic substances than the green morph; exposure to irradiation may be a key factor for accumulation of these phenolic compounds. Transmission electron microscopy of the purple morph showed massive vacuolization with homogenous medium electron‐dense content in the cell periphery, which possibly contains the secondary compounds. In contrast, the green morph had smaller, electron‐translucent vacuoles. The ecophysiological data on photosynthesis and desiccation tolerance indicated that increasing photon fluence densities led to much higher relative electron transport rates (rETR) in the purple than in the green morph. These data suggest that the secondary metabolites in the purple morph are important for light acclimation in high‐alpine habitats. However, the green morph recovered better after 4 d of rehydration following desiccation stress.     

Involvement of virulence properties and antibiotic resistance in Escherichia coli strains causing pyelonephritis in children

A total of 145 Escherichia coli strains causing pyelonephritis in children were investigated for the prevalence of genes encoding the following virulence factors (VFs): P fimbria (67.6?%), S fimbria (53.8?%), AFA adhesins (2.8?%), cytotoxic necrotizing factor 1 (37.9?%), ??-hemolysin (41.4?%), and aerobactin (71.7?%). One hundred and thirty-six (93.8?%) isolates harbored at least one of the virulence genes detected in the present study. Statistically significant co-occurrent presence of two VF genes was found for ??-hly?Ccnf1, ??-hly?Csfa, cnf1?Csfa (p?<?0.001), and ??-hly?Cpap (p?=?0.001). Twenty-six profiles of VF genes were detected in this study. The combinations of aer?Cpap and aer?Cpap?Csfa?C??-hly?Ccnf1 were presented with the highest frequency??both of them in 28 isolates (19.3?%). All E. coli strains included in the study were susceptible to meropenem, amikacin, and tobramycin; the highest frequency resistance was found toward ampicillin (43.4?%), piperacillin (31.7?%), tetracycline (15.9?%), and trimethoprim/sulfamethoxazole (11.7?%). The resistance to the other tested antimicrobial drugs did not exceed 3?% incidence. Overall, 55.9?% strains were susceptible to all tested anti-infective agents. Antimicrobial resistance of E. coli strains toward trimethoprim/sulfamethoxazole statistically significantly correlated with the presence of ??-hly (p?<?0.001), sfa (p?<?0.01), and cnf1 (p?<?0.05).     

Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (Part I)

A series of structurally novel aryl ureas was derived from optimization of the HTS lead as selective histamine H₃ receptor (H₃R) antagonists. The SAR was explored and the data obtained set up the starting point and foundation for further optimization. The most potent tool compounds, as exemplified by compounds 2l, 5b, 5d, and 5e, displayed antagonism potencies in the subnanomolar range in in vitro human-H₃R FLIPR assays and rhesus monkey H₃R binding assays.     

Signatures of diversifying selection at EST‐SSR loci and association with climate in natural Eucalyptus populations

Understanding the environmental parameters that drive adaptation among populations is important in predicting how species may respond to global climatic changes and how gene pools might be managed to conserve adaptive genetic diversity. Here, we used Bayesian F_ST outlier tests and allele–climate association analyses to reveal two Eucalyptus EST‐SSR loci as strong candidates for diversifying selection in natural populations of a southwestern Australian forest tree, Eucalyptus gomphocephala (Myrtaceae). The Eucalyptus homolog of a CONSTANS‐like gene was an F_ST outlier, and allelic variation showed significant latitudinal clinal associations with annual and winter solar radiation, potential evaporation, summer precipitation and aridity. A second F_ST outlier locus, homologous to quinone oxidoreductase, was significantly associated with measures of temperature range, high summer temperature and summer solar radiation, with important implications for predicting the effect of temperature on natural populations in the context of climate change. We complemented these data with investigations into neutral population genetic structure and diversity throughout the species range. This study provides an investigation into selection signatures at gene‐homologous EST‐SSRs in natural Eucalyptus populations, and contributes to our understanding of the relationship between climate and adaptive genetic variation, informing the conservation of both putatively neutral and adaptive components of genetic diversity.     

phenoSeeder - A Robot System for Automated Handling and Phenotyping of Individual Seeds

