A multilocus approach to assessing co-evolutionary relationships between <Emphasis Type=Italic>Steinernema</Emphasis> spp. (Nematoda: Steinernematidae) and their bacterial symbionts <Emphasis Type=Italic>Xenorhabdus</Emphasis> spp. (γ-Proteobacteria: Enterobacteriaceae)

Nematodes of the genus Steinernema Travassos, 1927 (Nematoda: Steinernematidae) and their associated bacteria, Xenorhabdus spp. (γ-Proteobacteria), are an emergent model of terrestrial animal-microbe symbiosis. Interest in this association initially arose out of their potential as biocontrol agents against insect pests, but, despite advances in their field application and the growing popularity of this model system, relatively little has been published to uncover the evolutionary facets of this beneficial partnership. This study adds to the body of knowledge regarding nematode-bacteria symbiosis by proposing a possible scenario for their historical association in the form of a cophylogenetic hypothesis. Topological and likelihood based testing methods were employed to reconstruct a history of association between 30 host-symbiont pairs and to gauge the level of similarity between their inferred phylogenetic patterns.     

Does scavenging extend the host range of entomopathogenic nematodes (Nematoda: Steinernematidae)?

Living and freeze-killed natural and laboratory hosts, with different susceptibility to entomopathogenic nematodes, were exposed to the larvae of Steinernema affine and Steinernema kraussei in two different experimental arenas (Eppendorf tubes, Petri dishes), and the success of the colonisation and eventual progeny production were observed. Both nematodes were able to colonise both living and dead larvae of Galleria mellonella (Lepidoptera) and adult Blatella germanica (Blattodea) even though the progeny production in dead hosts was lower on average. Living carabid beetles, Poecilus cupreus, and elaterid larvae (Coleoptera) were resistant to the infection, however, both nematodes were able to colonise and multiply in several dead P. cupreus and in a majority of dead elaterid larvae. By scavenging, EPNs can utilise cadavers of insects that are naturally resistant to EPN infection, and so broaden their host range.     

Hidden diversity uncovered in Hygrophorus sect. Aurei (Hygrophoraceae), including the Mediterranean H. meridionalis and the North American H. boyeri, spp. nov.

For many years, the binomial Hygrophorus hypothejus was widely applied to collections from various geographical regions in different continents, assuming a circum-boreal and circum-mediterranean distribution for this species. This hypothesis, however, had never been put to the test. To assess the diversity and species-limits within this complex of yellow-coloured waxcaps, a phylogenetic, morphological and taxonomical investigation into Hygrophorus sect. Aurei and similar species in sect. Olivaceoumbrini was carried out, including material of pan-European origin, as well as the east and west coasts of North America. Following sequencing of the ITS rDNA locus, nine lineages are confirmed in sect. Aurei, most of them highly continentalised. Of these, two are new to science, introduced here as Hygrophorus boyeri sp. nov., from Pinus banksiana and P. rigida forests in eastern North America and from P. muricata and P. contorta forests in western North America, and Hygrophorus meridionalis sp. nov., from Pinus brutia and Pinus halepensis forests in the island of Cyprus and mainland Greece. H. hypothejus is lectotypified and epitypified, and here resolved as a strictly European species, with the old forgotten taxon Hygrophorus siccipes revived as its North American vicariant. The placement of Hygrophorus fuligineus in sect. Aurei is phylogenetically confirmed and detailed comparisons between morphologically similar and phylogenetically affiliated taxa in sect. Aurei and sect. Olivaceoumbrini are provided. The chronic confusion associated with Hygrophorus fuscoalbus, a highly controversial taxon described from Germany nearly two centuries ago and variously interpreted since, is discussed, concluding that this name is too ambiguous to be applied to any currently recognized species.     

In vitro, ex vivo and in vivo models: A comparative analysis of Paracoccidioides spp. proteomic studies

Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.     

Characterization and Phylogenetic Relationships of Photorhabdus luminescens subsp. sonorensis (γ-Proteobacteria: Enterobacteriaceae), the Bacterial Symbiont of the Entomopathogenic Nematode Heterorhabditis sonorensis (Nematoda: Heterorhabditidae)

