Inhibition of fucosylation of cell wall components by 2‐fluoro 2‐deoxy‐l‐fucose induces defects in root cell elongation

Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2‐fluoro 2‐l ‐fucose (2F‐Fuc) reduces root growth at micromolar concentrations. The inability of 2F‐Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F‐Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N‐linked glycans is fully inhibited by 10 μm 2F‐Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F‐Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan‐II (RG‐II). At low concentrations, 2F‐Fuc induced a decrease in RG‐II dimerization. Both RG‐II dimerization and root growth were partially restored in 2F‐Fuc‐treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F‐Fuc was due to a deficiency of RG‐II dimerization. Closer investigation of the 2F‐Fuc‐induced growth phenotype demonstrated that cell division is not affected by 2F‐Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG‐II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG‐II cross‐linking, but that it might also be a signal molecule in the cell wall integrity‐sensing mechanism.     

Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites

Phosphorylation of the voltage-gated Na⁺ (Na_V) channel Na_V1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed Na_V1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on Na_V1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic Na_V1.5 mutants revealed the roles of three phosphosites in regulating Na_V1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface Na_V1.5 expression and peak Na⁺ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular Na_V1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of Na_V1.5 channels is highly complex, site specific, and dynamic.     

N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.     

Richness of ancient forest plant species indicates suitable habitats for macrofungi

Macrofungal species richness generally increases with forest continuity as does the richness of so-called ancient forest plant species (AFS). Based on this assumption, we examined the ability of AFS to indicate macrofungal diversity in six study areas covering a range of elevations and environments in the Czech Republic. In total, we used data from 106 sampling plots (2,500 m² each) distributed over six types of forest stands reflecting different intensities and temporal stages of forest management. Species composition of vascular plants and macrofungi was recorded by a single inventory and regular 2-year monitoring, respectively. In total, we found 71 AFS and 1,413 macrofungal species, of which 150 were red-listed macrofungal species. We documented that AFS show potential for being used in the prediction of macrofungi species richness, including endangered species, at the local scale (α-diversity). Additionally, we found significant differences in macrofungal species richness depending on study area and type of forest management, which did not, however, derogate the effect of AFS. Spatial congruence between species composition of AFS and macrofungi communities (β-diversity) increased with forest age and decreased with intensity of forest management. If we consider the simplicity of monitoring AFS in comparison to regular monitoring of macrofungi, we found a widely usable tool for estimating macrofungal diversity in all dominant types of managed forest in central Europe. However, we should be aware of the limited ability of AFS to capture macrofungal diversity across a broader spatial context (γ-diversity), especially in areas with a low diversity of AFS.     

Reduced rhinovirus-specific antibodies are associated with acute exacerbations of chronic obstructive pulmonary disease requiring hospitalisation

ABSTRACT: BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are often linked to respiratory infections. However, it is unknown if COPD patients who experience frequent exacerbations have impaired humoral immunity. The aim of this study was to determine if antibodies specific for common respiratory pathogens are associated with AECOPD. METHODS: Plasma was obtained from COPD patients when clinically stable. AECOPD requiring hospitalisation were recorded. IgG1 antibodies to H. Influenzae outer membrane protein 6 (P6), pneumococcal surface protein C (PspC) and the VP1 viral capsid protein of rhinovirus were measured. RESULTS: COPD patients who had an AECOPD (n = 32) had significantly lower anti-VP1 IgG1 antibody levels when stable compared to COPD patients who did not have an AECOPD (n = 28, p = 0.024). Furthermore, the number of hospitalisations was inversely proportional to anti-VP1 antibody levels (r = 0.331, p = 0.011). In contrast, antibodies specific for P6 and PspC were present at similar concentrations between groups. Plasma IL-21, a cytokine important for B-cell development and antibody synthesis, was also lower in COPD patients who had an AECOPD, than in stable COPD patients (p = 0.046). CONCLUSION: Deficient humoral immunity specific for rhinoviruses is associated with AECOPD requiring hospitalisation, and may partly explain why some COPD patients have an increased exacerbation risk following respiratory viral infections.     

