Demonstration of the Presence of the "Deleted" MIR122 Gene in HepG2 Cells

MicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells

Use of QSARs in international decision-making frameworks to predict ecologic effects and environmental fate of chemical substances

This article is a review of the use, by regulatory agencies and authorities, of quantitative structure–activity relationships (QSARs) to predict ecologic effects and environmental fate of chemicals. For many years, the U.S. Environmental Protection Agency has been the most prominent regulatory agency using QSARs to predict the ecologic effects and environmental fate of chemicals. However, as increasing numbers of standard QSAR methods are developed and validated to predict ecologic effects and environmental fate of chemicals, it is anticipated that more regulatory agencies and authorities will find them to be acceptable alternatives to chemical testing

Where do we go from here? An assessment of navigation performance using a compass versus a GPS unit

The Global Positioning System (GPS) looks set to replace the traditional map and compass for navigation tasks in military and civil domains. However, we may ask whether GPS has a real performance advantage over traditional methods. We present an exploratory study using a waypoint plotting task to compare the standard magnetic compass against a military GPS unit, for both expert and non-expert navigators. Whilst performance times were generally longer in setting up the GPS unit, once navigation was underway the GPS was more efficient than the compass. For mediumto long-term missions, this means that GPS could offer significant performance benefits, although the compass remains superior for shorter missions. Notwithstanding the performance times, significantly more errors, and more serious errors, occurred when using the compass. Overall, then, the GPS offers some clear advantages, especially for non-expert users. Nonetheless, concerns over the development of cognitive maps remain when using GPS technologies

The relative importance of large problems far away versus small problems closer to home: insights into limiting the spread of antimicrobial resistance in England

Background: To combat the spread of antimicrobial resistance (AMR), hospitals are advised to screen high-risk patients for carriage of antibiotic-resistant bacteria on admission. This often includes patients previously admitted to hospitals with a high AMR prevalence. However, the ability of such a strategy to identify introductions (and hence prevent onward transmission) is unclear, as it depends on AMR prevalence in each hospital, the number of patients moving between hospitals, and the number of hospitals considered ‘high risk’. / Methods: We tracked patient movements using data from the National Health Service of England Hospital Episode Statistics and estimated differences in regional AMR prevalences using, as an exemplar, data collected through the national reference laboratory service of Public Health England on carbapenemase-producing Enterobacteriaceae (CPE) from 2008 to 2014. Combining these datasets, we calculated expected CPE introductions into hospitals from across the hospital network to assess the effectiveness of admission screening based on defining high-prevalence hospitals as high risk. / Results: Based on numbers of exchanged patients, the English hospital network can be divided into 14 referral regions. England saw a sharp increase in numbers of CPE isolates referred to the national reference laboratory over 7 years, from 26 isolates in 2008 to 1649 in 2014. Large regional differences in numbers of confirmed CPE isolates overlapped with regional structuring of patient movements between hospitals. However, despite these large differences in prevalence between regions, we estimated that hospitals received only a small proportion (1.8%) of CPE-colonised patients from hospitals outside their own region, which decreased over time. / Conclusions: In contrast to the focus on import screening based on assigning a few hospitals as ‘high risk’, patient transfers between hospitals with small AMR problems in the same region often pose a larger absolute threat than patient transfers from hospitals in other regions with large problems, even if the prevalence in other regions is orders of magnitude higher. Because the difference in numbers of exchanged patients, between and within regions, was mostly larger than the difference in CPE prevalence, it would be more effective for hospitals to focus on their own populations or region to inform control efforts rather than focussing on problems elsewhere

Haemodynamic consequences of targeted single- and dual-site right ventricular pacing in adults with congenital heart disease undergoing surgical pulmonary valve replacement

