The phytoplasma associated with purple woodnettle witches'‐broom disease in Taiwan represents a new subgroup of the aster yellows phytoplasma group

In October 2013, a new disease affecting purple woodnettle, Oreocnide pedunculata, plants was found in Miaoli County, Taiwan. Diseased plants exhibited leaf yellowing and witches'‐broom symptoms. Molecular diagnostic tools and electron microscopic cell observation were used to investigate the possible cause of the disease with a specific focus on phytoplasmas. The result of polymerase chain reaction with universal primer pairs indicated that phytoplasmas were strongly associated with the symptomatic purple woodnettles. The virtual restriction fragment length polymorphism (RFLP) patterns and phylogenetic analysis based on 16S rDNA and ribosomal protein, rplV‐rpsC region revealed that purple woodnettle witches'‐broom phytoplasma (PWWB) belongs to a new subgroup of 16SrI and rpI group and was designated as 16SrI‐AH and rpI‐Q, respectively, herein. RFLP analysis based on tuf gene region revealed that the PWWB belongs to tufI‐B, but phylogenetic analysis suggested that PWWB should be delineated to a new subgroup under the tufI group. Taken together, our analyses based on 16S rRNA and rplV‐rpsC region gave a finer differentiation while classifying the subgroup of aster yellows group phytoplasmas. To our knowledge, this is the first report of a Candidatus Phytoplasma asteris‐related strain in 16SrI‐AH, rpI‐Q and tufI‐B subgroup affecting purple woodnettle, and of an official documentation of purple woodnettle as being a new host of phytoplasmas.     

Plankton dynamics under different climate conditions in tropical freshwater systems (a reply to the comment by Sarmento,Amado & Descy, )

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multiplierz v2.0: A Python‐based ecosystem for shared access and analysis of native mass spectrometry data

The continued evolution of modern mass spectrometry instrumentation and associated methods represents a critical component in efforts to decipher the molecular mechanisms which underlie normal physiology and understand how dysregulation of biological pathways contributes to human disease. The increasing scale of these experiments combined with the technological diversity of mass spectrometers presents several challenges for community‐wide data access, analysis, and distribution. Here we detail a redesigned version of multiplierz, our Python software library which leverages our common application programming interface (mzAPI) for analysis and distribution of proteomic data. New features include support for a wider range of native mass spectrometry file types, interfaces to additional database search engines, compatibility with new reporting formats, and high‐level tools to perform post‐search proteomic analyses. A GUI desktop environment, mzDesktop, provides access to multiplierz functionality through a user friendly interface. multiplierz is available for download from: https://github.com/BlaisProteomics/multiplierz ; and mzDesktop is available for download from: https://sourceforge.net/projects/multiplierz/     

The oldest cinctan carpoid (stem‐group Echinodermata), and the evolution of the water vascular system

A new cinctan ( Protocinctus mansillaensis gen. et sp. nov. ), from the early Middle Cambrian of the Iberian Chains (north‐east Spain), is described with the aid of X‐ray microtomography and three‐dimensional computer models. Investigation in this manner was possible because of the unusual condition of the fossils, which are preserved as recrystallized calcite. Protocinctus gen nov. possesses an elongate body with a single left anterior feeding groove and an open posterior marginal frame (in ventral view): this combination of characters is unique amongst cinctans. Through the study of original specimens and ‘virtual fossils’ it was possible to reconstruct the palaeobiology of Protocinctus gen. nov. : cinctans are interpreted as pharyngeal basket feeders with a U‐shaped gut, using their posterior appendage to aid stability on the sediment surface. Cinctans are critical to understanding the evolutionary history of the echinoderm stem group, as they illustrate the transition from a paired water vascular system (basal) to one constructed from just the left hydrocoel (derived). The former condition is also observed in another group of stem‐group echinoderms, the ctenocystoids, and is hypothesized for the latest common ancestor of the echinoderms and hemichordates.     

