DESI–MS as a tool for direct lipid analysis in cultured cells

Desorption electrospray ionization may be used as a fast and convenient method for analysis and identification of lipids in the cell culture. Oxidative stress, which usually involves changes in lipids, was used as a model of pathology to show the utility of this analysis methodology. This paper addresses the surface preparation of cell culture slides, induction of oxidative stress, and cell monolayer culture preparation as well as optimization of the analysis. Advantages and drawbacks of the method were also discussed.     

Experimental management control of Opuntia dillenii Haw. and Agave americana L. in Teno Rural Park,Canary Islands

Invasion biology is an important element of global environmental change and represents one of the main threats to biodiversity. American species were introduced to Tenerife after the Spanish conquest during the eighteenth century, as is the case for Agave americana and Opuntia dillenii. The long period of naturalization and adaptation of these species has led them to become two of the most dispersed introduced species of the archipelago. We analyzed several eradication management processes in an area intensively invaded by both O. dillenii and A. americana. Three treatments were randomly applied: mechanical removal, use of herbicide (glyphosate at 10% volume), and mechanical and herbicide applied together. Both the effectiveness of the treatments to remove the target exotic species biovolume and the impact of the eradication methods on species richness and species composition of the area were analyzed. We found that the treatments had an impact on species composition but not on species richness. Species composition was mainly affected by mechanical treatment. The effect caused by the mechanical removal of the exotic target species in species composition is minor after 4 years, and is related to a higher dominance of shrub species typical of coastal shrubland and of annual or pioneer species. The control of O. dillenii and A. americana is evident from insignificant recovery 4 years after treatment application. A mechanical and herbicide treatment together, allowed not only the immediate removal of large individuals but also the herbicidal control of smaller ones.     

Genetic Diversities of Cymbidium mosaic virus and Odontoglossum ringspot virus Isolates Based on the Coat Protein Genes from Orchids in Guangdong Province,China

Orchids are some of the most important ornamental flowers. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most prevalent and economically important viruses affecting orchids in China. In this study, 20 CymMV and 28 ORSV isolates were selected for genetic diversity analysis. The CymMV isolates shared 84.6–100% and 89.5–100% identities of coat protein (CP) at the nucleotide (nt) and amino acid (aa) levels, respectively. The identities of ORSV isolates were 96.4–100% (nt) and 92.5–99.4% (aa). The CP genes of CymMV were found to have genetic diversity, and the CP genes of ORSV were genetically conservative. These results can aid in designing effective disease‐control strategies.     

Sex pheromone of the cloaked pug moth,Eupithecia abietaria (Lepidoptera: Geometridae), a pest of spruce cones

The sex pheromone of the cloaked pug moth, Eupithecia abietaria Götze, an important cone‐feeding pest in spruce seed orchards in Europe, was investigated. Chemical and electrophysiological analyses of pheromone gland extracts of female moths and analogous analyses of synthetic hydrocarbons and epoxides of chain length C₁₉ and C₂₁ revealed (3Z,6Z,9Z)‐3,6,9‐nonadecatriene (3Z,6Z,9Z‐19:H) and 3Z,6Z‐cis‐9,10‐epoxynonadecadiene (3Z,6Z‐cis‐9,10‐epoxy‐19:H) as candidate pheromone components, which were found in a gland extract in a ratio of 95 : 5. In field trapping experiments, conspecific males were only attracted to a combination of 3Z,6Z,9Z‐19:H and the (9S,10R)‐enantiomer of 3Z,6Z‐cis‐9,10‐epoxy‐19:H. The (9R,10S)‐enantiomer was not attractive, which is in agreement with studies on other Eupithecia species, for which males have only been attracted by the (9S,10R)‐enantiomer of epoxides. Subsequent experiments showed that E. abietaria males were attracted to a wide range of ratios of the two active compounds and that trap catches increased with increasing dose of the binary blend. A two‐component bait containing 300 μg 3Z,6Z,9Z‐19:H and 33 μg of the (9S,10R)‐enantiomer of 3Z,6Z‐cis‐9,10‐epoxy‐19:H was efficient for monitoring E. abietaria in spruce seed orchards in southern Sweden, where this species has probably been overlooked as an important pest in the past. With sex pheromones recently identified for two other moths that are major pests on spruce cones, the spruce seed moth, Cydia strobilella L., and the spruce coneworm, Dioryctria abietella Denis & Schiffermüller, pheromone‐based monitoring can now be achieved for the whole guild of cone‐feeding moths in European spruce seed orchards.     

Head shape dimorphism in European glass eels (Anguilla anguilla)

The life cycle of the European eel (Anguilla anguilla) remained a mystery until the 20th century, when Schmidt discovered that the Sargasso Sea was its spawning area. However, many aspects of the eel's life cycle remain poorly understood. Among these is the bimodal distribution in head shape, with broad- and narrowheaded phenotypes reported in the yellow eel stage. Although this has been linked to dietary preferences of the yellow eels, very little is known about why, how and when this dimorphism arises during their ontogeny. To determine whether this dimorphism indeed appears in relation to trophic niche segregation, we examined head shape variation at an earlier ontogenetic stage, the glass eel stage, as at this stage eels are considered to be non-feeding. Head shape was studied in a large dataset, containing glass eels captured from the Yser river mouth, the Leopold Canal (Belgium) and from the rivers Severn, Trent and Parret (UK), by both taking measurements (head width/head length) and using an outline analysis. Our results show that there is already considerable variation in broadness and bluntness of the head at the glass eel stage. In most cases, equal support for a unimodal and bimodal head shape distribution is found, whereas some cases support head shape bimodality in glass eels, suggesting that glass eel head shape might be shifting from a unimodal to a bimodal distribution. This, in combination with the observation that variation in head width/head length ratios in non-feeding glass eels shows a similar range as in feeding yellow eels, indicates that head shape in European eel might be at least partially determined through other mechanisms than trophic segregation.     

Iron and copper in progressive demyelination – New lessons from Skogholt's disease

The pathophysiological mechanisms of progressive demyelinating disorders including multiple sclerosis are incompletely understood. Increasing evidence indicates a role for trace metals in the progression of several neurodegenerative disorders. The study of Skogholt disease, a recently discovered demyelinating disease affecting both the central and peripheral nervous system, might shed some light on the mechanisms underlying demyelination. Cerebrospinal fluid iron and copper concentrations are about four times higher in Skogholt patients than in controls. The transit into cerebrospinal fluid of these elements from blood probably occurs in protein bound form. We hypothesize that exchangeable fractions of iron and copper are further transferred from cerebrospinal fluid into myelin, thereby contributing to the pathogenesis of demyelination. Free or weakly bound iron and copper ions may exert their toxic action on myelin by catalyzing production of oxygen radicals. Similarities to demyelinating processes in multiple sclerosis and other myelinopathies are discussed.     

Network of long-range concerted chemical shift displacements upon ligand binding to human angiogenin

Molecular recognition models of both induced fit and conformational selection rely on coupled networks of flexible residues and/or structural rearrangements to promote protein function. While the atomic details of these motional events still remain elusive, members of the pancreatic ribonuclease superfamily were previously shown to depend on subtle conformational heterogeneity for optimal catalytic function. Human angiogenin, a structural homologue of bovine pancreatic RNase A, induces blood vessel formation and relies on a weak yet functionally mandatory ribonucleolytic activity to promote neovascularization. Here, we use the NMR chemical shift projection analysis (CHESPA) to clarify the mechanism of ligand binding in human angiogenin, further providing information on long-range intramolecular residue networks potentially involved in the function of this enzyme. We identify two main clusters of residue networks displaying correlated linear chemical shift trajectories upon binding of substrate fragments to the purine- and pyrimidine-specific subsites of the catalytic cleft. A large correlated residue network clusters in the region corresponding to the V₁ domain, a site generally associated with the angiogenic response and structural stability of the enzyme. Another correlated network (residues 40–42) negatively affects the catalytic activity but also increases the angiogenic activity. ¹⁵N-CPMG relaxation dispersion experiments could not reveal the existence of millisecond timescale conformational exchange in this enzyme, a lack of flexibility supported by the very low-binding affinities and catalytic activity of angiogenin. Altogether, the current report potentially highlights the existence of long-range dynamic reorganization of the structure upon distinct subsite binding events in human angiogenin.     

Vibrational entropy differences between mesophile and thermophile proteins and their use in protein engineering

We recently introduced ENCoM, an elastic network atomic contact model, as the first coarse-grained normal mode analysis method that accounts for the nature of amino acids and can predict the effect of mutations on thermostability based on changes vibrational entropy. In this proof-of-concept article, we use pairs of mesophile and thermophile homolog proteins with identical structures to determine if a measure of vibrational entropy based on normal mode analysis can discriminate thermophile from mesophile proteins. We observe that in around 60% of cases, thermophile proteins are more rigid at equivalent temperatures than their mesophile counterpart and this difference can guide the design of proteins to increase their thermostability through series of mutations. We observe that mutations separating thermophile proteins from their mesophile orthologs contribute independently to a decrease in vibrational entropy and discuss the application and implications of this methodology to protein engineering.     

Comparison of topological clustering within protein networks using edge metrics that evaluate full sequence,full structure,and active site microenvironment similarity

The development of accurate protein function annotation methods has emerged as a major unsolved biological problem. Protein similarity networks, one approach to function annotation via annotation transfer, group proteins into similarity-based clusters. An underlying assumption is that the edge metric used to identify such clusters correlates with functional information. In this contribution, this assumption is evaluated by observing topologies in similarity networks using three different edge metrics: sequence (BLAST), structure (TM-Align), and active site similarity (active site profiling, implemented in DASP). Network topologies for four well-studied protein superfamilies (enolase, peroxiredoxin (Prx), glutathione transferase (GST), and crotonase) were compared with curated functional hierarchies and structure. As expected, network topology differs, depending on edge metric; comparison of topologies provides valuable information on structure/function relationships. Subnetworks based on active site similarity correlate with known functional hierarchies at a single edge threshold more often than sequence- or structure-based networks. Sequence- and structure-based networks are useful for identifying sequence and domain similarities and differences; therefore, it is important to consider the clustering goal before deciding appropriate edge metric. Further, conserved active site residues identified in enolase and GST active site subnetworks correspond with published functionally important residues. Extension of this analysis yields predictions of functionally determinant residues for GST subgroups. These results support the hypothesis that active site similarity-based networks reveal clusters that share functional details and lay the foundation for capturing functionally relevant hierarchies using an approach that is both automatable and can deliver greater precision in function annotation than current similarity-based methods.     

Limited influence of local and landscape factors on finescale gene flow in two pond‐breeding amphibians

Dispersal and gene flow within animal populations are influenced by the composition and configuration of the landscape. In this study, we evaluated hypotheses about the impact of natural and anthropogenic factors on genetic differentiation in two amphibian species, the spotted salamander (Ambystoma maculatum) and the wood frog (Lithobates sylvaticus) in a commercial forest in central Maine. We conducted this analysis at two scales: a local level, focused on factors measured at each breeding pond, and a landscape level, focused on factors measured between ponds. We investigated the effects of a number of environmental factors in six categories including Productivity, Physical, Land Composition, Land Configuration, Isolation and Location. Embryos were sampled from 56 spotted salamander breeding ponds and 39 wood frog breeding ponds. We used a hierarchical Bayesian approach in the program GESTE at each breeding pond and a random forest algorithm in conjunction with a network analysis between the ponds. We found overall high genetic connectivity across distances up to 17 km for both species and a limited effect of natural and anthropogenic factors on gene flow. We found the null models best explained patterns of genetic differentiation at a local level and found several factors at the landscape level that weakly influenced gene flow. This research indicates multiscale investigations that incorporate local and landscape factors are valuable for understanding patterns of gene flow. Our findings suggest that dispersal rates in this system are high enough to minimize genetic structuring and that current forestry practices do not significantly impede dispersal.