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.     

Colorimetric detection of residual quaternary ammonium compounds on dry surfaces and prediction of antimicrobial activity using bromophenol blue

Controlling and monitoring the residual activity of quaternary ammonium compounds (QACs) are critical for maintaining safe yet effective levels of these agents in the environment. This study investigates the utility of bromophenol blue (BPB) as a safe, rapid and user-friendly indicator to detect in situ residual QACs dried on hard, non-porous surfaces, as well a means to assess their antimicrobial efficacy. At pH 7, BPB has a purple colour which turns blue upon its complexation with QACs such as didecyldimethylammonium chloride (DDAC). BPB itself has no antimicrobial properties up to 400 ppm. Within the range of 0–400 ppm, BPB colour change was tied to specific DDAC antimicrobial performances with a detection threshold of 100 ppm. BPB concentration and application volume could be adjusted such that a colour shift from purple to blue correlated with a set percent reduction (>99·9%) in test bacteria (Staphylococcus aureus and Klebsiella aerogenes). The BPB solutions developed in this study yielded similar colour shifts on polycarbonate and stainless steel surfaces and did not cross-react with chemical ingredients commonly found in sanitizers and disinfectant products. Overall, this study suggests that BPB provides a simple solution to safely monitor the post-application level and biocidal activity of residual dried QACs on surfaces.     

A hierarchical matrix model to assess the impact of habitat fragmentation on population dynamics: an elasticity analysis

To better understand the role of habitat quality and boundaries on population dynamics at the landscape scale, we develop a model combining a spatially implicit approach, a spatial population Leslie-type model and an implicit model of habitat fragmentation. An original approach of elasticity permits to identify which types of element and boundary influence the most population viability according to the wood fragmentation degree. The studied species is a corridor forest insect sensitive to fragmentation (Abax parallelepipedus, Coleoptera, Carabidae). We show that a single large patch of wood is better than several small patches for the population viability.     

Structural Changes in Senescing Oilseed Rape Leaves at Tissue and Subcellular Levels Monitored by Nuclear Magnetic Resonance Relaxometry through Water Status

Nitrogen use efficiency is relatively low in oilseed rape (Brassica napus) due to weak nitrogen remobilization during leaf senescence. Monitoring the kinetics of water distribution associated with the reorganization of cell structures, therefore, would be valuable to improve the characterization of nutrient recycling in leaf tissues and the associated senescence processes. In this study, nuclear magnetic resonance (NMR) relaxometry was used to describe water distribution and status at the cellular level in different leaf ranks of well-watered plants. It was shown to be able to detect slight variations in the evolution of senescence. The NMR results were linked to physiological characterization of the leaves and to light and electron micrographs. A relationship between cell hydration and leaf senescence was revealed and associated with changes in the NMR signal. The relative intensities and the transverse relaxation times of the NMR signal components associated with vacuole water were positively correlated with senescence, describing water uptake and vacuole and cell enlargement. Moreover, the relative intensity of the NMR signal that we assigned to the chloroplast water decreased during the senescence process, in agreement with the decrease in relative chloroplast volume estimated from micrographs. The results are discussed on the basis of water flux occurring at the cellular level during senescence. One of the main applications of this study would be for plant phenotyping, especially for plants under environmental stress such as nitrogen starvation.The main physiological outcome of leaf senescence is the recycling of organic resources and the provision of nutrients to sink organs such as storage and growing tissues (Buchanan-Wollaston, 1997; Hikosaka, 2005; Krupinska and Humbeck, 2008). In crop plants, senescence progresses from the lower older leaves to the younger top leaves. Macromolecular degradation and the mechanism of reallocation of breakdown products are mediated by the up-regulation of senescence-related genes (Lee et al., 2001) in close relationship with both developmental and environmental conditions (Gombert et al., 2006). This leads to remobilization of carbon and nitrogen (N) compounds mostly from plastidial compartments (Martínez et al., 2008; Guiboileau et al., 2012), involving proteolytic activity in plasts, vacuole, and cytosol (Adam and Clarke, 2002; Otegui et al., 2005), chlorophyll breakdown (Hoertensteiner, 2006), galactolipid recycling (Kaup et al., 2002) in the plastoglobules (Brehelin et al., 2007), and loading of Suc and amino acids into the phloem through appropriate transporters (Wingler et al., 2004; Masclaux-Daubresse et al., 2008). In terms of leaf senescence at the cell level, where chloroplasts are degraded sequentially, relative organelle volume does not seem to be greatly modified, the vacuole remains intact, and in darkness-induced senescence the number of chloroplasts per cell decreases only slightly (Keech et al., 2007). However, major changes in metabolic fluxes and cell water relationships are expected during the senescence program that may be associated with macromolecule catabolism, organic solute synthesis, transport and remobilization, and cell structure reconfiguration such as chloroplast evolution to gerontoplast (Hoertensteiner, 2006; Zhang et al., 2010) through the autophagy process (Wada et al., 2009), accumulation of senescence-associated vacuoles (Otegui et al., 2005), and cell wall degradation (Mohapatra et al., 2010).The senescent leaves of oilseed rape (Brassica napus), a major oleiferous crop, generally fall while still maintaining a high N content (about 2.5%–3% [w/w] of the dry matter; Malagoli et al., 2005). In addition to the environmental impact of this leaking of N out of the plant, the low capacity to remobilize foliar N is associated with a high requirement for N fertilization to meet the potential crop yield (Dreccer et al., 2000). In order to improve the nitrogen use efficiency (NUE), new genotypes are being selected for their ability to maintain high yields under limited N fertilization, mainly via the improvement of N uptake efficiency and N mobilization from the senescing leaves (Hirel et al., 2007). In Arabidopsis (Arabidopsis thaliana) and oilseed rape, N can be remobilized from old to expanding leaves at the vegetative stage during sequential senescence as well as from leaves to seeds at the reproductive stage during monocarpic senescence (Malagoli et al., 2005; Diaz et al., 2008; Lemaitre et al., 2008). Senescence can also be induced by environmental stress such as N starvation (Etienne et al., 2007) or water deficit (Reviron et al., 1992) and propagated from old to mature leaves and delayed in young leaves, suggesting finely tuned high regulation of metabolism at the whole-plant level with consequences for NUE (Desclos et al., 2008). One major challenge to understanding the efficiency of senescence-induced organic resource reallocation and to highlighting major molecular and mechanistic attributes of nutrient recycling is monitoring the kinetics of the structural reorganization of cell structures. This reorganization will provide nutrients remobilized through phloem loading. From a technological and phenotyping point of view, the measurement of N remobilization efficiency has already been addressed in crop species and oilseed rape, as it is a reliable trait to screen for the genetic variability of NUE (Franzaring et al., 2012). However, techniques such as stable isotope feeding are time consuming, destructive, and difficult to adapt to large genotype panels. Therefore, it is important to develop a technique for following changes in water distribution at the cell level in order to understand metabolic reconfigurations occurring throughout senescence.NMR relaxometry has been used in several studies to investigate plant cell structure and functioning (Hills and Duce, 1990; Van As, 1992). The ¹H-NMR signal originates almost entirely from water protons because other ¹H nuclei in the plant produce much less intense signals, as they correspond to molecules that are at a much lower concentration than water. The technique allows the measurement of longitudinal (T1) and transverse (T2) relaxation times and proton spin density. Water proton relaxation times are related to the rotational and translational mobility of water molecules (Van As, 2007). They are also modified by the mobility and structure of the surrounding macromolecules (i.e. starch, proteins, and polysaccharides) through proton exchange (chemical exchange). In plant cells, the water in different cell compartments has different chemical and physical properties and, therefore, different bulk T2 values. Moreover, relaxation times are affected by the exchange of molecules between different compartments that is determined by water diffusion and, therefore, by the compartment size and membrane permeability (Van der Weerd et al., 2002). The slow diffusion process between compartments results in multiexponential behavior of the relaxation signal. The multiexponential relaxation reflects water in cell compartments and, therefore, can be used to study changes in water distribution and properties at a subcellular level and, hence, can be used for the estimation of structural and volume transformations in cell compartments. The T2 relaxation time is more sensitive to small variations in water content and chemical exchange processes than T1 and, therefore, is usually preferred. Indeed, differences in T1 for the different compartments are relatively small, resulting in an averaging effect that results in poor discrimination between water compartments (Van As, 2007).To date, NMR relaxometry has mainly been used for the characterization of fruit and vegetable tissues and has been shown to be effective in providing valuable information about cell organization (Sibgatullin et al., 2007). However, although a number of studies have contributed to the interpretation of the NMR results (Snaar and Van As, 1992; Hills and Nott, 1999; Marigheto et al., 2009), this is still not always straightforward, as the NMR signal depends both on the nature of the plant tissue and on the NMR measurement protocol. The situation is even more complex in the case of leaves, because leaves contain different tissue types characterized by different cell sizes and structures (Teixeira et al., 2005), and only a few studies involving NMR relaxometry in leaves have been reported. Changes in T2 in response to high temperature were investigated in wheat (Triticum aestivum; Maheswari et al., 1999) in order to develop a method for the detection of heat injury. McCain (1995) measured the T2 relaxation time of chloroplast and nonchloroplast water in maple (Acer platanoides) leaves by separating corresponding peaks in an NMR spectrum without taking into account the compartmentalization of nonchloroplast water. Oshita et al. (2006) investigated cell membrane permeability to water in spinach (Spinacia oleracea) leaves by measuring the T1 relaxation time of the leaf protoplasts without consideration of the subcellular structure. Qiao et al. (2005) attempted to associate NMR signal components with different chive (Allium schoenoprasum) cells using combined transverse relaxation and restricted diffusion measurements. Finally, Capitani et al. (2009) recently used a portable unilateral NMR instrument to detect the water status of leaves of herbaceous crops, mesophyllous trees, and natural Mediterranean vegetation under field conditions. Further investigations are necessary to improve leaf characterization by NMR, especially in the attribution of NMR signal components to the tissue and subcellular compartments. Progress in this field would make it possible to use the full potential of noninvasive NMR relaxometry in plant research and phenotyping.Using NMR relaxometry, we describe here the differences in water status that occurred at tissue and cellular levels through different leaf ranks of well-irrigated oilseed rape plants, from the young leaves at the top of the canopy to the senescing older leaves at the bottom of the plant. The aim of the study was to show that changes that occur in the leaves while senescing can be related to changes in water distribution and cell structure. As these changes are directly linked to the modifications in cell compartment organization, especially those occurring in the chloroplast, vacuole, and cell wall due to macromolecule degradation and N and carbon reallocation processes, this study was designed to contribute to the understanding of these physiological processes.     

Functional landscape heterogeneity and animal biodiversity in agricultural landscapes

Biodiversity in agricultural landscapes can be increased with conversion of some production lands into 'more-natural'- unmanaged or extensively managed - lands. However, it remains unknown to what extent biodiversity can be enhanced by altering landscape pattern without reducing agricultural production. We propose a framework for this problem, considering separately compositional heterogeneity (the number and proportions of different cover types) and configurational heterogeneity (the spatial arrangement of cover types). Cover type classification and mapping is based on species requirements, such as feeding and nesting, resulting in measures of 'functional landscape heterogeneity'. We then identify three important questions: does biodiversity increase with (1) increasing heterogeneity of the more-natural areas, (2) increasing compositional heterogeneity of production cover types and (3) increasing configurational heterogeneity of production cover types? We discuss approaches for addressing these questions. Such studies should have high priority because biodiversity protection globally depends increasingly on maintaining biodiversity in human-dominated landscapes.