Photorhabdus are motile Gram-negative bacteria that have a mutualistic association with Heterorhabditis nematodes (Heterorhabditidae). These bacteria possess peculiar biochemical characteristics such as inability to reduce nitrates, and the capacity to ferment only a limited number of carbohydrates. Heterorhabditis nematodes vector the bacteria from one insect host to another and also provide shelter to the bacteria from soil stressors and antagonists. Once inside the insect host, the bacterial symbionts are released and produce toxins and secondary metabolites and broad-spectrum antibiotics, which kill the host by septicemia within 48 h. At present, three Photorhabdus spp. have been identified: P. luminescens, P. temperata, and P. asymbiotica, and many subspecies have also been described. Characterization of new species and subspecies has been based on sequence data, mostly of the 16S rDNA, and also of a selection of protein coding genes. In addition to this, phenotypic traits including temperature growth, colony morphology, color, light production, carbohydrate response, and assimilation, among others, have been considered. In this study, we characterize the bacterial symbiont of Heterorbabditis sonorensis, a recently discovered entomopathogenic nematode species form the Sonoran desert in Arizona, USA. A selection of classic biochemical and molecular methods including sequence data of six genes: 16s rDNA, and four protein coding genes: gyrB, recA, gltX, and dnaN were considered. Evolutionary relationships of this new Photorhabdus subsp. were inferred considering maximum parsimony and Bayesian analyses.     

The larval silk of Hypera spp. (Coleoptera: Curculionidae). A new example of the cross-β protein conformation in an insect silk

X-ray diffraction studies on the larval cocoon silk of the weevils Hypera postica and H. rumicis (Coleoptera: Curculionidae) indicate that it is a new example of the cross-β conformation in proteins. This silk is secreted by Malpighian tubules and stored in the rectum. Chemically it is more complex, with a greater range of constituent amino acids, than Chrysopa (Neuroptera) silk (the first well-documented example of a naturally occurring cross-β protein). There is a relatively high proline content, an unusual feature for an arthropod silk. X-ray data, and direct measurement of dispersed ribbons, suggest that the micellar width is about 3.0 compared to 2.5 nm in Chrysopa silk. This latter silk is the ‘type structure’ for naturally occurring cross-β protein conformations. Hypera silk can be considered the ‘next one up’ in a possible hierarchy of cross-β structures.     

Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

Background

The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.

Methods

Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.

Results

Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.

Conclusion

The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.

General significance

The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

Effectiveness of different species of entomopathogenic nematodes for biocontrol of the Mediterranean flatheaded rootborer, Capnodis tenebrionis (Linné) (Coleoptera: Buprestidae) in potted peach tree

The susceptibility of larvae of the Mediterranean flatheaded rootborer (Capnodis tenebrionis) to 13 isolates of entomopathogenic nematodes was examined using GF-677 potted trees (peachxalmond hybrid) as the host plant. The nematode strains tested included nine Steinernema feltiae, one S. affine, one S. carpocapsae and two Heterorhabditis bacteriophora. Nematodes showed the ability to locate and kill larvae of C. tenebrionis just after they enter into the roots of the tree. S. feltiae strains provided an efficacy ranging from 79.68% to 88.24%. H. bacteriophora strains resulted in control of 71.66-76.47%. S. carpocapsae (B14) and S. affine (Gspe3) caused lower control of C. tenebrionis larvae (62.03% and 34.76%, respectively). The influence of foraging strategy and the use of autochthonous nematodes to control C. tenebrionis larvae inside the roots is discussed.     

Complete mitochondrial genomes offer insights into the evolutionary relationships and comparative genetic diversity of New Zealand’s iconic kiwi (Apteryx spp.)

ABSTRACT

Kiwis (Apterygidae) are an enigmatic family of flightless birds endemic to New Zealand. Apterygidae is made up of a single genus, Apteryx with five species, four of which are characterised as at risk of greater by the New Zealand Department of Conservation. These five species are further separated into two morphologically and genetically distinguishable clades, containing A.haastii, and A.owenii in one and A.rowi, A.mantelli, and A.australis in the other. We reconstructed 17 kiwi mitochondrial genomes from previously published genomic data, nine from A.rowi and eight from A.owenii. Mitochondrial diversity analyses uncovered low levels of genetic diversity consistent with their reduced ranges and conservation concern. We further used one of the assembled A.rowi mitochondrial genomes together with mitochondrial genomes from A.haastii, A.owenii, A.mantelli, and several other individuals from Palaeognathae to estimate the within and between clade divergence times of kiwis. Our study exemplifies how available published data can be used in novel ways to provide new and complementary evolutionary insights to previous studies.     

Evaluation of a rapid molecular screening approach for the detection of toxigenic Clostridium difficile in general and subsequent identification of the tcdC Δ117 mutation in human stools

We have developed and validated a rapid molecular screening protocol for toxigenic Clostridium difficile, that also enables the identification of the hypervirulent epidemic 027/NAP1 strain. We describe a multiplex real-time PCR assay, which detects the presence of the tcdA and tcdB genes directly in stool samples. In case of positive PCR results, a separate multiplex real-time PCR typing assay was performed targeting the tcdC gene frame shift mutation at position 117. We prospectively compared the results of the screening PCR with those of a cytotoxicity assay (CTA), and a rapid immuno-enzyme assay for 161 stool samples with a specific request for diagnosis of C. difficile infection (CDI). A total of 16 stool samples were positive by CTA. The screening PCR assay confirmed all 16 samples, and gave a PCR positive signal in eight additional samples. The typing PCR assay detected the tcdC Δ117 mutation in 2/24 samples suggesting the presence of the epidemic strain in these samples. This was confirmed by PCR ribotyping and sequencing of the tcdC gene. Using CTA as the “gold standard”, the sensitivity, specificity, positive predictive value, and negative predictive value, for the screening PCR were 100%, 94.4%, 66.7%, and 100%, respectively. In conclusion, PCR may serve as a rapid negative screening assay for patients suspected of having CDI, although the low PPV hamper the use of PCR as a standalone test. However, PCR results may provide valuable information for patient management and minimising the spread of the epidemic 027/NAP1 strain.     

Application of the phase-space distribution approach of Monte Carlo for radiation contamination dose estimation from the (n,γ), (γ,n) nuclear reactions and linac leakage photons in the megavoltage radiotherapy facility

AimThe aim of this study was to characterize the radiation contamination inside and outside the megavoltage radiotherapy room.BackgroundRadiation contamination components in the 18 MV linac room are the secondary neutron, prompt gamma ray, electron and linac leakage radiation.Materials and MethodsAn 18 MV linac modeled in a typical bunker employing the MCNPX code of Monte Carlo. For fast calculation, phase-space distribution (PSD) file modeling was applied and the calculations were conducted for the radiation contamination components dose and spectra at 6 locations inside and outside the bunker.ResultsThe results showed that the difference of measured and calculated percent depth-dose (PDD) and photo beam-profile (PBP) datasets were lower than acceptable values. At isocenter, the obtained photon dose and neutron fluence were 2.4 × 10⁻¹⁴ Gy/initial e° and 2.22 × 10^-8 n°/cm², respectively. Then, neutron apparent source strength (Q_N) value was found as 1.34 × 10¹² n°/Gy X at isocenter and the model verified to photon and neutron calculations. A surface at 2 cm below the flattening filter was modeled as phase-space (PS) file for PDD and PBP calculations. Then by use of a spherical cell in the center of the linac target as a PS surface, contaminant radiations dose, fluence and spectra were estimated at 6 locations in a considerably short time, using the registered history of all particles and photons in the 13GB PSD file as primary source in the second step.ConclusionDesigning the PSD file in MC modeling helps user to solve the problems with complex geometry and physics precisely in a shorter run-time.     

A critical reappraisal of Phytolacca acinosa Roxb. (Phytolaccaceae) – A manganese-hyperaccumulating plant

Phytolacca acinosa Roxb. (Phytolaccaceae) was reported in 2004 to be a manganese-hyperaccumulating plant. Phytolacca americana L., growing at the same site was then independently reported in 2007 to have the same property. An apparent confusion in the taxonomy of the genus Phytolacca and interest in the Mn hyperaccumulative trait reported in P. acinosa has led to an extended field sampling, glasshouse cultivation and comparison with herbarium materials of P. americana s.l. Our specific aim was to identify and name the Mn hyperaccumulator plant correctly. P. acinosa is closely related to P. americana but it differs from the former in having an erect infructescence with usually eight distinct carpels, while from the latter has a nutant infructescence usually with 10 concrescent carpels. Our study indicated that the Mn hyperaccumulator plant P. acinosa which we first reported from the Xiangtan Mn tailings wasteland must now be reduced to P. americana as a synonym in order to prevent further confusion in identity and nomenclatural redundancy.     

A study of the pollen grains of Amaranthus spinosus Linné and A. Dubius Mart ex Thellung and their hybrids

A pollen-morphology study of Amaranthus spinosus, A. dubius, and their hybrids has been carried out. Three pollen types have been observed, namely (1) Type A: micrograins; (2) Type B: grains with smaller pores; and (3) Type C: grains with larger pores. Type B is characteristic of A. spinosus, Type C of A. dubius, and the micrograins of the hybrids. Pollen size range, and frequency of the various morphotypes serve to throw light on the biosystematics of the plants studied.     

pH Variation of midpoint potential for three photosynthetic bacterial cytochromes c′. A link between physical and functional properties

Midpoint redox potential (E_M) versus pH curves are reported over the pH range 5 to 10 for the cytochromes c′ from three species of purple photosynthetic bacteria: Rhodospirillum rubrum, Rhodopseudomonas palustris and Chromatium vinosum. In each case, theoretical curves are fitted to the data and pK values for the reduced (pH 5–5.5) and oxidized (pH 8–8.5) forms of the protein are found to influence the midpoint redox potentials. The oxidized form pK values in each case are found to correlate with previously determined pK values for variation in physical and/or spectroscopic properties. This correlation of functional and physical observables is discussed in terms of a possible mechanism of control of midpoint redox potential through heme iron-ligand bonding as moderated by the protein conformation in response to solution conditions. The reduced form pK values are discussed in terms of a mechanism which would alter the polarity of the heme environment, thereby influencing redox potentials.     

A chemoenzymatic approach to the synthesis of enantiomerically pure (S)-3-hydroxy-γ-butyrolactone

Optically pure (S)-3-hydroxy-gamma-butyrolactone, an important chiral building block in the pharmaceutical industry, was synthesized from L: -malic acid by combining a selective hydrogenation and a lipase-catalyzed hydrolysis. Lipase from Candida rugosa was found to be the most efficient enzyme for the hydrolysis of (S)-beta-benzoyloxy-gamma-butyrolactone. The use of organic solvent-aqueous two-phase system was employed to extract benzoic acid generated from enzymatic hydrolysis of the substrate. Tert-butyl methyl ether as an organic solvent was effective to extract the reaction product, benzoic acid, and stably maintained the enzyme activity of Lipase OF immobilized on polymeric supports Amberlite XAD-7. The immobilization made the recovery of the product simpler and prevented the formation of the emulsion. The pH adjustment was unnecessary with the immobilized Lipase OF. The scale-up of the enzymatic hydrolysis of S-BBL at 1,850-kg scale was carried out without problems to give 728.5 kg of S-HGB at 80% isolated yield. The scale-up results are similar to those of bench scale reactions. Racemic (R,S)-beta-benzoyloxy-gamma-butyrolactone was prepared from D-, L: -malic acid and was found to be hydrolyzed nonselectively by the enzyme.     

Depth-dependent reproductive output of the green sea urchin, Strongylocentrotus droebachiensis (O.F. Müller), in relation to the nature and availability of food

Reproductive output of green sea urchins (Strongylocentrotus droebachiensis O.F. Müller) in the field was highest at depths where preferred macro-algae were abundant, and lowest at depths where preferred macro-algae were overgrazed or replaced by non-preferred species (Agarum and Ptilota). Feeding rate and gonad indices of sea urchins in the laboratory were highest on a diet of preferred algae (Fucusdistichus L. subsp. edentatus (Pyl.) Powell, Laminaria longicruris Pyl., Desmarestia spp. and Saccorhizadermatodea (Pyl.) J. Ag.), and lowest on the less preferred Agarum cribrosum (Mert.) Bory, Ptilota serrata Kütz., and crustose corallines. Gamete production/unit area in overgrazed habitats was as great or greater than in kelp beds because of the higher biomass of urchins in overgrazed areas. Gonad weight and reproductive output of urchins from habitats poor in food can be increased by providing preferred foods.     

Genetic relationships between European and Siberian larch,Larix spp. (Pinaceae), studied by allozymes. Is the Polish larch a hybrid between these two species?

Thirteen populations ofLarix decidua subsp.decidua and subsp.polonica, and three populations ofL. sibirica were investigated by starch-gel electrophoresis. In the populations assayed 61 alleles at 17 loci were revealed. The allozyme data support the earlier observations about close relationships between these two larch species. Nei's genetic distances betweenL. decidua andL. sibirica were relatively small (D = 0.057), however, almost five times larger, on average, than those between populations of the same species. Results obtained in this study disagree withBobrov's hypothesis about the hybrid origin of the Polish larch and suggest a direct origin from the European larch.     

Cordaites schatzlarensis sp. nov. and Samaropsis newberryi (Andrews) Seward from the Westphalian (Carboniferous) of the ?aclé? area (Czech Republic)

The monotonous cordaitalean leaves are usually difficult to determine as leaf shape and venation can be similar in many species. Therefore cuticular analysis is an important method for distinguishing cordaitalean leaves. Pennsylvanian Cordaites schatzlarensis nov. sp. comes from the ?aclé? locality in the Intrasudetic Basin, Czech Republic. It has been found in mudstone accompanying the upper coal seams No. 9 and 10 of the Jan Šverma Coals, Lampertice Member, ?aclé? Formation and are late Duckmantian in age. The leaves of Cordaites schatzlarensis are lanceolate, amphistomatic. Stomata of the adaxial epidermis are scarce, isolated, or in very short stomatal rows. In contrast, the density of stomata on the abaxial cuticle is high and stomata are arranged into single or double stomatal rows. The cuticles of C. schatzlarensis are comparable with the Chinese Upper Permian C. baodeensis Ge. Leaves can be narrow, comparable to French Bolsovian Dorycordaites zeilleri Ledran, or relatively wide. Accompanying big seeds more than 5 cm in diameter are attributed to Samaropsis newberryi (Andrews) Seward. The pith casts Artisia Sternberg were found in sandy channel fill deposits.