Discovery of a fourth evolutionary lineage of Phytophthora ramorum: EU2

Phytophthora ramorum is a recently introduced, aggressive Phytophthora species that has caused extensive mortality of oak and tanoak trees in the western USA and Japanese larch trees in the UK. P. ramorum is also present on Rhododendron, Camellia, and Viburnum in the nursery industry, which is thought to have been the pathway for its spread into new geographic regions including forests and natural ecosystems. Three lineages of P. ramorum have been described, informally designated EU1, NA1, and NA2, and each lineage is believed to originate from an as yet unknown exotic centre of origin. Preliminary SSR and sequence analysis of isolates from a UK P. ramorum survey revealed seven isolates with profiles that did not match the previously known lineages. Detailed SSR and multilocus sequence analysis of these isolates are presented, allowing us to assign these isolates to a new P. ramorum lineage, designated EU2. Although the known geographical origin of these isolates is currently limited to Northern Ireland and western Scotland, the EU2 lineage isolates have been obtained from four different host plants, including Japanese larch. All isolates are of A1 compatibility type, which implies that this finding does not increase the risk of outcrossing with the EU1 lineage isolates already present in the UK. The oldest EU2 strain was isolated in 2007 but no SSR-based intraEU2 lineage genotypic diversity was detected. The combination of these elements points to a recent introduction, despite emergency phytosanitary measures to control introduction and spread. A PCR-RFLP method for the rapid identification of EU2 lineage isolates is presented.     

Toll-like receptor adaptor signaling molecule MyD88 on intervertebral disk homeostasis: in vitro, ex vivo studies

MyD88 is an adapter protein that links toll-like receptors (TLRs) and Interleukin-1 receptors (IL-1Rs) with downstream signaling molecules. The MyD88 has been found to be an essential mediator in the development of osteoarthritis in articular cartilage. However, the role of the MyD88 pathway has yet to be elucidated in the intervertebral disk (IVD). Using in vitro techniques, we analyzed the effect of MyD88 pathway-specific inhibition on the potent inflammatory and catabolic mediator LPS and IL-1 in bovine and human nucleus pulposus (NP) cells by assessing matrix-degrading enzyme expression, including matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family). We also analyzed inhibition of MyD88 in the regulation of inducible nitric oxide synthase and TLR-2. Finally, we used an ex vivo organ culture model to assess the effects of MyD88 inhibitor (MyD88i) on catabolic factor-induced disk degeneration in mice lumbar disks. In bovine NP cells, MyD88i potently antagonizes LPS- or IL-1-mediated induction of cartilage-degrading enzyme production, including MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. MyD88i also attenuates the LPS- or IL-1-mediated induction of iNOS and TLR-2 gene expression. Our ex vivo findings reveal inhibition of MyD88 via counteraction of IL-1-mediated proteoglycan depletion. The findings from this study demonstrate the potent anti-inflammatory and anti-catabolic effects of inhibition of MyD88 pathway inhibition on IVD homeostasis, suggesting a potential therapeutic benefit of a MyD88i in degenerative disk disease in the future.     

Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

     

Monomeric and oligomeric flavanols are agonists of membrane androgen receptors

The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone-protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action.     

Repression of Clostridium population in young broiler chickens after administration of a probiotic mixture

The influence of the administration of a mixed probiotic on zootechnical performances and the microbial composition of the feces of young broilers, up 42 days old, was investigated. In a six weeks trial the feed was supplemented with 1(w/w)% of a mixture of five potentially probiotic strains belonging to the genera Lactobacillus and Enterococcus. No effect on the weight of the broilers was observed. Plate counting of fecal samples showed a decrease in the Clostridium population with more than 4 10log units after 5 days and 2 10log units after 14 days of treatment. The DGGE pattern obtained after nested PCR, revealed a significant shift in the Lactobacillus population. This study shows that the application of probiotics in the feed of broiler chickens considerably lowers the Clostridium population in broilers, hereby also lowering the risk of spreading in the housing through fecal contamination.     

Detection and persistence of environmental DNA from an invasive,terrestrial mammal

Invasive Sus scrofa, a species commonly referred to as wild pig or feral swine, is a destructive invasive species with a rapidly expanding distribution across the United States. We used artificial wallows and small waterers to determine the minimum amount of time needed for pig eDNA to accumulate in the water source to a detectable level. We removed water from the artificial wallows and tested eDNA detection over the course of 2 weeks to understand eDNA persistence. We show that our method is sensitive enough to detect very low quantities of eDNA shed by a terrestrial mammal that has limited interaction with water. Our experiments suggest that the number of individuals shedding into a water system can affect persistence of eDNA. Use of an eDNA detection technique can benefit management efforts by providing a sensitive method for finding even small numbers of individuals that may be elusive using other methods.     

Alkaloids of Alstonia lanceolata

Thirteen alkaloids were isolated from the stem bark of Alstonia lanceolata. They were lochnericine, gentianine, 10,11-dimethoxy-1-methyl-deacetylpi