Solution structure of mu-conotoxin PIIIA,a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels

Mu-conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of mu-conotoxins PIIIA and PIIIA-(2-22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2-22), and mu-conotoxins GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. (1)H NMR studies found that PIIIA adopts two conformations in solution due to cis/trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of mu-conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective mu-conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by mu-conotoxins.     

Competition between SsrA tagging and translational termination at weak stop codons in Escherichia coli

SsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA-tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C-terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging.     

A unified view of the DNA-damage checkpoint

Recent investigation of the DNA-damage checkpoint in several organisms has highlighted the conservation of this pathway. The checkpoint's signal transduction pathway consists of four conserved classes of molecules: two large protein kinases having homology to phosphatidylinositol 3-kinases, three "sensor" proteins with homology to proliferating cell nuclear antigen, two serine/threonine (S/T) kinases, and two adaptors for the S/T kinases. This review compares the role of these four classes of checkpoint proteins in humans and model organisms.     

Asymmetric loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment

Spindle alignment is the process in which the two spindle poles are directed toward preselected and opposite cell ends. In budding yeast, the APC-related molecule Kar9 is required for proper alignment of the spindle with the mother-bud axis. We find that Kar9 localizes to the prospective daughter cell spindle pole. Kar9 is transferred from the pole to cytoplasmic microtubules, which are then guided in a myosin-dependent manner to the bud. Clb4/Cdc28 kinase phosphorylates Kar9 and accumulates on the pole destined to the mother cell. Mutations that block phosphorylation at Cdc28 consensus sites result in localization of Kar9 to both poles and target them both to the bud. Thus, Clb4/Cdc28 prevents Kar9 loading on the mother bound pole. In turn, asymmetric distribution of Kar9 ensures that only one pole orients toward the bud. Our results indicate that Cdk1-dependent spindle asymmetry ensures proper alignment of the mitotic spindle with the cell division axis.     

The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.     

The Contribution of Occult Precipitation to Nutrient Deposition on the West Coast of South Africa

The Strandveld mediterranean-ecosystem of the west coast of South Africa supports floristically diverse vegetation growing on mostly nutrient-poor aeolian sands and extending from the Atlantic Ocean tens of kilometers inland. The cold Benguela current upwelling interacts with warm onshore southerly winds in summer causing coastal fogs in this region. We hypothesized that fog and other forms of occult precipitation contribute moisture and nutrients to the vegetation. We measured occult precipitation over one year along a transect running inland in the direction of the prevailing wind and compared the nutrient concentrations with those in rainwater. Occult deposition rates of P, N, K, Mg, Ca, Na, Al and Fe all decreased with distance from the ocean. Furthermore, ratios of cations to Na were similar to those of seawater, suggesting a marine origin for these. In contrast, N and P ratios in occult precipitation were higher than in seawater. We speculate that this is due to marine foam contributing to occult precipitation. Nutrient loss in leaf litter from dominant shrub species was measured to indicate nutrient demand. We estimated that occult precipitation could meet the demand of the dominant shrubby species for annual N, P, K and Ca. Of these species, those with small leaves intercepted more moisture and nutrients than those with larger leaves and could take up foliar deposits of glycine, NO₃-, NH₄⁺ and Li (as tracer for K) through leaf surfaces. We conclude that occult deposition together with rainfall deposition are potentially important nutrient and moisture sources for the Strandveld vegetation that contribute to this vegetation being floristically distinct from neighbouring nutrient-poor Fynbos vegetation.     

“I Spent a Full Month Bleeding,I Thought I Was Going to Die…” A Qualitative Study of Experiences of Women Using Modern Contraception in Wakiso District,Uganda

Introduction

There is high unmet need for family planning (FP) in Uganda as well as high contraceptive discontinuation rates. These contribute to the high fertility rates that in part are due to unplanned pregnancies. There are gaps in knowledge about experiences that couples go through while using contraceptives in their lives. This study explored women’s experiences during the course of their contraceptive use.

Methods

We conducted a qualitative study involving 30 women who had used modern contraception for at least one year in Wakiso district, central Uganda. We used in-depth interviews to obtain their personal accounts. Index women were approached through health officers at four health centres in the district. All ethical approvals and informed consent were obtained. We used conventional content analysis; identifying codes through open coding, on which basis categories were developed and grouped into overarching themes.

Results

Women’s accounts were summarised in the following themes: negative experiences with modern contraceptive use, motivation to continue using FP in spite of these negative experiences, the role of influential people, and discontinuation of use. Negative accounts dominated the experiences of most women but they expressed strong desire to continue using modern contraception even amidst all challenges. Health workers emerged as the most influential people that played a vital role in women’s decisions.

Conclusion

Varied negative experiences with modern contraception and misperceptions exist amidst a determination to continue use. Partner engagement, health service strengthening to improve side effects management and health worker skills, and engaging older women that have successfully used contraception as community champions, are potential strategies to support women’s contraceptive decisions.     

Endogenous Murine BST-2/Tetherin Is Not a Major Restriction Factor of Influenza A Virus Infection

BST-2 (tetherin, CD317, HM1.24) restricts virus growth by tethering enveloped viruses to the cell surface. The role of BST-2 during influenza A virus infection (IAV) is controversial. Here, we assessed the capacity of endogenous BST-2 to restrict IAV in primary murine cells. IAV infection increased BST-2 surface expression by primary macrophages, but not alveolar epithelial cells (AEC). BST-2-deficient AEC and macrophages displayed no difference in susceptibility to IAV infection relative to wild type cells. Furthermore, BST-2 played little role in infectious IAV release from either AEC or macrophages. To examine BST-2 during IAV infection in vivo, we infected BST-2-deficient mice. No difference in weight loss or in viral loads in the lungs and/or nasal tissues were detected between BST-2-deficient and wild type animals. This study rules out a major role for endogenous BST-2 in modulating IAV in the mouse model of infection.     

Assessment of the Incremental Benefit of Computer-Aided Detection (CAD) for Interpretation of CT Colonography by Experienced and Inexperienced Readers

Objectives

To quantify the incremental benefit of computer-assisted-detection (CAD) for polyps, for inexperienced readers versus experienced readers of CT colonography.

Methods

10 inexperienced and 16 experienced radiologists interpreted 102 colonography studies unassisted and with CAD utilised in a concurrent paradigm. They indicated any polyps detected on a study sheet. Readers’ interpretations were compared against a ground-truth reference standard: 46 studies were normal and 56 had at least one polyp (132 polyps in total). The primary study outcome was the difference in CAD net benefit (a combination of change in sensitivity and change in specificity with CAD, weighted towards sensitivity) for detection of patients with polyps.

Results

Inexperienced readers’ per-patient sensitivity rose from 39.1% to 53.2% with CAD and specificity fell from 94.1% to 88.0%, both statistically significant. Experienced readers’ sensitivity rose from 57.5% to 62.1% and specificity fell from 91.0% to 88.3%, both non-significant. Net benefit with CAD assistance was significant for inexperienced readers but not for experienced readers: 11.2% (95%CI 3.1% to 18.9%) versus 3.2% (95%CI -1.9% to 8.3%) respectively.

Conclusions

Concurrent CAD resulted in a significant net benefit when used by inexperienced readers to identify patients with polyps by CT colonography. The net benefit was nearly four times the magnitude of that observed for experienced readers. Experienced readers did not benefit significantly from concurrent CAD.