Polypyrimidine tract binding protein and poly r(C) binding protein 1 interact with the BAG-1 IRES and stimulate its activity in vitro and in vivo

The 5′-untranslated region of Bag-1 mRNA contains an internal ribosome entry segment (IRES) and the translation of Bag-1 protein can be initiated by both cap-dependent and cap-independent mechanisms. In general, cellular IRESs require non-canonical trans-acting factors for their activity, however, very few of the proteins that act on cellular IRESs have been identified. Proteins that interact with viral IRESs have also been shown to stimulate the activity of cellular IRESs and therefore the ability of a range of known viral trans-acting factors to stimulate the Bag-1 IRES was tested. Two proteins, poly r(C) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB), were found to increase the activity of the Bag-1 IRES in vitro and in vivo. The regions of the Bag-1 IRES RNA to which they bind have been determined, and it was shown that PCBP1 binds to a short 66 nt section of RNA, whilst PTB interacts with a number of sites over a larger area. The minimum section of the RNA that still retained activity was determined and both PCBP1 and PTB interacted with this region suggesting that these proteins are essential for Bag-1 IRES function.     

Rebecca Worthylake: the flooding of science

Starting up a brand new lab is difficult enough. But when a hurricane soon forces you thousands of miles from your new research home, a whole new layer of complications arises, as Rebecca Worthylake recently found.     

Assessing structural variation in a personal genome—towards a human reference diploid genome

Background

Characterizing large genomic variants is essential to expanding the research and clinical applications of genome sequencing. While multiple data types and methods are available to detect these structural variants (SVs), they remain less characterized than smaller variants because of SV diversity, complexity, and size. These challenges are exacerbated by the experimental and computational demands of SV analysis. Here, we characterize the SV content of a personal genome with Parliament, a publicly available consensus SV-calling infrastructure that merges multiple data types and SV detection methods.

Results

We demonstrate Parliament’s efficacy via integrated analyses of data from whole-genome array comparative genomic hybridization, short-read next-generation sequencing, long-read (Pacific BioSciences RSII), long-insert (Illumina Nextera), and whole-genome architecture (BioNano Irys) data from the personal genome of a single subject (HS1011). From this genome, Parliament identified 31,007 genomic loci between 100 bp and 1 Mbp that are inconsistent with the hg19 reference assembly. Of these loci, 9,777 are supported as putative SVs by hybrid local assembly, long-read PacBio data, or multi-source heuristics. These SVs span 59 Mbp of the reference genome (1.8%) and include 3,801 events identified only with long-read data. The HS1011 data and complete Parliament infrastructure, including a BAM-to-SV workflow, are available on the cloud-based service DNAnexus.

Conclusions

HS1011 SV analysis reveals the limits and advantages of multiple sequencing technologies, specifically the impact of long-read SV discovery. With the full Parliament infrastructure, the HS1011 data constitute a public resource for novel SV discovery, software calibration, and personal genome structural variation analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1479-3) contains supplementary material, which is available to authorized users.     

Selective enrichment,isolation and molecular detection of <Emphasis Type="Italic">Salinibacter</Emphasis> and related extremely halophilic <Emphasis Type="Italic">Bacteria</Emphasis> from hypersaline environments

Salinibacter is a genus of red, extremely halophilic Bacteria. Thus far the genus is represented by a single species, Salinibacter ruber, strains of which have been isolated from saltern crystallizer ponds in Spain and on the Balearic Islands. Both with respect to its growth conditions and its physiology, Salinibacter resembles the halophilic Archaea of the order Halobacteriales. We have designed selective enrichment and isolation techniques to obtain Salinibacter and related red extremely halophilic Bacteria from different hypersaline environments, based on their resistance to anisomycin and bacitracin, two antibiotics that are potent inhibitors of the halophilic Archaea. Using direct plating on media containing bacitracin, we found Salinibacter-like organisms in numbers between 1.4×10³ and 1.4×10⁶ml⁻¹ in brines collected from the crystallizer ponds of the salterns in Eilat, Israel, being equivalent to 1.8–18% of the total colony counts obtained on identical media without bacitracin. A number of strains from Eilat were subjected to a preliminary characterization, and they proved similar to the type strain of S. ruber. We also report here the isolation and molecular detection of Salinibacter-like organisms from an evaporite crust on the bottom of salt pools at the Badwater site in Death Valley, CA. These isolates and environmental 16S rRNA gene sequences differ in a number of properties from S. ruber, and they may represent a new species of Salinibacter or a new related genus. Guest Editor: John M. Melack Saline Waters and their Biota     

