Elevated basal slippage mutation rates among the Canidae

The remarkable responsiveness of dog morphology to selection is a testament to the mutability of mammals. The genetic sources of this morphological variation are largely unknown, but some portion is due to tandem repeat length variation in genes involved in development. Previous analysis of tandem repeats in coding regions of developmental genes revealed fewer interruptions in repeat sequences in dogs than in the orthologous repeats in humans, as well as higher levels of polymorphism, but the fragmentary nature of the available dog genome sequence thwarted attempts to distinguish between locus-specific and genome-wide origins of this disparity. Using whole-genome analyses of the human and recently completed dog genomes, we show that dogs possess a genome-wide increase in the basal germ-line slippage mutation rate. Building on the approach that gave rise to the initial observation in dogs, we sequenced 55 coding repeat regions in 42 species representing 10 major carnivore clades and found that a genome-wide elevated slippage mutation rate is a derived character shared by diverse wild canids, distinguishing them from other Carnivora. A similarly heightened slippage profile was also detected in rodents, another taxon exhibiting high diversity and rapid evolvability. The correlation of enhanced slippage rates with major evolutionary radiations suggests that the possession of a "slippery" genome may bestow on some taxa greater potential for rapid evolutionary change.     

Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta

Irradiation of the heart and vasculature can cause a spectrum of cardiovascular complications, including increased risk of myocardial infarction or coronary heart disease. Although irradiation is implicated in oxidant stress and chronic inflammation, the underlying molecular mechanisms have not been elucidated. We tested the hypothesis that irradiation-initiated upregulation of xanthine oxidase (XO), a primary source of cardiovascular reactive oxygen species, contributes to endothelial dysfunction and increased vascular stiffness. Twenty-two, 3-month-old Sprague–Dawley male rats were gamma-irradiated at the following doses: 0, 50, 160, and 500 cGy. Rats exposed to 500 cGy showed a significant increase in endothelial XO expression and a twofold increase in XO activity, compared to the 0 cGy controls. Endothelial function was investigated ex vivo through vascular tension dose–responses to the endothelial dependent vasodilator, acetylcholine. Endothelial-dependent relaxation in aorta of the 500 cGy exposed rats was significantly attenuated from the control group. Remarkably, specific inhibition of XO with oxypurinol restored the relaxation response to that of the control. Furthermore, these ex vivo results are reflected in vivo through alterations in vascular stiffness, as measured by pulse wave velocity (PWV). As early as 1-day post-exposure, rats exhibited a significant increase in PWV from pre-exposure. The PWV of irradiated rats (50, 160, and 500 cGy) were greater than those of 0 cGy control rats at 1 day, 1 and 2 weeks. The sham and irradiated rats possessed equivalent pre-exposure PWV, with sham showing no change over 2 weeks. Thus, these findings suggest that early upregulation of XO contributes to oxidative stress and endothelial nitro-redox imbalance with resultant endothelial dysfunction and altered vascular mechanics. Furthermore, these data identify XO as a potential molecular target for attenuating irradiation-induced cardiovascular injury.     

Air trapping on chest CT is associated with worse ventilation distribution in infants with cystic fibrosis diagnosed following newborn screening

Background

In school-aged children with cystic fibrosis (CF) structural lung damage assessed using chest CT is associated with abnormal ventilation distribution. The primary objective of this analysis was to determine the relationships between ventilation distribution outcomes and the presence and extent of structural damage as assessed by chest CT in infants and young children with CF.

Methods

Data of infants and young children with CF diagnosed following newborn screening consecutively reviewed between August 2005 and December 2009 were analysed. Ventilation distribution (lung clearance index and the first and second moment ratios [LCI, M₁/M₀ and M₂/M₀, respectively]), chest CT and airway pathology from bronchoalveolar lavage were determined at diagnosis and then annually. The chest CT scans were evaluated for the presence or absence of bronchiectasis and air trapping.

Results

Matched lung function, chest CT and pathology outcomes were available in 49 infants (31 male) with bronchiectasis and air trapping present in 13 (27%) and 24 (49%) infants, respectively. The presence of bronchiectasis or air trapping was associated with increased M₂/M₀ but not LCI or M₁/M₀. There was a weak, but statistically significant association between the extent of air trapping and all ventilation distribution outcomes.

Conclusion

These findings suggest that in early CF lung disease there are weak associations between ventilation distribution and lung damage from chest CT. These finding are in contrast to those reported in older children. These findings suggest that assessments of LCI could not be used to replace a chest CT scan for the assessment of structural lung disease in the first two years of life. Further research in which both MBW and chest CT outcomes are obtained is required to assess the role of ventilation distribution in tracking the progression of lung damage in infants with CF.     

A three-hybrid system to probe in vivo protein-protein interactions: application to the essential proteins of the RD1 complex of M. tuberculosis

Background

Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes.

Methodology/Principal Findings

The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3.

Conclusions/Significance

The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.     

The integrin alpha L beta 2 hybrid domain serves as a link for the propagation of activation signal from its stalk regions to the I-like domain

Integrin activation involves global conformational changes as demonstrated by various functional and structural analyses. The integrin beta hybrid domain is proposed to be involved in the propagation of this activation signal. Our previous study showed that the integrin beta(2)-specific monoclonal antibody 7E4 abrogates monoclonal antibody KIM185-activated but not Mg(2+)/EGTA-activated leukocyte function-associated antigen-1 (LFA-1; alpha(L)beta(2))-mediated adhesion to ICAM-1. Here we investigated the allosteric inhibitory property of 7E4. By using human/mouse chimeras and substitution mutations, the epitope of 7E4 was mapped to Val(407), located in the mid-region of the beta(2) hybrid domain. Two sets of constitutively active LFA-1 variants were used to examine the effect of 7E4 on LFA-1/ICAM-1 binding. 7E4 attenuated the binding of variants that have modifications to regions membrane proximal with respect to the beta(2) hybrid domain. In contrast, the inhibitory effect was minimal on variants with alterations in the alpha(L) I- and beta(2) I-like domains preceding the hybrid domain. Furthermore, 7E4 abrogated LFA-1/ICAM-1 adhesion of phorbol 12-myristate 13-acetate-treated MOLT-4 cells. Our data demonstrate that interaction between the hybrid and I-like domain is critical for the regulation of LFA-1-mediated adhesion.