Translating Sleeping Beauty transposition into cellular therapies: Victories and challenges

Recent results confirm that long‐term expression of therapeutic transgenes can be achieved by using a transposon‐based system in primary stem cells and in vivo. Transposable elements are natural DNA transfer vehicles that are capable of efficient genomic insertion. The latest generation, Sleeping Beauty transposon‐based hyperactive vector (SB100X), is able to address the basic problem of non‐viral approaches – that is, low efficiency of stable gene transfer. The combination of transposon‐based non‐viral gene transfer with the latest improvements of non‐viral delivery techniques could provide a long‐term therapeutic effect without compromising biosafety. The new challenges of pre‐clinical research will focus on further refinement of the technology in large animal models and improving the safety profile of SB vectors by target‐selected transgene integration into genomic “safe harbors.” The first clinical application of the SB system will help to validate the safety of this approach.     

Examining carry‐over effects of winter habitat on breeding phenology and reproductive success in prairie warblers Setophaga discolor

Winter habitat quality can influence breeding phenology and reproductive success of migratory birds. Using stable isotope ratios of carbon (δ¹³C) from bird claws and red blood cells collected in Massachusetts, USA, we assessed if winter habitat occupancy carried over to affect prairie warbler Setophaga discolor breeding arrival dates, body condition upon arrival, pairing success, first‐egg dates and reproductive success. In two of three years (in 2011 and 2012, but not in 2013), after‐second‐year (ASY) males wintering in drier habitat, as indicated by enriched δ¹³C values, arrived later on the breeding grounds. Based on the North Atlantic Oscillation index, there was likely less rainfall in the Caribbean wintering grounds during the winters of 2011 and 2012 compared to the winter of 2013, suggesting increased winter rainfall in 2013 may have diminished the influence of winter habitat occupancy on arrival date. We did not find any effects of winter habitat on breeding season phenomena for second‐year (SY) males or females, but our sample sizes for these age/sex classes were relatively low. Although winter habitat quality influenced arrival dates of ASY males, there was no evidence that it affected reproductive performance, perhaps because of high rates of nest depredation in our system. Our study adds to a growing body of research that shows the influence of carry‐over effects can differ among species and within populations, and also can be modulated by other environmental conditions. This information enriches our understanding of the role of carry‐over effects in population limitation for migratory birds.     

Human fear acquisition deficits in relation to genetic variants of the corticotropin‐releasing hormone receptor 1 and the serotonin transporter — revisited

We recently showed that a genetic polymorphism (rs878886) in the human corticotropin‐releasing hormone receptor 1 (CRHR1) is associated with reduced fear‐conditioned responses to a threat cue. This is a potentially important finding considering that the failure to acquire fear contingencies can leave an individual in a maladaptive state of more generalized anxiety. Consistent with that idea, the CRHR1‐dependent fear acquisition deficit translated into heightened contextual anxiety when taking genetic variability within the serotonin transporter long polymorphic region (5‐HTTLPR) into account. To replicate our previous findings, we conducted a replication study in 224 healthy medication‐free human subjects using the exact same cue and context virtual reality fear‐conditioning procedure as in study by Heitland et al. (2013). In the replication study, consistent with the original findings, CRHR1 rs878886 G‐allele carriers showed reduced acquisition of cue‐specific fear‐conditioned responses compared with C/C homozygotes. Also, in this larger sample the cue acquisition deficit of G‐allele carriers translated into heightened contextual anxiety, even independent of 5‐HTT gene variation. In contrast to our earlier findings, there was an additional interaction effect of CRHR1 rs878886 and the triallelic 5‐HTTLPR/rs25531 variant on cued fear acquisition. In summary, this study replicated the initially reported association of the CRHR1 rs878886 G‐allele with cued fear acquisition deficits, albeit with a different pattern of results regarding the interaction with 5‐HTT variation. This further supports the notion that the human corticotropin‐releasing hormone plays a role in the acquisition of fears.     

Enantioselective Liquid–Liquid Extraction of Zopiclone With Mandelic Acid Ester Derivatives

Enantioselective liquid–liquid extraction of zopiclone was conducted by employing a series of (R)‐mandelic acid esters as chiral extractants. The effects of concentration of extractant, concentration of zopiclone, type of organic solvent, pH value, and temperature on the extraction efficiency were investigated. (R)‐o‐chloromandelic acid propyl ester was demonstrated to be an efficient chiral extractant for zopiclone resolution with a maximum enantioselectivity of 1.6. Chirality 25:952–956, 2013. © 2013 Wiley Periodicals, Inc.     

