Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 30 end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode

The mechanism of the Einstellung (set) effect: A pervasive source of cognitive bias

Copyright @ The Authors 2010The eye movements of expert players trying to solve a chess problem show that the first idea that comes to mind directs attention towards sources of information consistent with itself and away from inconsistent information. This bias continues unconsciously even when the player believes he is looking for alternatives. The result is that alternatives to the first idea are ignored. This mechanism for biasing attention ensures a speedy response in familiar situations but it can lead to errors when the first thought that comes to mind is not appropriate. We propose that this mechanism is the source of many cognitive biases from phenomena in problem solving and reasoning, to perceptual errors and failures in memory

A comparison of high-throughput techniques for assaying circadian rhythms in plants

Over the last two decades, the development of high-throughput techniques has enabled us to probe the plant circadian clock, a key coordinator of vital biological processes, in ways previously impossible. With the circadian clock increasingly implicated in key fitness and signalling pathways, this has opened up new avenues for understanding plant development and signalling. Our tool-kit has been constantly improving through continual development and novel techniques that increase throughput, reduce costs and allow higher resolution on the cellular and subcellular levels. With circadian assays becoming more accessible and relevant than ever to researchers, in this paper we offer a review of the techniques currently available before considering the horizons in circadian investigation at ever higher throughputs and resolutions

Heparin versus citrate anticoagulation for continuous renal replacement therapy in intensive care: the RRAM observational study

Background: In the UK, 10% of admissions to intensive care units receive continuous renal replacement therapy with regional citrate anticoagulation replacing systemic heparin anticoagulation over the last decade. Regional citrate anticoagulation is now used in > 50% of intensive care units, despite little evidence of safety or effectiveness. Aim: The aim of the Renal Replacement Anticoagulant Management study was to evaluate the clinical and health economic impacts of intensive care units moving from systemic heparin anticoagulation to regional citrate anticoagulation for continuous renal replacement therapy. Design: This was an observational comparative effectiveness study. Setting: The setting was NHS adult general intensive care units in England and Wales. Participants: Participants were adults receiving continuous renal replacement therapy in an intensive care unit participating in the Intensive Care National Audit & Research Centre Case Mix Programme national clinical audit between 1 April 2009 and 31 March 2017. Interventions: Exposure – continuous renal replacement therapy in an intensive care unit after completion of transition to regional citrate anticoagulation. Comparator – continuous renal replacement therapy in an intensive care unit before starting transition to regional citrate anticoagulation or had not transitioned. Outcome measures: Primary effectiveness – all-cause mortality at 90 days. Primary economic – incremental net monetary benefit at 1 year. Secondary outcomes – mortality at hospital discharge, 30 days and 1 year; days of renal, cardiovascular and advanced respiratory support in intensive care unit; length of stay in intensive care unit and hospital; bleeding and thromboembolic events; prevalence of end-stage renal disease at 1 year; and estimated lifetime incremental net monetary benefit. Data sources: Individual patient data from the Intensive Care National Audit & Research Centre Case Mix Programme were linked with the UK Renal Registry, Hospital Episode Statistics (for England), Patient Episodes Data for Wales and Civil Registrations (Deaths) data sets, and combined with identified periods of systemic heparin anticoagulation and regional citrate anticoagulation (survey of intensive care units). Staff time and consumables were obtained from micro-costing. Continuous renal replacement therapy system failures were estimated from the Post-Intensive Care Risk-adjusted Alerting and Monitoring data set. EuroQol-3 Dimensions, three-level version, health-related quality of life was obtained from the Intensive Care Outcomes Network study. Results: Out of the 188 (94.9%) units that responded to the survey, 182 (96.8%) use continuous renal replacement therapy. After linkage, data were available from 69,001 patients across 181 intensive care units (60,416 during periods of systemic heparin anticoagulation use and 8585 during regional citrate anticoagulation use). The change to regional citrate anticoagulation was not associated with a step change in 90-day mortality (odds ratio 0.98, 95% confidence interval 0.89 to 1.08). Secondary outcomes showed step increases in days of renal support (difference in means 0.53 days, 95% confidence interval 0.28 to 0.79 days), advanced cardiovascular support (difference in means 0.23 days, 95% confidence interval 0.09 to 0.38 days) and advanced respiratory support (difference in means, 0.53 days, 95% CI 0.03 to 1.03 days) with a trend toward fewer bleeding episodes (odds ratio 0.90, 95% confidence interval 0.76 to 1.06) with transition to regional citrate anticoagulation. The micro-costing study indicated that regional citrate anticoagulation was more expensive and was associated with an estimated incremental net monetary loss (step change) of –£2376 (95% confidence interval –£3841 to –£911). The estimated likelihood of cost-effectiveness at 1 year was less than 0.1%. Limitations: Lack of patient-level treatment data means that the results represent average effects of changing to regional citrate anticoagulation in intensive care units. Administrative data are subject to variation in data quality over time, which may contribute to observed trends. Conclusions: The introduction of regional citrate anticoagulation has not improved outcomes for patients and is likely to have substantially increased costs. This study demonstrates the feasibility of evaluating effects of changes in practice using routinely collected data. Future work: (1) Prioritise other changes in clinical practice for evaluation and (2) methodological research to understand potential implications of trends in data quality. Trial registration: This trial is registered as ClinicalTrials.gov NCT03545750

