PCR-RFLP analysis of chitinase genes enables efficient genotyping of Metarhizium anisopliae var. anisopliae

A new genotyping tool has been developed and evaluated for Metarhizium anisopliae var. anisopliae. The tool is based on Restriction Fragment Length Polymorphism (RFLP) analysis of three chitinase genes that are functionally linked to insect-pathogenicity of this fungus. It allowed for discrimination of 14 genotypes among 22 M. anisopliae var. anisopliae strains of a world wide collection. Analyses revealed that the approach may also be applicable to other Metarhizium varieties. The new tool will be useful for genetic characterization of M. anisopliae var. anisopliae strains, and it is applicable for laboratories with limited access to molecular diagnostic equipment.     

Structure and thermodynamic characterization of the EphB4/Ephrin-B2 antagonist peptide complex reveals the determinants for receptor specificity

The Eph receptor tyrosine kinases and their ligands, the ephrins, regulate numerous biological processes in developing and adult tissues and have been implicated in cancer progression and in pathological forms of angiogenesis. We report the crystal structure of the EphB4 receptor in complex with a highly specific antagonistic peptide at a resolution of 1.65 angstroms. The peptide is situated in a hydrophobic cleft of EphB4 corresponding to the cleft in EphB2 occupied by the ephrin-B2 G-H loop, consistent with its antagonistic properties. Structural analysis identifies several residues within the EphB4 binding cleft that likely determine the ligand specificity of this receptor, while isothermal titration calorimetry experiments with truncated forms of the peptide define the amino acid residues of the peptide that are critical for receptor binding. These studies reveal structural features that will aid drug discovery initiatives to develop EphB4 antagonists for therapeutic applications.     

Structural and biophysical characterization of the EphB4*ephrinB2 protein-protein interaction and receptor specificity

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.     

Introgression in peripheral populations and colonization shape the genetic structure of the coastal shrub Armeria pungens

The coastal shrub Armeria pungens has a disjunct Atlantic-Mediterranean distribution. The historic range expansion underlying this distribution was investigated using the nuclear internal transcribed spacer region, three plastid regions (namely trnL-F, trnS-fM and matK) and morphometric data. A highly diverse ancestral lineage was identified in southwest Portugal. More recently, two areas have been colonized: (1) Corsica and Sardinia, where disjunct Mediterranean populations have been established as a result of the long-distance dispersal of Portuguese genotypes, and (2) the southern part of the Atlantic range, Gulf of Cadiz, where a distinct lineage showing no genetic differentiation among populations occurs. Genetic consequences of colonization seem to have been more severe in the Gulf of Cadiz than in Corsica-Sardinia. Although significant genetic divergence is associated with low plastid diversity in the Gulf of Cadiz, in Corsica-Sardinia, the loss of plastid haplotypes was not accompanied by divergence from disjunct Portuguese source populations. In addition, in its northernmost and southernmost populations, A. pungens exhibited evidence for ancient or ongoing introgression from sympatric congeners. Introgression might have created novel genotypes able to expand beyond the latitudinal margins of the species or, alternatively, these genotypes may be the result of surfing of alleles from other species in demographic equilibrium into peripheral populations of A. pungens. Our results highlight the evolutionary significance of genetic drift following the colonization of new areas and the key role of introgression in range expansion.     

A PCR-based tool for cultivation-independent detection and quantification of Metarhizium clade 1

The entomopathogenic fungus Metarhizium anisopliae and sister species are some of the most widely used biological control agents for insects. Availability of specific monitoring and quantification tools are essential for the investigation of environmental factors influencing their environmental distribution. Naturally occurring as well as released Metarhizium strains in the environment traditionally are monitored with cultivation-dependent techniques. However, specific detection and quantification may be limited due to the lack of a defined and reliable detection range of such methods. Cultivation-independent PCR-based detection and quantification tools offer high throughput analyses of target taxa in various environments. In this study a cultivation-independent PCR-based method was developed, which allows for specific detection and quantification of the defined Metarhizium clade 1, which is formed by the species Metarhizium majus, Metarhizium guizhouense, Metarhizium pingshaense, Metarhizium anisopliae, Metarhizium robertsii and Metarhiziumbrunneum, formerly included in the M. anisopliae cryptic species complex. This method is based on the use of clade-specific primers, i.e. Ma 1763 and Ma 2097, that are positioned within the internal transcribed spacer regions 1 and 2 of the nuclear ribosomal RNA gene cluster, respectively. BLAST similarity searches and empirical specificity tests performed on target and non-target species, as well as on bulk soil DNA samples, demonstrated specificity of this diagnostic tool for the targeted Metarhizium clade 1. Testing of the primer pair in qPCR assays validated the diagnostic method for specific quantification of Metarhizium clade 1 in complex bulk soil DNA samples that significantly correlated with cultivation-dependent quantification. The new tool will allow for highly specific and rapid detection and quantification of the targeted Metarhizium clade 1 in the environment. Habitat with high Metarhizium clade 1 densities can then be analyzed for habitat preferences in greater detail using cultivation-dependent techniques and genetic typing of isolates.     

An Aggressive Hypoxia Related Subpopulation of Melanoma Cells is TRP-2 Negative

Despite existing vaccination strategies targeting TRP-2, its function is not yet fully understood. TRP-2 is an enzyme involved in melanin biosynthesis and therefore discussed as a differentiation antigen. However, in mice Trp-2 was shown to be expressed in melanocyte stem cells of the hair follicle and therefore also considered as an indicator of stemness. A proper understanding of the TRP-2 function is crucial, considering a vaccination targeting cells with stemness properties would be highly effective in contrast to a therapy targeting differentiated melanoma cells.Analysing over 200 melanomas including primaries, partly matched metastases and patients’ cell cultures we show that TRP-2 is correlated with Melan A expression and decreases with tumor progression. In mice it is expressed in differentiated melanocytes as well as in stem cells. Furthermore, we identify a TRP-2 negative, proliferative, hypoxia related cell subpopulation which is significantly associated with tumor thickness and diseases progression. Patients with a higher percentage of those cells have a less favourable tumor specific survival.Our findings underline that TRP-2 is a differentiation antigen, highlighting the importance to combine TRP-2 vaccination with other strategies targeting the aggressive undifferentiated hypoxia related subpopulation.     

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.     

The oak browsing index correlates linearly with roe deer density: a new indicator for deer management?

Increasing populations of large herbivores during the last decades have had a major impact on vegetation. While several studies have looked for quantifying this impact in terms of plant biomass, plant survival or financial costs, the potential benefit of using the response of the vegetation to changes in browsing pressure by large herbivores to monitor their populations has been poorly investigated. As getting accurate estimates of density in populations of large herbivores is problematic, the use of indicators measuring the intensity of browsing might offer reliable alternative to managers. From the intensive monitoring of a roe deer population subject to an experimental manipulation of density, we looked for assessing the response of oak to changes of roe deer population size. Using a simple browsing index calculated from field data over 10 years, we found that this oak browsing index linearly increased with increasing population size of roe deer. This suggests that such an oak browsing index might be a reliable “indicator of ecological change” for monitoring roe deer populations in oak forests with natural regeneration.     

The amino acid sequence of ferredoxin from the alga Mastigocladuslaminosus

Ferredoxin was purified from the thermophilic blue-green alga, Mastigocladuslaminosus. The physicochemical properties of this ferredoxin are similar to those of other [2Fe-2S] plant ferredoxins except for its unusual thermal stability. The primary structure of the protein was determined and consists of 98 amino acid residues, 5 of which are cysteines. The positions of 4 cysteines which bind the iron atoms of the active centre are identical to those in other ferredoxins. The primary structure of the ferredoxin does not reveal any special features to account for its high thermal stability.     

Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion

We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of the large repeats fit the repeat of the underlying DNA sequence. The short repeats are between 170 and 180 bp, and after H1 depletion the short repeat in the BR 2.2 gene is 160 bp. Our most favoured interpretation of these data is that in intact chromatin the nucleosomes in the BR genes are phased with respect to the repeating DNA sequence, whereas micrococcus nuclease digestion leads to loss of a nucleosome-positioning constraint and hence to rearrangement of the nucleosomes. Our results imply a possible artefact of nuclease digestion of chromatin, which has to be taken into account in mapping nucleosome positions.