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.

     

Quantitative Analysis of BTF3, HINT1, NDRG1 and ODC1 Protein Over-Expression in Human Prostate Cancer Tissue

Prostate carcinoma is the most common cancer in men with few, quantifiable, biomarkers. Prostate cancer biomarker discovery has been hampered due to subjective analysis of protein expression in tissue sections. An unbiased, quantitative immunohistochemical approach provided here, for the diagnosis and stratification of prostate cancer could overcome this problem. Antibodies against four proteins BTF3, HINT1, NDRG1 and ODC1 were used in a prostate tissue array (> 500 individual tissue cores from 82 patients, 41 case pairs matched with one patient in each pair had biochemical recurrence). Protein expression, quantified in an unbiased manner using an automated analysis protocol in ImageJ software, was increased in malignant vs non-malignant prostate (by 2-2.5 fold, p<0.0001). Operating characteristics indicate sensitivity in the range of 0.68 to 0.74; combination of markers in a logistic regression model demonstrates further improvement in diagnostic power. Triple-labeled immunofluorescence (BTF3, HINT1 and NDRG1) in tissue array showed a significant (p<0.02) change in co-localization coefficients for BTF3 and NDRG1 co-expression in biochemical relapse vs non-relapse cancer epithelium. BTF3, HINT1, NDRG1 and ODC1 could be developed as epithelial specific biomarkers for tissue based diagnosis and stratification of prostate cancer.     

Founder populations and the current status of exotic parrots in South Africa

Parrots (families Psittacidae and Cacatuidae) are one of the most threatened taxa of birds, with a combination of threats to wild populations, including capture for the cage bird market, habitat modification and destruction, persecution, disease, and threats from introduced species. As a result of the group's popularity as a cage bird, and the transport of vast numbers of individuals across the globe (both legally and illegally), the establishment of populations beyond their natural ranges has likely been enhanced. This review reports on c. 30 parrot species that have been observed outside of their natural ranges in South Africa, most likely derived, and supplemented, from both intentional and unintentional releases. Of these, the Rose-ringed Parakeet Psittacula krameri and at least one Agapornis species have become established as breeding wild populations. Like other invasive species they may pose threats to local biodiversity; however, all are strongly associated with major urban centres where significant changes to natural biota have already occurred.     

Secretarybird Sagittarius serpentarius Population Trends and Ecology: Insights from South African Citizen Science Data

Data from two long-term citizen science projects were used to examine the status and ecology of a Red List species, the Secretarybird Sagittarius serpentarius (Vulnerable), in South Africa. The first phase of the Southern African Bird Atlas Project operated from 1987 until 1992, and the second phase began in 2007. The Coordinated Avifaunal Roadcounts (CAR) project began in 1993 and by 1998 had expanded to cover much of the south-eastern half of the country. Data submitted up until April 2013 were used. A new method of comparing reporting rates between atlas projects was developed. Changing reporting rates are likely to reflect changes in abundance; in this instance the data suggest that the Secretarybird population decreased across much of South Africa between the two atlas projects, with a widespread important decrease in the Kruger National Park. Habitat data from the CAR project were analysed to gain insight into the ecology of the species. Secretarybirds tended to avoid transformed habitats across much of the area covered by the CAR project. In the winter rainfall region of the Western Cape, which is characterised by heavily transformed fynbos vegetation, at least 50% of Secretarybirds recorded were in transformed environments. This implies that in the Fynbos biome, at least, Secretarybirds have adapted to transformed environments to some degree. However, in the rest of the country it is likely that habitat loss, largely through widespread bush encroachment but also through agriculture, afforestation, and urbanisation, is a major threat to the species. The methods developed here represent a new approach to analysing data from long-term citizen science projects, which can provide important insights into a species'' conservation status and ecology.     

Seasonal occurrence and local movements of the grey-headed (brown-necked) parrot Poicephalus fuscicollis suahelicus in southern Africa

Seasonal movements of grey‐headed (brown‐necked) parrots were recorded in parts of its range and are likely a response to breeding and availability of specific food sources. Breeding occurred in the northern Kruger National Park and lowveld near the Mutale–Luvhuvhu river confluence from April to August. Aggregations and movements of birds occurred during the post‐breeding season (August–December) in response to seasonally abundant food sources. In north‐eastern South Africa, grey‐headed parrots occurred at Levubu, following the breeding season and their arrival in the area was correlated with the availability of unripe Mabola Plum, Parinari curatellifolia fruit. Similar regional movements occurred in Zimbabwe, the Caprivi of northern Namibia and Zambia. During these movements, flocks of up to 50 individuals were observed, whilst during breeding months singletons and pairs were more frequently seen. This increased abundance in time and space suggests that seasonal migratory movements occur.