Aims The purpose of this study was to create an epicardial electroanatomic map of the right ventricle (RV) and then apply post-operative-targeted single- and dual-site RV temporary pacing with measurement of haemodynamic parameters. Cardiac resynchronization therapy is an established treatment for symptomatic left ventricular (LV) dysfunction. In congenital heart disease, RV dysfunction is a common cause of morbidity—little is known regarding the potential benefits of CRT in this setting. Methods and results Sixteen adults (age = 32 ± 8 years; 6 M, 10 F) with right bundle branch block (RBBB) and repaired tetralogy of Fallot (n = 8) or corrected congenital pulmonary stenosis (n = 8) undergoing surgical pulmonary valve replacement (PVR) for pulmonary regurgitation underwent epicardial RV mapping and haemodynamic assessment of random pacing configurations including the site of latest RV activation. The pre-operative pulmonary regurgitant fraction was 49 ± 10%; mean LV end-diastolic volume (EDV) 85 ± 19 mL/min/m2 and RVEDV 183 ± 89 mL/min/m2 on cardiac magnetic resonance imaging. The mean pre-operative QRS duration is 136 ± 26 ms. The commonest site of latest activation was the RV free wall and DDD pacing here alone or combined with RV apical pacing resulted in significant increases in cardiac output (CO) vs. AAI pacing (P < 0.01 all measures). DDDRV alternative site pacing significantly improved CO by 16% vs. AAI (P = 0.018), and 8.5% vs. DDDRV apical pacing (P = 0.02). Conclusion Single-site RV pacing targeted to the region of latest activation in patients with RBBB undergoing PVR induces acute improvements in haemodynamics and supports the concept of ‘RV CRT’. Targeted pacing in such patients has therapeutic potential both post-operatively and in the long term

R-process enrichment from a single event in an ancient dwarf galaxy

Elements heavier than zinc are synthesized through the (r)apid and (s)low neutron-capture processes. The main site of production of the r-process elements (such as europium) has been debated for nearly 60 years. Initial studies of chemical abundance trends in old Milky Way halo stars suggested continual r-process production, in sites like core-collapse supernovae. But evidence from the local Universe favors r-process production mainly during rare events, such as neutron star mergers. The appearance of a europium abundance plateau in some dwarf spheroidal galaxies has been suggested as evidence for rare r-process enrichment in the early Universe, but only under the assumption of no gas accretion into the dwarf galaxies. Cosmologically motivated gas accretion favors continual r-process enrichment in these systems. Furthermore, the universal r-process pattern has not been cleanly identified in dwarf spheroidals. The smaller, chemically simpler, and more ancient ultra-faint dwarf galaxies assembled shortly after the first stars formed, and are ideal systems with which to study nucleosynthesis events such as the r-process. Reticulum II is one such galaxy. The abundances of non-neutron-capture elements in this galaxy (and others like it) are similar to those of other old stars. Here, we report that seven of nine stars in Reticulum II observed with high-resolution spectroscopy show strong enhancements in heavy neutron-capture elements, with abundances that follow the universal r-process pattern above barium. The enhancement in this "r-process galaxy" is 2-3 orders of magnitude higher than that detected in any other ultra-faint dwarf galaxy. This implies that a single rare event produced the r-process material in Reticulum II. The r-process yield and event rate are incompatible with ordinary core-collapse supernovae, but consistent with other possible sites, such as neutron star mergers.Comment: Published in Nature, 21 Mar 2016: http://dx.doi.org/10.1038/nature1742

Comparative prebiotic activity of mixtures of cereal grain polysaccharides

The main components of the non-starch polysaccharide (NSP) fraction of wheat flour are arabinoxylan (AX) and β-glucan. These are also present in other cereal grains, but their proportions vary with AX being the major component in wheat and rye and β-glucan in barley and oats. Therefore, it was hypothesised that these NSPs could act synergistically when fermented in vitro at the ratios present in the major foods consumed, resulting in increased prebiotic activity. AX and β-glucan were therefore tested in in vitro fermentation studies to assess their prebiotic activity when used individually and/or in different ratios. Short-chain fatty-acids (SCFAs) produced from in vitro fermentation were measured using HPLC and bacterial populations were measured using flow cytometry with fluorescence in situ hybridisation (Flow-FISH). Fermentation of AX alone resulted in a significant bifidogenic activity and increased concentrations of SCFAs, mainly acetate, after 8-24 h of fermentation, however β-glucan alone did not show prebiotic activity. The greatest prebiotic activity, based on concentration of total SCFAs and increases in total bacteria as well as beneficial Bifidobacterium and Clostridium coccoides/Eubacterium groups, was observed when AX and β-glucan were combined at a 3:1 ratio, which corresponds to their ratios in wheat flour which is major source of cereal fibre in the diet. This indicates that the population of bacteria in the human GI tract may be modulated by the composition of the fibre in the diet, to maximise the prebiotic potential

Identification of hip fracture patients from radiographs using Fourier analysis of the trabecular structure: a cross-sectional study

Peer reviewedPublisher PD

Structural insights into Clostridium perfringens delta toxin pore formation

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins