In-cell click labelling of small molecules to determine subcellular localisation

Small molecule fluorometric boron dipyrromethene probes were developed to bind hepatitis C virus-encoded NS5A protein and aid subcellular distribution studies. These molecules did not co-locate with NS5A, therefore alternative ‘silent’ azide reporters were used to obtain a more relevant picture of their distribution. Following pre-incubation with replicon cells, click chemistry was used to append a fluorophore to the azide that confirmed the co-localisation of the small molecule with the NS5A protein, thus providing greater insight into the antiviral mode of action of this chemotype.

Electronic supplementary material

The online version of this article (doi:10.1007/s12154-010-0047-1) contains supplementary material, which is available to authorized users.     

Genomewide association scan of suicidal thoughts and behaviour in major depression

Background

Suicidal behaviour can be conceptualised as a continuum from suicidal ideation, to suicidal attempts to completed suicide. In this study we identify genes contributing to suicidal behaviour in the depression study RADIANT.

Methodology/Principal Findings

A quantitative suicidality score was composed of two items from the SCAN interview. In addition, the 251 depression cases with a history of serious suicide attempts were classified to form a discrete trait. The quantitative trait was correlated with younger onset of depression and number of episodes of depression, but not with gender. A genome-wide association study of 2,023 depression cases was performed to identify genes that may contribute to suicidal behaviour. Two Munich depression studies were used as replication cohorts to test the most strongly associated SNPs. No SNP was associated at genome-wide significance level. For the quantitative trait, evidence of association was detected at GFRA1, a receptor for the neurotrophin GDRA (p = 2e-06). For the discrete trait of suicide attempt, SNPs in KIAA1244 and RGS18 attained p-values of <5e-6. None of these SNPs showed evidence for replication in the additional cohorts tested. Candidate gene analysis provided some support for a polymorphism in NTRK2, which was previously associated with suicidality.

Conclusions/Significance

This study provides a genome-wide assessment of possible genetic contribution to suicidal behaviour in depression but indicates a genetic architecture of multiple genes with small effects. Large cohorts will be required to dissect this further.     

Solving the Problem: Genome Annotation Standards before the Data Deluge

The promise of genome sequencing was that the vast undiscovered country would be mapped out by comparison of the multitude of sequences available and would aid researchers in deciphering the role of each gene in every organism. Researchers recognize that there is a need for high quality data. However, different annotation procedures, numerous databases, and a diminishing percentage of experimentally determined gene functions have resulted in a spectrum of annotation quality. NCBI in collaboration with sequencing centers, archival databases, and researchers, has developed the first international annotation standards, a fundamental step in ensuring that high quality complete prokaryotic genomes are available as gold standard references. Highlights include the development of annotation assessment tools, community acceptance of protein naming standards, comparison of annotation resources to provide consistent annotation, and improved tracking of the evidence used to generate a particular annotation. The development of a set of minimal standards, including the requirement for annotated complete prokaryotic genomes to contain a full set of ribosomal RNAs, transfer RNAs, and proteins encoding core conserved functions, is an historic milestone. The use of these standards in existing genomes and future submissions will increase the quality of databases, enabling researchers to make accurate biological discoveries.     

Despotism and risk of infanticide influence grizzly bear den-site selection

Given documented social dominance and intraspecific predation in bear populations, the ideal despotic distribution model and sex hypothesis of sexual segregation predict adult female grizzly bears (Ursus arctos) will avoid areas occupied by adult males to reduce risk of infanticide. Under ideal despotic distribution, juveniles should similarly avoid adult males to reduce predation risk. Den-site selection and use is an important component of grizzly bear ecology and may be influenced by multiple factors, including risk from conspecifics. To test the role of predation risk and the sex hypothesis of sexual segregation, we compared adult female (n = 142), adult male (n = 36), and juvenile (n = 35) den locations in Denali National Park and Preserve, Alaska, USA. We measured elevation, aspect, slope, and dominant land cover for each den site, and used maximum entropy modeling to determine which variables best predicted den sites. We identified the global model as the best-fitting model for adult female (area under curve (AUC) = 0.926) and elevation as the best predictive variable for adult male (AUC = 0.880) den sites. The model containing land cover and elevation best-predicted juvenile (AUC = 0.841) den sites. Adult females spatially segregated from adult males, with dens characterized by higher elevations ( = 1,412 m, SE = 52) and steeper slopes ( = 21.9°, SE = 1.1) than adult male (elevation:  = 1,209 m, SE = 76; slope:  = 15.6°, SE = 1.9) den sites. Juveniles used a broad range of landscape attributes but did not avoid adult male denning areas. Observed spatial segregation by adult females supports the sex hypothesis of sexual segregation and we suggest is a mechanism to reduce risk of infanticide. Den site selection of adult males is likely related to distribution of food resources during spring.     

RECENT ECOLOGICAL SELECTION ON REGULATORY DIVERGENCE IS SHAPING CLINAL VARIATION IN SENECIO ON MOUNT ETNA

The hybrid zone on Mount Etna (Sicily) between Senecio aethnensis and Senecio chrysanthemifolius (two morphologically and physiologically distinct species) is a classic example of an altitudinal cline. Hybridization at intermediate altitudes and gradients in phenotypic and life‐history traits occur along altitudinal transects of the volcano. The cline is considered to be a good example of ecological selection with species differences arising by divergent selection opposing gene flow. However, the possibility that the cline formed from recent secondary contact following an allopatric phase is difficult to exclude. We demonstrate a recent split between S. aethnensis and S. chrysanthemifolius (as recent as ～32,000 years ago) and sufficient gene flow (2Nm > 1) to have prevented divergence (implicating a role for diversifying selection in the maintenance of the cline). Differentially expressed genes between S. aethnensis and S. chrysanthemifolius exhibit significantly higher genetic divergence relative to “expression invariant” controls, suggesting that species differences may in part be mediated by divergent selection on differentially expressed genes involved with altitude‐related adaptation. The recent split time and the absence of fixed differences between these two ecologically distinct species suggest the rapid evolution to an altitudinal cline involving selection on both sequence and expression variation.     

Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages

Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/β and not PKCδ, -ϵ, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages.     

Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca²⁺ permeant cation channels (MscCa) and large conductance (200 pS) Ca²⁺-sensitive K⁺ (BK_Ca2+) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK_Ca2+ channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca²⁺-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca²⁺] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.