Brassinosteroid-Mediated Stress Responses

Brassinosteroids (BRs) are a group of naturally occurring plant steroidal compounds with wide-ranging biological activity that offer the unique possibility of increasing crop yields through both changing plant metabolism and protecting plants from environmental stresses. In recent years, genetic and biochemical studies have established an essential role for BRs in plant development, and on this basis BRs have been given the stature of a phytohormone. A remarkable feature of BRs is their potential to increase resistance in plants to a wide spectrum of stresses, such as low and high temperatures, drought, high salt, and pathogen attack. Despite this, only a few studies aimed at understanding the mechanism by which BRs promote stress resistance have been undertaken. Studies of the BR signaling pathway and BR gene-regulating properties indicate that there is cross-talk between BRs and other hormones, including those with established roles in plant defense responses such as abscisic acid, jasmonic acid, and ethylene. Recent studies aimed at understanding how BRs modulate stress responses suggest that complex molecular changes underlie BR-induced stress tolerance in plants. Analyses of these changes should generate exciting results in the future and clarify whether the ability of BRs to increase plant resistance to a range of stresses lies in the complex interactions of BRs with other hormones. Future studies should also elucidate if BRI1, an essential component of the BR receptor, directly participates in stress response signaling through interactions with ligands and proteins involved in plant defense responses.     

Joint generalized models for multidimensional outcomes: A case study of neuroscience data from multimodalities

This paper is motivated from the analysis of neuroscience data in a study of neural and muscular mechanisms of muscle fatigue. Multidimensional outcomes of different natures were obtained simultaneously from multiple modalities, including handgrip force, electromyography (EMG), and functional magnetic resonance imaging (fMRI). We first study individual modeling of the univariate response depending on its nature. A mixed‐effects beta model and a mixed‐effects simplex model are compared for modeling the force/EMG percentages. A mixed‐effects negative‐binomial model is proposed for modeling the fMRI counts. Then, I present a joint modeling approach to model the multidimensional outcomes together, which allows us to not only estimate the covariate effects but also to evaluate the strength of association among the multiple responses from different modalities. A simulation study is conducted to quantify the possible benefits by the new approaches in finite sample situations. Finally, the analysis of the fatigue data is illustrated with the use of the proposed methods.     

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

Ostreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins.     

Divergent selection drives the adaptive radiation of crossbills

Abstract Knowledge of how phenotype influences fitness is necessary if we are to understand the basis of natural selection and how natural selection contributes to adaptive radiations. Here I quantify selection on a wild population of red crossbills ( Loxia curvirostra complex) in the South Hills, Idaho. Bill depth is the target of selection and selection on bill depth is stabilizing. I then show how fitness is related to both bill depth and performance. I use these and previously published relationships to estimate a fitness surface for five species of red crossbills that are part of an ongoing adaptive radiation in western North America. The fitness surface for crossbills has distinct peaks and valleys, with each crossbill species residing on or very near the summits. This work strongly supports a key tenet of the ecological theory of adaptive radiations; namely, divergent selection for utilizing alternative resources is the ultimate cause of adaptive radiations.     

Enzyme synthesis of oligosaccharides using cashew apple juice as substrate

The use of agriculture substrates in industrial biotechnological processes has been increasing because of their low cost. In this work, the use of clarified cashew apple juice was investigated as substrate for enzyme synthesis of prebiotic oligosaccharide. The results showed that cashew apple juice is a good source of reducing sugars and can be used as substrate for the production of dextransucrase by Leuconostoc citreum B-742 for the synthesis of oligosaccharides using the crude enzyme. Optimal oligosaccharide yield (approximately 80%) was obtained for sucrose concentrations lower than 60 g/L and reducing sugar concentrations higher than 100 g/L.     

Gill morphometrics of the thresher sharks (Genus Alopias): Correlation of gill dimensions with aerobic demand and environmental oxygen

Gill morphometrics of the three thresher shark species (genus Alopias) were determined to examine how metabolism and habitat correlate with respiratory specialization for increased gas exchange. Thresher sharks have large gill surface areas, short water–blood barrier distances, and thin lamellae. Their large gill areas are derived from long total filament lengths and large lamellae, a morphometric configuration documented for other active elasmobranchs (i.e., lamnid sharks, Lamnidae) that augments respiratory surface area while limiting increases in branchial resistance to ventilatory flow. The bigeye thresher, Alopias superciliosus, which can experience prolonged exposure to hypoxia during diel vertical migrations, has the largest gill surface area documented for any elasmobranch species studied to date. The pelagic thresher shark, A. pelagicus, a warm‐water epi‐pelagic species, has a gill surface area comparable to that of the common thresher shark, A. vulpinus, despite the latter's expected higher aerobic requirements associated with regional endothermy. In addition, A. vulpinus has a significantly longer water–blood barrier distance than A. pelagicus and A. superciliosus, which likely reflects its cold, well‐oxygenated habitat relative to the two other Alopias species. In fast‐swimming fishes (such as A. vulpinus and A. pelagicus) cranial streamlining may impose morphological constraints on gill size. However, such constraints may be relaxed in hypoxia‐dwelling species (such as A. superciliosus) that are likely less dependent on streamlining and can therefore accommodate larger branchial chambers and gills. J. Morphol. 276:589–600, 2015. © 2015 Wiley Periodicals, Inc.     

Functional assessment of cryopreserved pig aortas for pharmaceutical research

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached.After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.     

Antibody fingerprints in lyme disease deciphered with high density peptide arrays

Lyme disease is the most common tick‐borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface‐exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE‐positive patients by mapping the protein as overlapping peptides and subsequent in‐depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE‐positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein‐Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.     

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry

Background

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.

Method

Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.

Results

The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10⁵ and (0.85 ± 0.11) × 10⁵, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.

Conclusion

Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-43) contains supplementary material, which is available to authorized users.     

Real-time and Label-free Bio-sensing of Molecular Interactions by Surface Plasmon Resonance: A Laboratory Medicine Perspective

Radioactive, chromogenic, fluorescent and other labels have long provided the basis of detection systems for biomolecular interactions including immunoassays and receptor binding studies. However there has been unprecedented growth in a number of powerful label free biosensor technologies over the last decade. While largely at the proof-of-concept stage in terms of clinical applications, the development of more accessible platforms may see surface plasmon resonance (SPR) emerge as one of the most powerful optical detection platforms for the real-time monitoring of biomolecular interactions in a label-free environment.In this review, we provide an overview of SPR principles and current and future capabilities in a diagnostic context, including its application for monitoring a wide range of molecular markers of disease. The advantages and pitfalls of using SPR to study biomolecular interactions are discussed, with particular emphasis on its potential to differentiate subspecies of analytes and the inherent ability for quantitation through calibration-free concentration analysis (CFCA). In addition, recent advances in multiplex applications, high throughput arrays, miniaturisation, and enhancements using noble metal nanoparticles that promise unprecedented sensitivity to the level of single molecule detection, are discussed.In summary, while SPR is not a new technique, technological advances may see SPR quickly emerge as a highly powerful technology, enabling rapid and routine analysis of molecular interactions for a diverse range of targets, including those with clinical applicability. As the technology produces data quickly, in real-time and in a label-free environment, it may well have a significant presence in future developments in lab-on-a-chip technologies including point-of-care devices and personalised medicine.