Structural mechanism of synergistic activation of Aurora kinase B/C by phosphorylated INCENP

Aurora kinases B and C (AURKB/AURKC) are activated by binding to the C-terminal domain of INCENP. Full activation requires phosphorylation of two serine residues of INCENP that are conserved through evolution, although the mechanism of this activation has not been explained. Here we present crystal structures of the fully active complex of AURKC bound to INCENP, consisting of phosphorylated, activated, AURKC and INCENP phosphorylated on its TSS motif, revealing the structural and biochemical mechanism of synergistic activation of AURKC:INCENP. The structures show that TSS motif phosphorylation stabilises the kinase activation loop of AURKC. The TSS motif phosphorylations alter the substrate-binding surface consistent with a mechanism of altered kinase substrate selectivity and stabilisation of the protein complex against unfolding. We also analyse the binding of the most specific available AURKB inhibitor, BRD-7880, and demonstrate that the well-known Aurora kinase inhibitor VX-680 disrupts binding of the phosphorylated INCENP TSS motif

Ceramic on ceramic bearing fractures in total hip arthroplasty : an analysis of data from the national joint registry

Aims: Ceramic-on-ceramic (CoC) bearings in total hip arthroplasty (THA) are commonly used but concerns exist regarding ceramic fracture. This study aims to report the risk of revision for fracture of modern CoC bearings and identify factors that might influence this risk, using data from the National Joint Registry (NJR). Patients and Methods: We analysed data on 111,681 primary CoC THA’s and 182 linked revisions for bearing fracture recorded in NJR. We used implant codes to identify ceramic bearing composition and generated Kaplan-Meier estimates for implant survivorship. Logistic regression analyses were performed for implant size and patient specific variables to determine any associated risks for revision. Results: 99.8% of bearings were CeramTec Biolox® products. Revisions for fracture were linked to 7 of 79,442 (0.009%) Biolox® Delta heads, 38 of 31,982 (0.119%) Biolox® Forte heads, 101 of 80,170 (0.126%) Biolox® Delta liners and 35 of 31,258 (0.112%) Biolox® Forte liners. Regression analysis of implant size revealed smaller heads had significantly higher odds of fracture (χ2=68.0, p<0.0001). The highest fracture risk were observed in the 28mm Biolox® Forte subgroup (0.382%). There were no fractures in the 40mm head group for either ceramic type. Liner thickness was not predictive of fracture (p=0.67). BMI was independently associated with revision for both head fractures (OR 1.09 per unit increase, p=0.031) and liner fractures (OR 1.06 per unit increase, p=0.006). Conclusions: We report the largest study of CoC bearing fractures to date. The risk of revision for CoC bearing fracture is very low, however previous studies have underestimated this risk. There is good evidence that the latest generation of ceramic has greatly reduced the odds of head fracture but not of liner fracture. Small head size and high patient BMI are associated with an increased risk of ceramic bearing fracture

The actin-myosin regulatory MRCK kinases: regulation, biological functions and associations with human cancer

The contractile actin-myosin cytoskeleton provides much of the force required for numerous cellular activities such as motility, adhesion, cytokinesis and changes in morphology. Key elements that respond to various signal pathways are the myosin II regulatory light chains (MLC), which participate in actin-myosin contraction by modulating the ATPase activity and consequent contractile force generation mediated by myosin heavy chain heads. Considerable effort has focussed on the role of MLC kinases, and yet the contributions of the myotonic dystrophy-related Cdc42-binding kinases (MRCK) proteins in MLC phosphorylation and cytoskeleton regulation have not been well characterized. In contrast to the closely related ROCK1 and ROCK2 kinases that are regulated by the RhoA and RhoC GTPases, there is relatively little information about the CDC42-regulated MRCKα, MRCKβ and MRCKγ members of the AGC (PKA, PKG and PKC) kinase family. As well as differences in upstream activation pathways, MRCK and ROCK kinases apparently differ in the way that they spatially regulate MLC phosphorylation, which ultimately affects their influence on the organization and dynamics of the actin-myosin cytoskeleton. In this review, we will summarize the MRCK protein structures, expression patterns, small molecule inhibitors, biological functions and associations with human diseases such as cancer

Dentin Bonding: SEM Comparison of the Resin-Dentin Interface in Primary and Permanent Teeth

Previous studies have suggested minor differences between primary and permanent teeth in terms of dentin composition and morphology. Other reports indicated lower bond strengths of resin composites to dentin of primary teeth compared with dentin of permanent teeth; however, no information is available regarding differences in the micromorphology of the resin-dentin interface that may explain these lower bond strengths. Therefore, the purpose of the present study was to compare primary and permanent teeth in terms of the thickness of the hybrid layer developed with two bonding systems. Our hypothesis was that bonding differences previously reported between primary and permanent dentin would be reflected in hybrid layer differences observable in SEM analyses. Twenty human extracted and non-carious teeth were divided into 4 groups: 5 primary and 5 permanent teeth restored with All-Bond 2/Bisfil P system; and 5 primary and 5 permanent teeth restored with Scotchbond Multi-Purpose/ZlOO. The sample area available on each tooth was divided for the two dentin conditioning times (7 and 15 sec). Measurements of hybrid layer thickness were performed by means of SEM at xl3,000. The results of this study indicated that the hybrid layer produced is significantly thicker in primary than in permanent teeth (p = 0.0001), suggesting that primary tooth dentin is more reactive to acid conditioning. No difference was observed in the hybrid layers produced by the two adhesive systems (p = 0.7920). The increased thickness of the hybrid layer in primary teeth (25 to 30%) and the subsequent lack of complete penetration of adhesive resin into previously demineralized dentin may contribute to the lower bond strengths to primary dentin reported in the literature. If a narrower hybrid layer more uniformly infused with resin is the goal of dentin bonding, it is concluded that a differentiated protocol for bonding to primary dentin (with shorter time for dentin conditioning) can be used as a means to reproduce the hybrid layer thickness seen in permanent teeth.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/67017/2/10.1177_00220345960750061101.pd

Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases

Soil-Transmitted Helminth Reinfection after Drug Treatment: A Systematic Review and Meta-Analysis

Infections with soil-transmitted helminths (the roundworm Ascaris lumbricoides, the whipworm Trichuris trichiura, and hookworm) affect over 1 billion people, particularly rural communities in the developing world. The global strategy to control soil-transmitted helminth infections is ‘preventive chemotherapy’, which means large-scale administration of anthelmintic drugs to at-risk populations. However, because reinfection occurs after treatment, ‘preventive chemotherapy’ must be repeated regularly. Our systematic review and meta-analysis found that at 3, 6, and 12 months after treatment, A. lumbricoides prevalence reached 26% (95% confidence interval (CI): 16–43%), 68% (95% CI: 60–76%) and 94% (95% CI: 88–100%) of pretreatment levels, respectively. For T. trichiura, respective reinfection prevalence at these time points were 36% (95% CI: 28–47%), 67% (95% CI: 42–100%), and 82% (95% CI: 62–100%); and for hookworm, 30% (95% CI: 26–34%), 55% (95% CI: 34–87%), and 57% (95% CI: 49–67%). Prevalence and intensity of reinfection were positively correlated with pretreatment infection status. Our results suggest a frequent anthelmintic drug administration to maximize the benefit of preventive chemotherapy. Moreover, an integrated control strategy, consisting of preventive chemotherapy combined with health education and environmental sanitation is needed to interrupt transmission of soil-transmitted helminths

Binding Free Energy Landscape of Domain-Peptide Interactions

Peptide recognition domains (PRDs) are ubiquitous protein domains which mediate large numbers of protein interactions in the cell. How these PRDs are able to recognize peptide sequences in a rapid and specific manner is incompletely understood. We explore the peptide binding process of PDZ domains, a large PRD family, from an equilibrium perspective using an all-atom Monte Carlo (MC) approach. Our focus is two different PDZ domains representing two major PDZ classes, I and II. For both domains, a binding free energy surface with a strong bias toward the native bound state is found. Moreover, both domains exhibit a binding process in which the peptides are mostly either bound at the PDZ binding pocket or else interact little with the domain surface. Consistent with this, various binding observables show a temperature dependence well described by a simple two-state model. We also find important differences in the details between the two domains. While both domains exhibit well-defined binding free energy barriers, the class I barrier is significantly weaker than the one for class II. To probe this issue further, we apply our method to a PDZ domain with dual specificity for class I and II peptides, and find an analogous difference in their binding free energy barriers. Lastly, we perform a large number of fixed-temperature MC kinetics trajectories under binding conditions. These trajectories reveal significantly slower binding dynamics for the class II domain relative to class I. Our combined results are consistent with a binding mechanism in which the peptide C terminal residue binds in an initial, rate-limiting step

The Fischer 344 Rat Reflects Human Susceptibility to Francisella Pulmonary Challenge and Provides a New Platform for Virulence and Protection Studies

Background: The pathogenesis of Francisella tularensis, the causative agent of tularemia, has been primarily characterized in mice. However, the high degree of sensitivity of mice to bacterial challenge, especially with the human virulent strains of F. tularensis, limits this animal model for screening of defined attenuated vaccine candidates for protection studies. Methods and Findings: We analyzed the susceptibility of the Fischer 344 rat to pulmonary (intratracheal) challenge with three different subspecies (subsp) of F. tularensis that reflect different levels of virulence in humans, and characterized the bacterial replication profile in rat bone marrow-derived macrophages (BMDM). In contrast to the mouse, Fischer 344 rats exhibit a broader range of sensitivity to pulmonary challenge with the human virulent subsp. tularensis and holarctica. Unlike mice, Fischer rats exhibited a high degree of resistance to pulmonary challenge with LVS (an attenuated derivative o

Modulation of hepatic PPAR expression during Ft LVS LPS-induced protection from Francisella tularensis LVS infection

<p>Abstract</p> <p>Background</p> <p>It has been shown previously that administration of <it>Francisella tularensis </it>(<it>Ft</it>) Live Vaccine Strain (LVS) lipopolysaccharide (LPS) protects mice against subsequent challenge with <it>Ft </it>LVS and blunts the pro-inflammatory cytokine response.</p> <p>Methods</p> <p>To further investigate the molecular mechanisms that underlie <it>Ft </it>LVS LPS-mediated protection, we profiled global hepatic gene expression following <it>Ft </it>LVS LPS or saline pre-treatment and subsequent <it>Ft </it>LVS challenge using Affymetrix arrays.</p> <p>Results</p> <p>A large number of genes (> 3,000) were differentially expressed at 48 hours post-infection. The degree of modulation of inflammatory genes by infection was clearly attenuated by pre-treatment with <it>Ft </it>LVS LPS in the surviving mice. However, <it>Ft </it>LVS LPS alone had a subtle effect on the gene expression profile of the uninfected mice. By employing gene set enrichment analysis, we discovered significant up-regulation of the fatty acid metabolism pathway, which is regulated by peroxisome proliferator activated receptors (PPARs).</p> <p>Conclusions</p> <p>We hypothesize that the LPS-induced blunting of pro-inflammatory response in mouse is, in part, mediated by PPARs (α and γ).</p