The Views and Experiences of Smokers Who Quit Smoking Unassisted. A Systematic Review of the Qualitative Evidence

BackgroundUnassisted cessation – quitting without pharmacological or professional support – is an enduring phenomenon. Unassisted cessation persists even in nations advanced in tobacco control where cessation assistance such as nicotine replacement therapy, the stop-smoking medications bupropion and varenicline, and behavioural assistance are readily available. We review the qualitative literature on the views and experiences of smokers who quit unassisted.MethodWe systematically searched for peer-reviewed qualitative studies reporting on smokers who quit unassisted. We identified 11 studies and used a technique based on Thomas and Harden’s method of thematic synthesis to discern key themes relating to unassisted cessation, and to then group related themes into overarching concepts.FindingsThe three concepts identified as important to smokers who quit unassisted were: motivation, willpower and commitment. Motivation, although widely reported, had only one clear meaning, that is ‘the reason for quitting’. Willpower was perceived to be a method of quitting, a strategy to counteract cravings or urges, or a personal quality or trait fundamental to quitting success. Commitment was equated to seriousness or resoluteness, was perceived as key to successful quitting, and was often used to distinguish earlier failed quit attempts from the final successful quit attempt. Commitment had different dimensions. It appeared that commitment could be tentative or provisional, and also cumulative, that is, commitment could be built upon as the quit attempt progressed.ConclusionA better understanding of what motivation, willpower and commitment mean from the smoker’s perspective may provide new insights and direction for smoking cessation research and practice.     

Comparative Evaluation of Nephrotoxicity and Management by Macrophages of Intravenous Pharmaceutical Iron Formulations

BackgroundThere is a significant clinical need for effective treatment of iron deficiency. A number of compounds that can be administered intravenously have been developed. This study examines how the compounds are handled by macrophages and their relative potential to provoke oxidative stress.MethodsHuman kidney (HK-2) cells, rat peritoneal macrophages and renal cortical homogenates were exposed to pharmaceutical iron preparations. Analyses were performed for indices of oxidative stress and cell integrity. In addition, in macrophages, iron uptake and release and cytokine secretion was monitored.ResultsHK-2 cell viability was decreased by iron isomaltoside and ferumoxytol and all compounds induced lipid peroxidation. In the renal cortical homogenates, lipid peroxidation occurred at lowest concentrations with ferric carboxymaltose, iron dextran, iron sucrose and sodium ferric gluconate. In the macrophages, iron sucrose caused loss of cell viability. Iron uptake was highest for ferumoxytol and iron isomaltoside and lowest for iron sucrose and sodium ferric gluconate. Iron was released as secretion of ferritin or as ferrous iron via ferroportin. The latter was blocked by hepcidin. Exposure to ferric carboxymaltose and iron dextran resulted in release of tumor necrosis factor α.ConclusionsExposure to iron compounds increased cell stress but was tissue and dose dependent. There was a clear difference in the handling of iron from the different compounds by macrophages that suggests in vivo responses may differ.     

High Throughput Screening of Signal Transduction Mutants With Luciferase Imaging

A detailed procedure for high throughput genetic screening of hormone and environmental stress signal transduction mutants of Arabidopsis thaliana is described. The screen was carried out with mutagenized plants expressing the firefly luciferase reporter under control of a cold, osmotic stress, and absciscic acid responsive promoter. A thermoelectrically cooled CCD camera was used to detect luminescence emitted by the plants in response to stresses or ABA. Advantages of the screening procedure include high throughput, capability to identify low as well as high expression mutants and employment of a highly sensitive but affordable imaging system and software. This procedure can be used to study complex signal transduction networks in higher plants.