Overcoming HERG affinity in the discovery of the CCR5 antagonist maraviroc

The discovery of maraviroc 17 is described with particular reference to the generation of high selectivity over affinity for the HERG potassium channel. This was achieved through the use of a high throughput binding assay for the HERG channel that is known to show an excellent correlation with functional effects.     

Assessing Causality in the Association between Child Adiposity and Physical Activity Levels: A Mendelian Randomization Analysis

Background

Cross-sectional studies have shown that objectively measured physical activity is associated with childhood adiposity, and a strong inverse dose–response association with body mass index (BMI) has been found. However, few studies have explored the extent to which this association reflects reverse causation. We aimed to determine whether childhood adiposity causally influences levels of physical activity using genetic variants reliably associated with adiposity to estimate causal effects.

Methods and Findings

The Avon Longitudinal Study of Parents and Children collected data on objectively assessed activity levels of 4,296 children at age 11 y with recorded BMI and genotypic data. We used 32 established genetic correlates of BMI combined in a weighted allelic score as an instrumental variable for adiposity to estimate the causal effect of adiposity on activity.In observational analysis, a 3.3 kg/m² (one standard deviation) higher BMI was associated with 22.3 (95% CI, 17.0, 27.6) movement counts/min less total physical activity (p = 1.6×10⁻¹⁶), 2.6 (2.1, 3.1) min/d less moderate-to-vigorous-intensity activity (p = 3.7×10⁻²⁹), and 3.5 (1.5, 5.5) min/d more sedentary time (p = 5.0×10⁻⁴). In Mendelian randomization analyses, the same difference in BMI was associated with 32.4 (0.9, 63.9) movement counts/min less total physical activity (p = 0.04) (∼5.3% of the mean counts/minute), 2.8 (0.1, 5.5) min/d less moderate-to-vigorous-intensity activity (p = 0.04), and 13.2 (1.3, 25.2) min/d more sedentary time (p = 0.03). There was no strong evidence for a difference between variable estimates from observational estimates. Similar results were obtained using fat mass index. Low power and poor instrumentation of activity limited causal analysis of the influence of physical activity on BMI.

Conclusions

Our results suggest that increased adiposity causes a reduction in physical activity in children and support research into the targeting of BMI in efforts to increase childhood activity levels. Importantly, this does not exclude lower physical activity also leading to increased adiposity, i.e., bidirectional causation. Please see later in the article for the Editors'' Summary     

Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice

Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation.     

Factors influencing detection of density dependence in British birds

If censuses are taken at less than generation intervals, the number of successive censuses in which a given individual is recorded will depend on longevity. Repeatedly recording the same individuals could produce under-estimates of population variability and influence detection of density dependence. We investigated this possibility in 60 time series of abundances of British birds compiled from the Common Birds Census data and then used simple population models to illustrate the proposed mechanism. Species had average lifespans of 2–10 years and were censused annually. Density dependence was detected (at P<0.05) much more frequently in bird species with long lifespans than in those with short lifespans; 75% of the 12 longest-lived species showed density dependence compared to 46% of all species. Population variability measured in annual censuses (termed annual variability) was lower in bird species with longer lifespans. We used discrete time models based on difference equations to demonstrate how longevity influences population variability and detection of density dependence in series of annual censuses. A model in which only first-year birds experienced density dependence was rejected because annual variability was greater and detection of density dependence was less likely when longevity was greater, the opposite of the observed effects of longevity in birds. A model in which all age classes experienced density dependence gave time series with lower annual variability and in which density dependence was detected more frequently when longevity was greater, which is the pattern observed in British birds. Analysis of data from this model showed that the amount of density dependence actually present caused only small changes in annual variability, whereas detection of density dependence from simulated series was strongly influenced by annual variability. The high annual variability of series from short-lived bird species could mask any density dependence that was present. Correcting for trends lead us to detect density dependence in 75% of the 12 longest lived bird species. There is no reason to believe that this rate is not also representative of short-lived species.     

Visualization of Mycobacterium avium in Crohn's tissue by oil-immersion microscopy

Studies seeking Mycobacterium avium subsp. paratuberculosis in Crohn's disease by PCR have generated inconsistent findings. As an alternative, microscopy offers a number of advantages, including direct visualization of organisms in tissue. Experimental infections have demonstrated that M. avium organisms can be seen by both acid-fast staining and species-specific in situ hybridization, but because they are smaller than M. tuberculosis, oil-immersion microscopy (x 1,000 magnification) is needed. We performed a blinded search for M. avium in paraffin-embedded surgical resections from Crohn's and control subjects at two centres. Specimens were coded and subjected to acid-fast staining and ribosomal RNA in situ hybridization for M. avium rRNA. Agreement between these two methods was good (42/52 patients, kappa=0.60) and similar results were observed for patients from two centers. Together, both methods provided positive results in 10 of 17 Crohn's subjects (59%, 95% CI: 36-78), contrasting with only 5 of 35 control subjects (Odds ratio for Crohn's vs. controls=8.6, p=0.002). M. avium organisms had an intracellular localization within inflammatory lesions, but were often observed as lone organisms outside of granulomas. Using two assays in two settings, presence of M. avium organisms was strongly associated with Crohn's disease.     

Purification, crystallisation and X-ray diffraction study of fully functional laccases from two ligninolytic fungi

Laccase isozymes from the white-rot basidiomycete fungi Trametes versicolor and Pycnoporus cinnabarinus were purified to apparent iso-electric homogeneity and crystallised. T. versicolor laccase crystallises in two crystal forms, both with the orthorhombic space group P2(1)2(1)2(1), which diffract to 1.9 and 2.95 A resolution, respectively. The crystals of P. cinnabarinus laccase belong to the monoclinic space group C2 and diffract to at least 2.2 A resolution. All the laccase crystals are suitable for X-ray structure determination and contain a full complement of copper ions.     

The ShdA adhesin binds to the cationic cradle of the fibronectin 13FnIII repeat module: evidence for molecular mimicry of heparin binding

Introduction of Salmonella enterica serotype Typhimurium into food products results from its ability to persist in the intestine of healthy livestock by mechanisms that are poorly understood. The non-fimbrial adhesin ShdA is a fibronectin binding protein required for persistent intestinal carriage of S. Typhimurium. We further investigated the molecular mechanism of ShdA-mediated intestinal persistence by determining the binding-site of this receptor in fibronectin. Analysis of ShdA binding to fibronectin proteolytic fragments and to recombinant fibronectin fusion proteins identified the (13)FnIII repeat module of the Hep-2 domain as the primary binding site for this adhesin. The (13)FnIII repeat module of fibronectin contains a cationic cradle formed by six basic residues (R6, R7, R9, R23, K25 and R54) that is a high affinity heparin-binding site conserved among fibronectin sequences from frogs to man. Binding of ShdA to the (13)FnIII repeat module of fibronectin and to a second extracellular matrix protein, Collagen I, could be inhibited by heparin. Furthermore, binding of ShdA to the Hep-2 domain was sensitive to the ionic buffer strength, suggesting that binding involved ionic interactions. We therefore determined whether amino acid substitutions of basic residues in the cationic cradle of the Hep-2 domain that inhibit heparin binding also abrogate binding of ShdA. Combined substitution of R6S and R7S strongly reduced ShdA binding to (13)FnIII. These data suggest that ShdA binds the Hep-2 domain of fibronectin by a mechanism that may mimic binding of the host polysaccharide heparin.     

The monitoring of bovine pregnancies derived from transfer of in vitro produced embryos

Both an increased rate of embryonic, foetal and perinatal losses, and the occurrence of deviations in foetal and placental development are associated with bovine pregnancies obtained from in vitro produced embryos. This thus requires for a more accurate and frequent monitoring of foetal and maternal functions during pregnancies. Such approaches will enable to establish the period during which these losses and deviations in development occur and to plan possible clinical interventions. This paper reviews some recent data on return rates, late embryonic and foetal losses in recipients after the transfer of either MOET, IVF or nuclear transfer embryos. Special attention is paid to the diagnostic value of measurements of pregnancy specific/associated proteins and progesterone in maternal plasma. Possibilities to measure foetal body sizes, size of placentomes and foetal heart rate by means of transrectal or transabdominal ultrasonography are illustrated with data from the literature and with recent results from our own large field study with MOET, IVP-co-culture and IVP-SOF embryos.     

Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis

The synthetic alkyl-lysophospholipid (ALP), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, is an antitumor agent that acts on cell membranes and can induce apoptosis. We investigated how ALP is taken up by cells, how it affects de novo biosynthesis of phosphatidylcholine (PC), and how critical this is to initiate apoptosis. We compared an ALP-sensitive mouse lymphoma cell line, S49, with an ALP-resistant variant, S49(AR). ALP inhibited PC synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step in S49 cells, but not in S49(AR) cells. Exogenous lysophosphatidylcholine, providing cells with an alternative way (acylation) to generate PC, rescued cells from ALP-induced apoptosis, indicating that continuous rapid PC turnover is essential for cell survival. Apoptosis induced by other stimuli that do not target PC synthesis remained unaffected by lysophosphatidylcholine. Using monensin, low temperature and albumin back-extraction, we demonstrated that ALP is internalized by endocytosis, a process defective in S49(AR) cells. This defect neither involved clathrin-coated pit- nor fluid-phase endocytosis, but depended on lipid rafts, because disruption of these microdomains with methyl-beta-cyclodextrin or filipin (sequestering cholesterol) or bacterial sphingomyelinase reduced uptake of ALP. Furthermore, ALP was found accumulated in isolated rafts and disruption of rafts also prevented the inhibition of PC synthesis and apoptosis induction in S49 cells. In summary, ALP is internalized by raft-dependent endocytosis to inhibit PC synthesis, which triggers apoptosis.     

Involvement of a plastid terminal oxidase in plastoquinone oxidation as evidenced by expression of the Arabidopsis thaliana enzyme in tobacco

Chlororespiration has been defined as a respiratory electron transport chain in interaction with photosynthetic electron transport involving both non-photochemical reduction and oxidation of plastoquinones. Different enzymatic activities, including a plastid-encoded NADH dehydrogenase complex, have been reported to be involved in the non-photochemical reduction of plastoquinones. However, the enzyme responsible for plasquinol oxidation has not yet been clearly identified. In order to determine whether the newly discovered plastid oxidase (PTOX) involved in carotenoid biosynthesis acts as a plastoquinol oxidase in higher plant chloroplasts, the Arabidopsis thaliana PTOX gene (At-PTOX) was expressed in tobacco under the control of a strong constitutive promoter. We showed that At-PTOX is functional in tobacco chloroplasts and strongly accelerates the non-photochemical reoxidation of plastoquinols; this effect was inhibited by propyl gallate, a known inhibitor of PTOX. During the dark to light induction phase of photosynthesis at low irradiances, At-PTOX drives significant electron flow to O(2), thus avoiding over-reduction of plastoquinones, when photo- synthetic CO(2) assimilation was not fully induced. We proposed that PTOX, by modulating the redox state of intersystem electron carriers, may participate in the regulation of cyclic electron flow around photosystem I.