Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure.

Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.     

Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus

Four mutants of Staphylococcus aureus strain Newman that were defective in the fibrinogen receptor (clumping factor) were isolated by transposon Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon-host DNA junction fragments, one of which was cloned and used to generate a probe to identify and clone the wild-type clumping factor locus (clfA). The mutants failed to form clumps in soluble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) coverslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fuily compiemented the ciumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen-coated PMMA. in addition, the cloned clfA gene on a shuttle plasmid aiiowed the weakiy ciumping strain 8325-4 to form clumps with the same avidity as the wild-type strain Newman and also significantly enhanced the adherence of 8325-4 strains. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid-phase fibrinogen. The clfA gene encodes a fibrinogen-binding protein with an apparent molecular mass of c. 130 kDa. The amino acid sequence of the protein was deduced from the DNA sequence; it was predicted that a 896 residue protein (molecular mass 92 kDa) would be expressed. The putative ClfA protein has features that suggest that it is associated with the ceil surface. Furthermore it contains a novel 308 residue region comprising dipeptide repeats predominantly of Asp and Ser ending 28 residues upstream from the LPXTG motif common to wall-associated proteins. Significant homology was found between the ClfA protein and the fibronectin-binding proteins of S. S. aureus, particularly in the N-and C-termini.     

Individual tree context and contrast dictate tree physiological features and arthropod biodiversity patterns across multiple trophic levels

1. The present study tested the hypothesis that tree context (natural, semi-natural or planted) and contrast (height of surrounding vegetation) affect tree physiological characters (leaf size, nutrient content, and stress-related factors), and also alter the arthropod biodiversity patterns either directly or indirectly. 2. Arthropods were collected from tree canopies using chemical fogging from the ecologically important South African native tree Podocarpus elongatus. 3. Low contrast trees had significantly larger leaves than those in high contrast environments and harboured an overall higher richness, abundance, and biomass of arthropods, although this was guild-dependent. 4. Trees in natural contexts had less foliar δ¹³C, suffered less from artificial nitrogen enrichment, and harboured significantly higher herbivore and predator arthropod richness and unique assemblages, compared with planted trees. 5. Semi-natural trees supported natural levels of arthropod richness, although these were mostly generalist species. 6. Tree context and contrast can therefore dictate associated biota at multiple trophic levels and native trees may fail to maintain natural biodiversity in transformed landscapes.     

Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of α-toxin than did the proliferative supernatants. Addition of α-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, δ-toxin or phenol soluble modulin α-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.     

DNA Methylation Mediates Persistent Epileptiform Activity In Vitro and In Vivo

Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5’ regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5’ region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5’ region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5’ region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5’ region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.     

Model Prediction and Validation of an Order Mechanism Controlling the Spatiotemporal Phenotype of Early Hepatocellular Carcinoma

Recently, hepatocyte–sinusoid alignment (HSA) has been identified as a mechanism that supports the coordination of hepatocytes during liver regeneration to reestablish a functional micro-architecture (Hoehme et al. in Proc Natl Acad Sci 107(23):10371–10376, 2010). HSA means that hepatocytes preferentially align along the closest micro-vessels. Here, we studied whether this mechanism is still active in early hepatocellular tumors. The same agent-based spatiotemporal model that previously correctly predicted HSA in liver regeneration was further developed to simulate scenarios in early tumor development, when individual initiated hepatocytes gain increased proliferation capacity. The model simulations were performed under conditions of realistic liver micro-architectures obtained from 3D reconstructions of confocal laser scanning micrographs. Interestingly, the established model predicted that initiated hepatocytes at first arrange in elongated patterns. Only when the tumor progresses to cell numbers of approximately 4000, does it adopt spherical structures. This prediction may have relevant consequences, since elongated tumors may reach critical structures faster, such as larger vessels, compared to a spherical tumor of similar cell number. Interestingly, this model prediction was confirmed by analysis of the spatial organization of initiated hepatocytes in a rat liver tumor initiation study using single doses of 250 mg/kg of the genotoxic carcinogen N-nitrosomorpholine (NNM). Indeed, small clusters of GST-P positive cells induced by NNM were elongated, almost columnar, while larger GDT-P positive foci of approximately the size of liver lobuli adopted spherical shapes. From simulations testing numerous possible mechanisms, only HSA could explain the experimentally observed initial deviation from spherical shape. The present study demonstrates that the architecture of small cell clusters of hepatocytes early after initiation is still controlled by physiological mechanisms. However, this coordinating influence is lost when the tumor grows to approximately 4000 cells, leading to further growth in spherical shape. Our findings stress the potential importance of organ micro-architecture in understanding tumor phenotypes.     

Identification of quantitative trait loci conferring resistance to tan spot in a biparental population derived from two Nebraska hard red winter wheat cultivars

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is a destructive foliar disease in all types of cultivated wheat worldwide. Genetics of tan spot resistance in wheat is complex, involving insensitivity to fungal-produced necrotrophic effectors (NEs), major resistance genes, and quantitative trait loci (QTL) conferring race-nonspecific and race-specific resistance. The Nebraska hard red winter wheat (HRWW) cultivar ‘Wesley’ is insensitive to Ptr ToxA and highly resistant to multiple Ptr races, but the genetics of resistance in this cultivar is unknown. In this study, we used a recombinant inbred line (RIL) population derived from a cross between Wesley and another Nebraska cultivar ‘Harry’ (Ptr ToxA sensitive and highly susceptible) to identify QTL associated with reaction to tan spot caused by multiple races/isolates. Sensitivity to Ptr ToxA conferred by the Tsn1 gene was mapped to chromosome 5B as expected. The Tsn1 locus was a major susceptibility QTL for the race 1 and race 2 isolates, but not for the race 2 isolate with the ToxA gene deleted. A second major susceptibility QTL was identified for all the Ptr ToxC-producing isolates and located to the distal end of the chromosome 1A, which likely corresponds to the Tsc1 locus. Three additional QTL with minor effects were identified on chromosomes 7A, 7B, and 7D. This work indicates that both Ptr ToxA-Tsn1 and Ptr ToxC-Tsc1 interactions are important for tan spot development in winter wheat, and Wesley is highly resistant largely due to the absence of the two tan spot sensitivity genes.