Effect of mechanical fatigue on commercial bioprosthetic TAVR valve mechanical and microstructural properties.

Valvular structural deterioration is of particular concern for transcatheter aortic valve replacements due to their suspected shorter longevity and increasing use in younger patient populations. In this work we investigated the mechanical and microstructural changes in commercial TAVR valves composed of both glutaraldehyde fixed bovine and porcine pericardium (GLBP and GLPP) following accelerated wear testing (AWT) as outlined in ISO 5840 standards. This provided greater physiological relevance to the loading compared to previous studies and by utilizing digital image correlation we were able to obtain strain contours for each leaflet pre and post fatigue and identify sites of fatigue damage. The areas of greatest change in mechanical strain for each leaflet were then further probed using biaxial tensile testing, confocal microscopy, and electron microscopy. It was observed that overall strain decreased in the GLPP valves following AWT of 200 million cycles while the GLBP valve showed an increase in overall strain. Biaxial tensile testing showed a statistically significant reduction in stress for GLPP while no significant changes were seen for GLBP. Both confocal and electron microscopy showed a disruption to the gross collagen organization and fibrillar structure, including fragmentation, for GLPP but only the former for GLBP. However, further test data is required to confirm these findings and to provide a better understanding of this fatigue pathway is required such that it can be incorporated into both valve design and selection processes to improve overall longevity for both GLPP and GLBP devices.

PIPKIγ targets to the centrosome and restrains centriole duplication.

Centriole biogenesis depends on the polo-like kinase (PLK4) and a small group of structural proteins. The spatiotemporal regulation of these proteins at pre-existing centrioles is essential to ensure that centriole duplication occurs once per cell cycle. Here, we report that phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C, hereafter referred to as PIPKIγ) plays an important role in centriole fidelity. PIPKIγ localized in a ring-like pattern in the intermediate pericentriolar materials around the proximal end of the centriole in G1, S and G2 phases, but not in M phase. This localization was dependent upon an association with centrosomal protein of 152 KDa (CEP152). Without detaining cells in S or M phase, the depletion of PIPKIγ led to centriole amplification in a manner that was dependent upon PLK4 and spindle assembly abnormal protein 6 homolog (SAS6). The expression of exogenous PIPKIγ reduced centriole amplification that occurred as a result of endogenous PIPKIγ depletion, hydroxyurea treatment or PLK4 overexpression, suggesting that PIPKIγ is likely to function at the PLK4 level to restrain centriole duplication. Importantly, we found that PIPKIγ bound to the cryptic polo-box domain of PLK4 and that this binding reduced the kinase activity of PLK4. Together, our findings suggest that PIPKIγ is a novel negative regulator of centriole duplication, which acts by modulating the homeostasis of PLK4 activity.

Chronic exposure to zinc chromate induces centrosome amplification and spindle assembly checkpoint bypass in human lung fibroblasts.

Hexavalent chromium (Cr(VI)) compounds are known human lung carcinogens. Solubility plays an important role in its carcinogenicity with the particulate or insoluble form being the most potent. Of the particulate Cr(VI) compounds, zinc chromate appears to be the most potent carcinogen; however, very few studies have investigated its carcinogenic mechanism. In this study, we investigated the ability of chronic exposure to zinc chromate to induce numerical chromosome instability. We found no increase in aneuploidy after a 24 h exposure to zinc chromate, but with more chronic exposures, zinc chromate induced concentration- and time-dependent increases in aneuploidy in the form of hypodiploidy, hyperdiploidy, and tetraploidy. Zinc chromate also induced centrosome amplification in a concentration- and time-dependent manner in both interphase and mitotic cells after chronic exposure, producing cells with centriolar defects. Furthermore, chronic exposure to zinc chromate induced concentration- and time-dependent increases in spindle assembly checkpoint bypass with increases in centromere spreading, premature centromere division, and premature anaphase. Last, we found that chronic exposure to zinc chromate induced a G2 arrest. All together, these data indicate that zinc chromate can induce chromosome instability after prolonged exposures.

CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability.

The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca2+-binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large (approximately 300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis.