Substitution of aspartate for glycine 1018 in the type III procollagen (COL3A1) gene causes type IV Ehlers-Danlos syndrome: the mutated allele is present in most blood leukocytes of the asymptomatic and mosaic mother.

A proband with arterial ruptures and skin changes characteristic of the type IV variant of Ehlers-Danlos syndrome was found to have a single-base mutation in the type III procollagen gene, which converted the codon for glycine at amino acid position 1018 to a codon for aspartate. (Amino acid positions are numbered by the standard convention in which the first glycine of the triple-helical domain of an alpha chain is number 1. The numbers of positions in the alpha 1(III) chains can be converted to positions in the human pro alpha(III) chain by adding 167.) Nucleotide sequencing of overlapping PCR products in which the two alleles were distinguished demonstrated that the mutation of glycine 1018 was the only mutation that changed the primary structure of type III procollagen. The glycine substitution markedly decreased the amount of type III procollagen secreted into the medium by cultured skin fibroblasts from the proband. It is surprising that the same mutation was found in about 94% of the peripheral blood leukocytes from the proband's asymptomatic 72-year-old mother. Other tissues from the mother contained the mutated allele; it was present in 0%-100% of different samples of hair cells and in about 40% of cells from the oral epithelium. Therefore, the mother was a mosaic for the mutation. Since the mutated allele was present in cells derived from all three germ layers, the results indicated that the mutation arose by the late blastocyst stage of development. The results also indicate that assays of blood leukocytes do not always reveal mosaicism or predict phenotypic involvement of tissues, such as blood vessels, that are derived from the same embryonic cells as are leukocytes.     

Age-specific breeding success in a wild mammalian population: selection, constraint, restraint and senescence

The Selection, Constraint, Restraint and Senescence Hypotheses predict how breeding success should vary with age. The Selection Hypothesis predicts between-individual variation arising from quality differences; the other hypotheses predict within-individual variation due to differing skills or physiological condition (Constraint), residual reproductive lifespan (Restraint), or somatic and reproductive investment (Senescence). Studies tend to focus on either the initial increase in breeding success or later decrease; however, both require consideration when unravelling the underlying evolutionary processes. Additionally, few studies present genetic fitness measures and rarely for both sexes. We therefore test these four hypotheses, which are not mutually exclusive, in a high-density population of European badgers Meles meles. Using an 18-year data set (including 22 microsatellite loci), we show an initial improvement in breeding success with age, followed by a later and steeper rate of reproductive senescence in male than in female badgers. Breeding success was skewed within age-classes, indicating the influence of factors other than age-class. This was partly attributable to selective appearance and disappearance of badgers (Selection Hypothesis). Individuals with a late age of last breeding showed a concave-down relationship between breeding success and experience (Constraint Hypothesis). There was no evidence of abrupt terminal effects; rather, individuals showed a concave-down relationship between breeding success and residual reproductive lifespan (Restraint Hypothesis), with an interaction with age of first breeding only in female badgers. Our results demonstrate the importance of investigating a comprehensive suite of factors in age-specific breeding success analyses, in both sexes, to fully understand evolutionary and population dynamics.     

Muramidases found in the foregut microbiome of the Tammar wallaby can direct cell aggregation and biofilm formation

We describe here the role of muramidases present in clones of metagenomic DNA that result in cell aggregation and biofilm formation by Escherichia coli. The metagenomic clones were obtained from uncultured Lachnospiraceae-affiliated bacteria resident in the foregut microbiome of the Tammar wallaby. One of these fosmid clones (p49C2) was chosen for more detailed studies and a variety of genetic methods were used to delimit the region responsible for the phenotype to an open reading frame of 1425 bp. Comparative sequence analysis with other fosmid clones giving rise to the same phenotype revealed the presence of muramidase homologues with the same modular composition. Phylogenetic analysis of the fosmid sequence data assigned these fosmid inserts to recently identified, but uncultured, phylogroups of Lachnospiraceae believed to be numerically dominant in the foregut microbiome of the Tammar wallaby. The muramidase is a modular protein containing putative N-acetylmuramoyl--alanine amidase and an endo-β-N-acetylglucosaminidase catalytic module, with a similar organization and functional properties to some Staphylococcal autolysins that also confer adhesive properties and biofilm formation. We also show here that the cloned muramidases result in the production of extracellular DNA, which appears to be the key for biofilm formation and autoaggregation. Collectively, these findings suggest that biofilm formation and cell aggregation in gut microbiomes might occur via the concerted action of carbohydrate-active enzymes and the production of extracellular DNA to serve as a biofilm scaffold.     

Optimization of microCT imaging and blood vessel diameter quantitation of preclinical specimen vasculature with radiopaque polymer injection medium

Vascular networks within a living organism are complex, multi-dimensional, and challenging to image capture. Radio-angiographic studies in live animals require a high level of infrastructure and technical investment in order to administer costly perfusion mediums whose signals metabolize and degrade relatively rapidly, diminishing within a few hours or days. Additionally, live animal specimens must not be subject to long duration scans, which can cause high levels of radiation exposure to the specimen, limiting the quality of images that can be captured. Lastly, despite technological advances in live-animal specimen imaging, it is quite difficult to minimize or prevent movement of a live animal, which can cause motion artifacts in the final data output. It is demonstrated here that through the use of postmortem perfusion protocols of radiopaque silicone polymer mediums and ex-vivo organ harvest, it is possible to acquire a high level of vascular signal in preclinical specimens through the use of micro-computed tomographic (microCT) imaging. Additionally, utilizing high-order rendering algorithms, it is possible to further derive vessel morphometrics for qualitative and quantitative analysis.     

More than just B-cell inhibition

Despite tremendous advances in the therapy of rheumatoid arthritis (RA), there remains interest in oral agents that may offer benefits that are similar to, or better than, those of biologic therapies. In their paper, Chang and colleagues demonstrate the effectiveness of a Bruton tyrosine kinase (Btk) inhibitor in two models of RA. Btk inhibition impacts several pathways affecting both B-cell and macrophage activation, making it a promising target in RA. However, other kinase inhibitors have failed to transition from animal models to human therapy, so it remains to be seen whether a Btk inhibitor will have a role in the RA treatment armamentarium.