Identification and characterisation of 2-aminopyridine inhibitors of checkpoint kinase 2

5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine inhibitors of CHK2 were identified from high throughput screening of a kinase-focussed compound library. Rapid exploration of the hits through straightforward chemistry established structure–activity relationships and a proposed ATP-competitive binding mode which was verified by X-ray crystallography of several analogues bound to CHK2. Variation of the 5-(hetero)aryl substituent identified bicyclic dioxolane and dioxane groups which improved the affinity and the selectivity of the compounds for CHK2 versus CHK1. The 3-(4-carboxamidophenyl) substituent could be successfully replaced by acyclic ω-aminoalkylamides, which made additional polar interactions within the binding site and led to more potent inhibitors of CHK2. Compounds from this series showed activity in cell-based mechanistic assays for inhibition of CHK2.     

Incomplete Lineage Sorting: Consistent Phylogeny Estimation from Multiple Loci

We introduce a simple computationally efficient algorithm for reconstructing phylogenies from multiple gene trees in the presence of incomplete lineage sorting, that is, when the topology of the gene trees may differ from that of the species tree. We show that our technique is statistically consistent under standard stochastic assumptions, that is, it returns the correct tree given sufficiently many unlinked loci. We also show that it can tolerate moderate estimation errors.     

Mycobacterium africanum--review of an important cause of human tuberculosis in West Africa

Mycobacterium africanum consists of two phylogenetically distinct lineages within the Mycobacterium tuberculosis complex, known as M. africanum West African 1 and M. africanum West African 2. These lineages are restricted to West Africa, where they cause up to half of human pulmonary tuberculosis. In this review we discuss the definition of M. africanum, describe the prevalence and restricted geographical distribution of M. africanum West African 1 and 2, review the occurrence of M. africanum in animals, and summarize the phenotypic differences described thus far between M. africanum and M. tuberculosis sensu stricto.     

An adhesion molecule in free-living Dictyostelium amoebae with integrin beta features

The study of free-living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin beta chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well-characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin beta chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.     

On the mechanism of mitochondrial uncoupling protein 1 function

Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography and identified by peptide mass mapping and tandem mass spectrometry. Native and expressed UCP 1 were reconstituted into liposomes, and proton flux through UCP 1 was shown to be fatty acid-dependent and GDP-sensitive. To investigate the mechanism of action of UCP 1, we determined whether hydrophilic modification of the omega-carbon of palmitate effected its transport function. We show that proton flux was greater through native UCP 1-containing proteoliposomes when facilitated by less hydrophilically modified palmitate (palmitate > omega-methoxypalmitate > omega-hydroxypalmitate with little or no proton flux due to glucose-O-omega-palmitate or undecanesulfonate). We show that non-proton-dependent charge transfer was greater when facilitated by less hydrophilically modified palmitate (palmitate/undecanesulfonate > omega-methoxypalmitate > omega-hydroxypalmitate, with no non-proton-dependent charge transfer flux due to glucose-O-omega-palmitate). We show that the GDP-inhibitable oxygen consumption rate in brown adipose tissue mitochondria was reversed by palmitate (as expected) but not by glucose-O-omega-palmitate. Our data are consistent with the model that UCP 1 flips long-chain fatty acid anions and contradict the "cofactor" model of UCP 1 function.     

Sleep and psychological factors are associated with meeting discharge criteria to return to sport following ACL reconstruction in athletes

This study aimed to determine if sleep quality and psychological factors were associated with time to meet the discharge criteria to return to sport (RTS) following anterior cruciate ligament reconstruction (ACL-R) among athletes. A cohort-study design included 89 athletes following ACL-R. Each participant completed a battery of questionnaires at 6 different time points: within 3 days of injury occurrence and at post-surgery (1.5 m, 3 m, 4.5 m, 6 m and when discharge criteria were met). Assessment included sleep quality and quantity, symptoms of depression, anxiety, stress, psychological readiness to RTS and fear of re-injury. The primary outcome was the time needed to meet all discharge criteria to RTS. Sleep parameters and psychological factors were not associated with time to meet the discharge criteria to RTS. However, athletes that had lower scores of anxiety (OR 1.2 (95% CI 1.0, 1.3) and insomnia (OR 1.2 (95% CI 1.0, 1.3) at baseline were more likely to meet the RTS discharge criteria. Athletes with better sleep quality at 3m, 4.5m and 6m were more likely to meet the RTS discharge criteria OR 1.3 (95% CI 1.1, 1.7), 2.0 (95% CI 1.1–3.4) and 1.4 (95% CI 1.0, 1.9) respectively. Sleep quality and psychological factors were not associated with time to meet the discharge criteria to RTS but impacted whether athletes adhered and completed their rehabilitation program or not. Monitoring sleep quality and psychological factors of athletes before and following ACL-R surgery is important to identify athletes who could have difficulties in adhering to and completing their rehabilitation program to RTS.     

Characterisation of recombinant unglycosylated human serum transferrin purified from Saccharomyces cerevisiae

Structural identity between a recombinant transferrin mutant (N413Q, N611Q) secreted from Saccharomyces cerevisiae and the native protein was shown by CD analysis and immunodiffusion assays against anti-hSTf. The ability of the recombinant protein to bind iron was confirmed by urea–PAGE and EPR analysis of the iron-saturated protein revealed the characteristic holo-transferrin spectrum, indicating conservation of both iron-binding sites. The integrity of the unglycosylated recombinant protein indicates that such protein could be a valuable tool not only for structure–function characterisation but also crystallisation assays. In addition, the recombinant transferrin was found to be as effective as native transferrin as a growth factor in cell culture medium.