Theoretical Study of N‐Heterocyclic Carbenes‐Catalyzed Cascade Annulation of Benzodienones and Enals

Growing attention in developing new N‐heterocyclic carbene (NHC)‐mediated reactions involving homoenolate intermediates has prompted our interest in exploring the mechanistic details of the related reactions. In this work, we carried out a detailed theoretical study for the NHC‐catalyzed annulation reaction of cinnamaldehyde ( A ) and benzodi(enone) ( B ) in the presence of 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU). By performing density functional theory calculations, we show clearly the detailed reaction mechanism and rationalize the experimental observation. The reaction of A and B falls into two stages: the formation of homoenolate intermediate and the annulation of homoenolate with B . In the homoenolate formation stage, three possible paths are characterized. The pathway involving the DBU‐assisted 1,2‐proton transfer with a stepwise mechanism is kinetically more favorable, and the DBU‐assisted C1 proton departure is the rate‐determining step of the total reaction. The annulation of homoenolate with B involves four elementary steps. The conformational difference of homoenolate (cis and trans) leads to two slightly different reaction processes. In the total reaction, the process involving cis‐conformation of A is kinetically more feasible. This can be clearly understood through the frontier molecular orbital analysis and the electronic inductive effect. The calculated results are expected to offer valuable information for further design and development of NHC‐mediated reactions. Chirality 25:521‐528, 2013. © 2013 Wiley Periodicals, Inc.     

Analysis of dental homologies and phylogeny of Paucituberculata (Mammalia: Marsupialia)

The Paucituberculata is an endemic group of South American marsupials, recorded from the early Cenozoic up to the present. In this report, the most comprehensive phylogenetic analysis of Paucituberculata to date is presented. Fifty‐seven terminal species were scored for 74 new and re‐examined characters. Homologies of dental characters used in previous systematic studies were critically reviewed to evaluate their inclusion in the analysis. Phylogenetic results corroborated two major paucituberculatan clades, Palaeothentoidea and Caenolestoidea, and the main palaeothentoid groupings: Pichipilidae, Palaeothentidae, and Abderitidae. Taxon sampling and reinterpretations of molar cusp and crest homologies played an important role in the generation of new phylogenetic hypotheses. The main differences with respect to previous phylogenies were focused on palaeothentoid relationships: Palaeothentes boliviensis and Pilchenia lucina are not members of Palaeothentidae but instead clustered with Pilchenia intermedia and P. antiqua, forming the sister‐group of a Palaeothentidae + Abderitidae clade, and Titanothentes simpsoni, previously considered a palaeothentine, is nested within the Acdestinae clade. Based on the time‐calibrated phylogeny, the following stages in the paucituberculatan evolutionary history are suggested: origin of the group, in the Paleocene to early Eocene at the latest, split of Caenolestoidea and Palaeothentoidea clades during the late early to middle Eocene, evolutionary radiation of palaeothentid and abderitid lineages near the Oligocene–Eocene boundary, and decreased diversity and extinction of palaeothentoids during the middle Miocene. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 441–465.     

Enantioselective Toxic Effects and Degradation of Myclobutanil Enantiomers in Scenedesmus obliquus

Research on the enantioselective environmental behavior of chiral pesticides has been a hot spot of environmental chemistry recently. In this study, the acute toxicity of myclobutanil enantiomers was investigated with the aquatic algae Scendesmus obliquus. After exposure for 96 h, the EC₅₀ values for (?)‐myclobutanil, rac‐myclobutanil and (+)‐myclobutanil were 3.951, 2.760, and 2.128 mg/L, respectively. The photosynthetic pigment (chlorophyll a, chlorophyll b, and carotenoids) and antioxidant enzyme activities catalase (CAT) were determined to evaluate the different toxic effects when S. obliquus were exposed to 1.5, 5 and 15 mg/L of rac‐myclobutanil, (?)‐myclobutanil, and (+)‐myclobutanil for 96 h, respectively. In addition, the degradation of myclobutanil enantiomers in S. obliquus was also studied. Myclobutanil in the medium inoculated with algae degraded faster than in the uninoculated medium. The degradation of (?)‐myclobutanil was faster than that of (+)‐myclobutanil at a concentration of 3 mg/L. On the basis of these data, the acute toxicity and toxic effects of myclobutanil against S. obliquus were concluded to be enantioselective, and such enantiomeric differences should be taken into consideration in pesticide risk assessment. Chirality 25:858–864, 2013. © 2013 Wiley Periodicals, Inc.     

Enantiomer‐Specific In Vitro Biotransformation of Select Pharmaceuticals in Rainbow Trout (Oncorhynchus mykiss)

The occurrence of pharmaceuticals in the environment represents a challenge of emerging concern. Many pharmaceuticals are chiral compounds; however, few studies have examined the relative toxicity of pharmaceutical enantiomers to wildlife. Further, our understanding of stereospecific pharmacokinetics remains largely informed by research on humans and a few well‐studied laboratory test animals, and not by studies conducted with environmentally relevant species, including fish. The objective of this study was to investigate whether rainbow trout display stereospecific in vitro metabolism of three common chiral pharmaceuticals. Metabolism by trout liver S9 fractions was evaluated using a substrate depletion approach, which provides an estimate of intrinsic hepatic clearance (CL_{IN VITRO,INT}). No biotransformation was observed for rac‐, R‐, or S‐fluoxetine. Ibuprofen, including both enantiomers and the racemic mixture, appeared to undergo slow metabolism, but the resulting substrate depletion curves did not differ significantly from those of inactive controls. Contrary to relative clearance rates in humans, S(?)‐propranolol was more rapidly cleared than the R(+)‐ enantiomer. This work demonstrates that relative clearance rates and the effects of racemic mixtures in trout could not have been predicted based on human data. Additional research describing species differences and exploring tools for species extrapolation in biomedical and environmental studies is needed. Chirality 25:763–767, 2013, © 2013 Wiley Periodicals, Inc.     

pH‐selective mutagenesis of protein–protein interfaces: In silico design of therapeutic antibodies with prolonged half‐life

Understanding the effects of mutation on pH‐dependent protein binding affinity is important in protein design, especially in the area of protein therapeutics. We propose a novel method for fast in silico mutagenesis of protein–protein complexes to calculate the effect of mutation as a function of pH. The free energy differences between the wild type and mutants are evaluated from a molecular mechanics model, combined with calculations of the equilibria of proton binding. The predicted pH‐dependent energy profiles demonstrate excellent agreement with experimentally measured pH‐dependency of the effect of mutations on the dissociation constants for the complex of turkey ovomucoid third domain (OMTKY3) and proteinase B. The virtual scanning mutagenesis identifies all hotspots responsible for pH‐dependent binding of immunoglobulin G (IgG) to neonatal Fc receptor (FcRn) and the results support the current understanding of the salvage mechanism of the antibody by FcRn based on pH‐selective binding. The method can be used to select mutations that change the pH‐dependent binding profiles of proteins and guide the time consuming and expensive protein engineering experiments. As an application of this method, we propose a computational strategy to search for mutations that can alter the pH‐dependent binding behavior of IgG to FcRn with the aim of improving the half‐life of therapeutic antibodies in the target organism. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Western European Quaternary lions: new working hypotheses

Cave lions (Panthera spelaea), which spread throughout Western Europe for several thousand years, disappeared approximately 14 000–14 500 years ago. They were supposedly replaced by modern lions (Panthera leo) approximately 8000 years ago. Modern lions reached the steppes of Ukraine and Hungary, without penetrating the forests of Central Europe. The present study focuses on Italian and Spanish findings that possibly bridge the alleged absence of these big cats from Europe for 6000 years. Fossil lion remains from reliably radiocarbon‐dated levels have been plotted against the δ¹⁸O curve and mapped. The accumulated evidence indicates that lions inhabited Western Europe uninterruptedly from the early Middle Pleistocene up to the Early Holocene. Moreover, all of the latest Pleistocene/early Holocene lion‐bearing localities do not range farther than the 44th parallel north and are located at relatively high altitudes. Two working hypotheses are formulated: one, which is less likely because it is not supported by palaeontological evidence indicating earlier migrations of lions from Africa, suggests that modern lions entered Western Europe prior to 8000 years ago; the second, which is more probable, suggests that P. spelaea (or an advanced offspring of the species) survived up until the latest Pleistocene. Panthera leo accessed Eastern Europe between 6000–6500 and 8000 years ago but was prevented from penetrating further west, probably because of the intrusive presence of their indigenous European relatives, and/or the increasing encroachment of modern human populations. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 66–77.     

Pharmmaker: Pharmacophore modeling and hit identification based on druggability simulations

Recent years have seen progress in druggability simulations, that is, molecular dynamics simulations of target proteins in solutions containing drug‐like probe molecules to characterize their drug‐binding abilities, if any. An important consecutive step is to analyze the trajectories to construct pharmacophore models (PMs) to use for virtual screening of libraries of small molecules. While considerable success has been observed in this type of computer‐aided drug discovery, a systematic tool encompassing multiple steps from druggability simulations to pharmacophore modeling, to identifying hits by virtual screening of libraries of compounds, has been lacking. We address this need here by developing a new tool, Pharmmaker, building on the DruGUI module of our ProDy application programming interface. Pharmmaker is composed of a suite of steps: (Step 1) identification of high affinity residues for each probe molecule type; (Step 2) selecting high affinity residues and hot spots in the vicinity of sites identified by DruGUI; (Step 3) ranking of the interactions between high affinity residues and specific probes; (Step 4) obtaining probe binding poses and corresponding protein conformations by collecting top‐ranked snapshots; and (Step 5) using those snapshots for constructing PMs. The PMs are then used as filters for identifying hits in structure‐based virtual screening. Pharmmaker, accessible online at http://prody.csb.pitt.edu/pharmmaker , can be used in conjunction with other tools available in ProDy.     

Does provenance matter? Fitness is not determined by genetic distance or the scale of pollen dispersal in Grevillea sphacelata (Proteaceae)

Understanding how the scale of pollen transfer determines the outcome of matings is important evolutionarily and a key issue in restoration ecology. We tested the effects of pollen transfer distance for the self‐incompatible shrub Grevillea sphacelata using (1) open pollination and transfer among (2) near neighbours, (3) neighbouring subpopulations and (4) populations separated by c. 4 km. We used AFLP markers to test for evidence of genetic differentiation within and among populations. Patterns of seed initiation suggest that open pollinated flowers were pollen limited, although in one subpopulation open seed set was greater than that achieved with pollen from near neighbours or other subpopulations. We detected no other effects of pollen source on seed initiation or seed and seedling development. In contrast, our genetic survey revealed significant spatial autocorrelation to 5 m, moderate differentiation of populations separated by up to 4 km and significant isolation by distance > 16 km. Our data suggest that, although dispersal of pollen may typically be localized, gene flow prevents localized adaptation or co‐adaptation and we detected no effects of inbreeding depression. In a restoration context, our results imply that movement of seed between populations separated by 4 km will not have detrimental consequences, despite significant differentiation at neutral genetic markers, and may be beneficial in maintaining genetic diversity and evolutionary potential. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 290–302.     

Identification of efflux proteins using efficient radial basis function networks with position‐specific scoring matrices and biochemical properties

Efflux proteins are membrane proteins, which are involved in the transportation of multidrugs. The annotation of efflux proteins in genomic sequences would aid to understand the function. Although the percentage of membrane proteins in genomes is estimated to be 25–30%, there is no information about the content of efflux proteins. For annotating such class of proteins it is necessary to develop a reliable method to identify efflux proteins from amino acid sequence information. In this work, we have developed a method based on radial basis function networks using position specific scoring matrices (PSSM) and amino acid properties. We noticed that the C‐terminal domain of efflux proteins contain vital information for discrimination. Our method showed an accuracy of 78 and 92% in discriminating efflux proteins from transporters and membrane proteins, respectively using fivefold cross‐validation. We utilized our method for annotating the genomes E. coli and P. aeruginosa and it predicted 8.7 and 9.2% of proteins as efflux proteins in these genomes, respectively. The predicted efflux proteins have been compared with available experimental data and we observed a very good agreement between them. Further, we developed a web server for classifying efflux proteins and it is freely available at http://rbf.bioinfo.tw/～sachen/EFFLUXpredict/Efflux‐RBF.php . We suggest that our method could be an effective tool for annotating efflux proteins in genomic sequences.Proteins 2013. © 2013 Wiley Periodicals, Inc.