Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis

To better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre-mRNA, which lacks a 3' splice site and 3' exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human B(act) complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to B(act) and B(act) to C transitions, and comparisons with the Saccharomyces cerevisiae B(act) complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to B(act) and B(act) to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human B(act) complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae B(act) complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.     

Morphological and physiological traits in the success of the invasive plant <Emphasis Type="Italic">Lespedeza cuneata</Emphasis>

To better understand the strategies and mechanisms of invading plants in tallgrass prairie, physiological and morphological characteristics of the invasive Lespedeza cuneata were compared to the dominant and abundant natives Ambrosia psilostachya and Andropogon gerardii. Gas exchange, chlorophyll fluorescence, plant water status, and total and specific leaf area were quantified in the field for each species both throughout daily sampling periods and across the growing season. Total and specific leaf area (cm² g⁻¹ of leaves) exceeded that of native species and may allow L. cuneata to successfully establish and dominate in tallgrass prairie, aiding in both resource acquisition and competitive exclusion. Gas exchange traits (e.g. net photosynthesis, stomatal conductance, and water use efficiency) of L. cuneata did not exceed other species, but remained constant throughout the daily sampling periods. The daily consistency of net photosynthesis and other gas exchange traits for L. cuneata reveal characteristics of stress tolerance. The combination of these characteristics and strategies may assist in the invasion of L. cuneata and also provide insight into general mechanisms responsible for successful invasions into tallgrass prairie.     

The phenology mismatch hypothesis: are declines of migrant birds linked to uneven global climate change?

1.  Migrant bird populations are declining and have been linked to anthropogenic climate change. The phenology mismatch hypothesis predicts that migrant birds, which experience a greater rate of warming in their breeding grounds compared to their wintering grounds, are more likely to be in decline, because their migration will occur later and they may then miss the early stages of the breeding season. Population trends will also be negatively correlated with distance, because the chances of phenology mismatch increase with number of staging sites.
2.  Population trends from the Palaearctic (1990–2000) and Nearctic (1980–2006) were collated for 193 spatially separate migrant bird populations, along with temperature trends for the wintering and breeding areas. An index of phenology mismatch was calculated as the difference between wintering and breeding temperature trends.
3.  In the Nearctic, phenology mismatch was correlated with population declines as predicted, but in the Palaearctic, distance was more important. This suggests that differential global climate change may be responsible for contributing to some migrant species' declines, but its effects may be more important in the Nearctic.
4.  Differences in geography and so average migration distance, migrant species composition and history of anthropogenic change in the two areas may account for the differences in the strength of the importance of phenology mismatch on migrant declines in the Nearctic and Palaearctic.     

Genetic clustering methods reveal bull trout (<Emphasis Type="Italic">Salvelinus confluentus</Emphasis>) fine-scale population structure as a spatially nested hierarchy

Many conservation genetics studies in fishes define populations based on capture location. In salmonid fishes, this traditional a priori designation is made by spawning stream, with subsequent post hoc approaches used to define units of conservation. In this study of bull trout from southwestern Alberta, we provide evidence that a model-based Bayesian genetic clustering method may provide a more parsimonious alternative to designating population structure and units of conservation in comparison to traditional methods. The clustering method captured a hierarchical model of population structure, in which seven local populations were nested within three genetic archipelagos. This was in contrast to using simple F _ST based approaches between thirteen a priori designated populations, which found significant differences for nearly every pairwise comparison. In addition, assignment tests results from Bayesian clustering revealed that movement may be common between sampling locations. These clustering methods are easy to use, intuitive and provide substantial information on populations of fish; this study provides an example of their utility for local fisheries management and conservation.     

BK channels mediate cholinergic inhibition of high frequency cochlear hair cells

Background

Outer hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K⁺ channels that control the membrane potential and also ligand-gated K⁺ channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea.

Methodology/Principal

Findings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K⁺ currents.

Conclusions/Significance

Our findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.     

Potential Role for IL-2 ELISpot in Differentiating Recent and Remote Infection in Tuberculosis Contact Tracing

Interferon (IFN)-γ release assays (IGRA) have improved tuberculosis contact tracing, but discrimination of recent from remote Mycobacterium tuberculosis contacts is not possible by IGRA alone. We present results of a tuberculosis contact investigation with a new early-secretory-antigenic-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 specific interleukin (IL)-2 ELISpot in addition to ESAT-6 and CFP-10 specific IFN-γ ELISpot and tuberculin skin testing (TST). Results of the TST, IFN-γ ELISpot and IL-2 ELISpot were positive in 6/172 (3.4%), 7/167 (4.2%) and 6/196 (3.1%) of contacts, respectively. Close contact (≥100 hours) to the index case increased the risk of positive results in the IFN-γ ELISpot, TST, and IL-2 ELISpot by 40.8, 19.3, and 2.5 times, respectively. Individuals with a positive IFN-γ ELISpot/negative IL-2 ELISpot result had a median (IQR) duration of index case exposure of 568 hours (133_1000) compared to individuals with a positive IFN-γ ELISpot/positive IL-2 ELISpot result (median = 24 hours; 20_130; p-value = 0.047). Combination of a M. tuberculosis specific IFN-γ ELISpot with a M. tuberculosis specific IL-2 ELISpot significantly improved the identification of individuals with the highest risk of recent M. tuberculosis infection and is a promising method that should be explored to target tuberculosis preventive chemotherapy.     

Mass-dependent predation risk as a mechanism for house sparrow declines?

House sparrow (Passer domesticus) numbers have declined rapidly in both rural and urban habitats across Western Europe over the last 30 years, leading to their inclusion on the UK conservation red list. The decline in farmland has been linked to a reduction in winter survival caused by reduced food supply. This reduction in food supply is associated with agricultural intensification that has led to the loss of seed-rich winter stubble and access to spilt grain. However, urban house sparrows have also declined, suggesting that reduced food supply in farmland is not the sole reason for the decline. Here, we show that changes in house sparrow mass and thus fat reserves are not regulated to minimize starvation risk, as would be expected if limited winter food were the only cause of population decline. Instead, the species appears to be responding to mass-dependent predation risk, with starvation risk and predation risk traded-off such that house sparrows may be particularly vulnerable to environmental change that reduces the predictability of the food supply.     

Multidimensional proteomics of human serum using parallel chromatography of native constituents and microplate technology

A versatile, multidimensional, and non-denaturing proteome separation procedure using microplate technology is presented, yielding a digitized image of proteome composition. In the first dimension, the sample under study is separated into 96 fractions by size exclusion chromatography (SEC). In the second dimension, the fractions of the first dimension are transferred by the liquid-handling device CyBi-Well (CyBio AG, Jena, Germany) to 96 parallel anion exchange chromatography columns. In this way the proteins are conserved in their native states and are distributed in 2400 liquid fractions with high recovery rates and sufficient reproducibility. The resulting fractions are subjected to protein quantitation and identification. Spectrophotometrical and immunological methods and enzyme activity measurements are used for quantitation. To identify proteins, the fractions are subjected to MALDI-MS, and their tryptic digests to both MALDI- and LC-ESI-MS/MS. All preparation steps except the first are applied in parallel to sets of multiples of 96 samples. The procedure may be refined by adding more separation steps and may be adapted to various protein amounts and to various proteomes. Moreover, the method offers the opportunity to investigate functional protein complexes. The method was applied to separate the normal human serum proteome. Within 255 fractions exhibiting the highest protein concentrations, 742 proteins were identified by LC-ESI-MS/MS peptide sequence tags.     

Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP

Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.