The enormous diversity of seed traits is an intriguing feature and critical for the overwhelming success of higher plants. In particular, seed mass is generally regarded to be key for seedling development but is mostly approximated by using scanning methods delivering only two-dimensional data, often termed seed size. However, three-dimensional traits, such as the volume or mass of single seeds, are very rarely determined in routine measurements. Here, we introduce a device named phenoSeeder, which enables the handling and phenotyping of individual seeds of very different sizes. The system consists of a pick-and-place robot and a modular setup of sensors that can be versatilely extended. Basic biometric traits detected for individual seeds are two-dimensional data from projections, three-dimensional data from volumetric measures, and mass, from which seed density is also calculated. Each seed is tracked by an identifier and, after phenotyping, can be planted, sorted, or individually stored for further evaluation or processing (e.g. in routine seed-to-plant tracking pipelines). By investigating seeds of Arabidopsis (Arabidopsis thaliana), rapeseed (Brassica napus), and barley (Hordeum vulgare), we observed that, even for apparently round-shaped seeds of rapeseed, correlations between the projected area and the mass of seeds were much weaker than between volume and mass. This indicates that simple projections may not deliver good proxies for seed mass. Although throughput is limited, we expect that automated seed phenotyping on a single-seed basis can contribute valuable information for applications in a wide range of wild or crop species, including seed classification, seed sorting, and assessment of seed quality.Seeds play a major role in keeping continuity between successive generations (Esau, 1977) and are key for the distribution and evolution (Moles et al., 2005) of higher plants. Fertile seeds carry an embryo and may contain nutrient storage tissues in cotyledons, endosperm, and/or perisperm, supporting germination and seedling development at early developmental stages. Although this is true for all seed plants, various traits of seeds, such as size, shape, weight, and chemical composition, can be very different between plant species or accessions. For example, the Arabidopsis (Arabidopsis thaliana) accession Cape Verde Islands was reported to yield on average 40% fewer seeds than Landsberg erecta, but they are almost twice as heavy (Alonso-Blanco et al., 1999). Considering today’s plant species, single-seed mass may vary over a range of 11.5 orders of magnitude (Moles et al., 2005). Seed mass is under strong genetic control, whereas the total number of seeds of a plant is largely affected by the environment (Paul-Victor and Turnbull, 2009). It has been demonstrated that the size, mass, and shape of Arabidopsis seeds may be regulated by brassinosteroid (Jiang et al., 2013), and it was shown recently that seed size in rice (Oryza sativa) can be influenced by the epiallele Epi-rav6 (Zhang et al., 2015). The ability of plants to switch between small and larger seeds may be understood as an adaptation to novel environments (Igea et al., 2016). However, it is still not fully understood whether, or to what extent, the variability of seed traits within plant species or genotypes has an impact on the development and further performance of a plant.When comparing biometric seed data of different dimensions such as length (one-dimensional), projected area (two-dimensional [2D]), or volume and mass (both three-dimensional [3D]), one can argue that mass is the most relevant parameter as a proxy for the amount of reserves a seed provides for the offspring. This might be true even when considering that the type of reserves, such as proteins, carbohydrates, or lipids (Rolletschek et al., 2015), and also different seed tissues, such as seed coat, embryo, or endosperm, may contribute differently to seed mass (Alonso-Blanco et al., 1999). While seed mass and time to germination (radicle protrusion) do not necessarily correlate (Norden et al., 2009), in particular under greenhouse conditions, higher seed mass may be advantageous for seedling establishment under adverse environmental conditions (Moles et al., 2005). For example, shade-tolerant species showed largely higher seed masses than cogeneric species growing in open habitats, indicating that seedlings under low-light conditions need more reserves than under good light (Salisbury, 1974). Seedlings of wild radish (Raphanus raphanistrum) emerged more likely from heavier seeds than from small seeds under field conditions but not in the greenhouse (Stanton, 1984), and for Arabidopsis, seed mass was reported to be higher in populations growing naturally at higher altitudes taken as a proxy for harsher conditions (Montesinos-Navarro et al., 2011).Seed mass can be measured individually (Stanton, 1984), but it is generally collected as an average value of batches of 50 to 1,000 seeds (Jako et al., 2001; Jofuku et al., 2005; Montesinos-Navarro et al., 2011; Tanabata et al., 2012). Alternatively, 2D scans are analyzed to determine parameters such as seed length, width, area, and perimeter length as a measure for seed size (Tanabata et al., 2012). This approach can be implemented in high-throughput facilities to obtain projected areas of seed grains combined with genome-wide association studies (Yang et al., 2014). Although projected seed area can easily be measured with a common office scanner (Herridge et al., 2011; Tanabata et al., 2012; Moore et al., 2013), it is not necessarily a precise or reliable measure of the true seed size because it may depend on the shape (Alonso-Blanco et al., 1999) and the orientation of a seed at scan (see “Results”). These issues also apply when using 2D projections to calculate length-to-width ratios as a simple shape factor (Tanabata et al., 2012). Projected seed area also has been used to calculate seed mass, assuming a fixed relationship between these parameters (de Jong et al., 2011; Herridge et al., 2011). This may hold with sufficient accuracy when averaging a large number of seeds but might be misleading when considering individual seeds.From a physical point of view, volume should be a much better proxy for mass than 2D traits. Although it has been stated that for 65 species analyzed seed masses can be compared easily with seed volumes (Moles et al., 2005), it is not clear how these seed volumes were determined. Volumes can be assessed using advanced methods such as x-ray computed tomography (CT) on fruits (Stuppy et al., 2003) or synchrotron radiation x-ray tomographic microscopy applied in paleobiological studies (e.g. on fruits and seed; Friis et al., 2014). Nuclear magnetic resonance (NMR) methods are used to measure water uptake in kidney beans (Phaseolus vulgaris) and adzuki beans (Vigna angularis; Kikuchi et al., 2006) or to estimate seed weight and content (Borisjuk et al., 2011; Rolletschek et al., 2015) rather than volumes. To our best knowledge, affordable methods to measure seed volumes directly are not achievable so far. For that reason, we have set up a volume-carving method for 3D seed shape reconstruction that is described briefly here and in more detail in a recent publication (Roussel et al., 2016).While traits derived from scanning procedures can easily be assigned to individual seeds (Herridge et al., 2011), further handling and processing of phenotyped single seeds is not as simple, in particular for tiny ones like those of Arabidopsis. The aim of this work was to develop an automated seed-handling system that can analyze single seeds of very different sizes or shapes, from Arabidopsis seeds up to barley (Hordeum vulgare) seeds or even bigger. The phenoSeeder system is designed to pick and place seeds, to achieve basic morphometric traits (one-dimensional and 2D data from projections, 3D reconstruction data, and mass) of each individual seed, and to store all analyzed seed traits in a database. Another goal is to use phenoSeeder for seed-to-plant tracking approaches and to analyze whether, or which, particular seed traits have an impact on plant development and performance under various environmental conditions. We describe the main features of the phenoSeeder technology and present results obtained with seeds of three accessions of Arabidopsis, rapeseed (Brassica napus), and barley, respectively. When analyzing the data, we focused particularly on correlations between projected seed area, seed volume, and seed mass, with the hypothesis that the respective seed volume may better correlate with mass than the projected area.     

Single giant vesicle rupture events reveal multiple mechanisms of glass-supported bilayer formation

The formation of supported lipid bilayers (SLBs) on glass from giant unilamellar vesicles (GUVs) was studied using fluorescence microscopy. We show that GUV rupture occurs by at least four mechanisms, including 1), spontaneous rupture of isolated GUVs yielding almost heart-shaped bilayer patches (asymmetric rupture); 2), spontaneous rupture of isolated GUVs yielding circular bilayer patches (symmetric rupture); 3), induced rupture of an incoming vesicle when it contacts a planar bilayer edge; and 4), induced rupture of an adsorbed GUV when a nearby GUV spontaneously ruptures. In pathway 1, the dominant rupture pathway for isolated GUVs, GUVs deformed upon adsorption to the glass surface, and planar bilayer patch formation was initiated by rupture pore formation near the rim of the glass-bilayer interface. Expanding rupture pores led to planar bilayer formation in approximately 10-20 ms. Rupture probability per unit time depended on the average intrinsic curvature of the component lipids. The membrane leaflet adsorbed to the glass surface in planar bilayer patches originated from the outer leaflet of GUVs. Pathway 2 was rarely observed. We surmise that SLB formation is predominantly initiated by pathway 1 rupture events, and that rupture events occurring by pathways 3 and 4 dominate during later stages of SLB formation.     

Plasma cell development in synovial germinal centers in patients with rheumatoid and reactive arthritis

Plasma cells are found surrounding the inflammatory infiltrates of macrophages, T, and B cells in the synovial tissue of patients with rheumatoid and reactive arthritis. This characteristic arrangement suggests that in the synovial tissue CD20+ B cells differentiate into plasma cells. To examine clonal relationships, we have used micromanipulation to separately isolate CD20+ B cells and plasma cells from single infiltrates. DNA was extracted, and from both populations the VH/VL gene repertoires was determined. The data show that in the inflamed synovial tissue activated B cells are clonally expanded. During proliferation in the network of follicular dendritic cells, V gene variants are generated by the hypermutation mechanism. Surprisingly, we do not find identical rearrangements between CD20+ B cells and plasma cells. Nevertheless, the finding of clonally related plasma cells within single infiltrates suggests that these cells underwent terminal differentiation in the synovial tissue. These results indicate that B cell differentiation in the synovial tissue is a dynamic process. Whereas CD20+ B cells may turnover rapidly, plasma cells may well be long lived and thus accumulate in the synovial tissue. The analysis of individual B cells recovered from synovial tissue opens a new way to determine the specificity of those cells that take part in the local immune reaction. This will provide new insights into the pathogenesis of chronic inflammatory diseases like rheumatoid or reactive arthritis.     

mau-2 acts cell-autonomously to guide axonal migrations in Caenorhabditis elegans

The gene mau-2 has been found to be required for the guidance of cellular and axonal migrations along both the anteroposterior and the dorsoventral body axes during the development of the nematode C. elegans. We show that mau-2 encodes a novel, previously uncharacterized protein that is highly conserved among animals. Maternal mau-2 gene expression is sufficient for normal development until the fourth larval stage, and a MAU-2::GFP fusion protein localizes to the cytoplasm of neurones. mau-2 is ubiquitously expressed in embryos by late gastrulation and becomes predominantly expressed in the nervous system as morphogenesis progresses. Expression of mau-2 within individual neurones rescues the guidance defects of mau-2 mutants, indicating that mau-2 functions cell-autonomously. Altering the activity of both the dorsal repellent slt-1 and mau-2 leads to the abnormal dorsal projection of the AVM axon, a phenotype that is novel and specific to the interaction of these two genes, indicating that mau-2 participates in the guidance of AVM by a slt-1-independent mechanism. Taken together, mau-2 defines a novel guidance factor that might be involved in the intracellular processing of guidance cues encountered by migrating cells and axons during development.