A Novel Genome-Wide Association Study Approach Using Genotyping by Exome Sequencing Leads to the Identification of a Primary Open Angle Glaucoma Associated Inversion Disrupting ADAMTS17

Closed breeding populations in the dog in conjunction with advances in gene mapping and sequencing techniques facilitate mapping of autosomal recessive diseases and identification of novel disease-causing variants, often using unorthodox experimental designs. In our investigation we demonstrate successful mapping of the locus for primary open angle glaucoma in the Petit Basset Griffon Vendéen dog breed with 12 cases and 12 controls, using a novel genotyping by exome sequencing approach. The resulting genome-wide association signal was followed up by genome sequencing of an individual case, leading to the identification of an inversion with a breakpoint disrupting the ADAMTS17 gene. Genotyping of additional controls and expression analysis provide strong evidence that the inversion is disease causing. Evidence of cryptic splicing resulting in novel exon transcription as a consequence of the inversion in ADAMTS17 is identified through RNAseq experiments. This investigation demonstrates how a novel genotyping by exome sequencing approach can be used to map an autosomal recessive disorder in the dog, with the use of genome sequencing to facilitate identification of a disease-associated variant

Effect of Sun and Planet-Bound Dark Matter on Planet and Satellite Dynamics in the Solar System

We apply our recent results on orbital dynamics around a mass-varying central body to the phenomenon of accretion of Dark Matter-assumed not self-annihilating-on the Sun and the major bodies of the solar system due to its motion throughout the Milky Way halo. We inspect its consequences on the orbits of the planets and their satellites over timescales of the order of the age of the solar system. It turns out that a solar Dark Matter accretion rate of \approx 10^-12 yr^-1, inferred from the upper limit \Delta M/M= 0.02-0.05 on the Sun's Dark Matter content, assumed somehow accumulated during last 4.5 Gyr, would have displaced the planets faraway by about 10^-2-10^1 au 4.5 Gyr ago. Another consequence is that the semimajor axis of the Earth's orbit, approximately equal to the Astronomical Unit, would undergo a secular increase of 0.02-0.05 m yr^-1, in agreement with the latest observational determinations of the Astronomical Unit secular increase of 0.07 +/- 0.02 m yr^-1 and 0.05 m yr^-1. By assuming that the Sun will continue to accrete Dark Matter in the next billions year at the same rate as in the past, the orbits of its planets will shrink by about 10^-1-10^1 au (\approx 0.2-0.5 au for the Earth), with consequences for their fate, especially of the inner planets. On the other hand, lunar and planetary ephemerides set upper bounds on the secular variation of the Sun's gravitational parameter GM which are one one order of magnitude smaller than 10^-12 yr^-1. Dark Matter accretion on planets has, instead, less relevant consequences for their satellites. Indeed, 4.5 Gyr ago their orbits would have been just 10^-2-10^1 km wider than now. (Abridged)Comment: LaTex2e, 17 pages, no figures, 7 tables, 61 references. Small problem with a reference fixed. To appear in Journal of Cosmology and Astroparticle Physics (JCAP

Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection of Colorectal Cancer

Colorectal cancer (CRC) is a common and treatable disease if diagnosed early. Current population screening programs are suboptimal, and consequently, there is a need for the development of new methodologies for early diagnosis of CRC. In the past 10 years, unprecedented technological advancements in the field of mass spectrometry (MS)-based proteomics have progressively increased the sophistication and utility of these investigations, leading to the draft mapping of the human proteome. These exciting studies have shaped our mechanistic understanding of the human genome and begun to provide us with a suite of novel biomarkers to predict the onset, progression and severity of many debilitating diseases. Thus, sophisticated MS workflows coupled with revolutionary protein quantification techniques hold promise for the field of MS-based plasma proteomics, particularly valuable in the context of early stage identification of curable CRC. However, within the last 40 years, no new plasma protein biomarkers of CRC have been translated into clinical practice. Here. we discuss the application of proteomic technologies within the field of CRC, highlighting contemporary MS-based plasma proteomic strategies that could be exploited to deliver on the promise of a panel of sensitive and specific plasma-based biomarkers with which to non-invasively detect early stage CRC

Aquaporins in Saccharomyces: Genetic and functional distinctions between laboratory and wild-type strains

Aquaporin water channel proteins mediate the transport of water across cell membranes in numerous species. The Saccharomyces genome data base contains an open reading frame (here designated AQY1) that encodes a protein with strong homology to aquaporins. AQY1 from laboratory and wild-type strains of Saccharomyces were expressed in Xenopus oocytes to determine the coefficients of osmotic water permeability (Pf). Oocytes injected with wild-type AQY1 cRNAs exhibit high Pf values, whereas oocytes injected with AQY1 cRNAs from laboratory strains exhibit low Pf values and have reduced levels of Aqy1p due to two amino acid substitutions. When the AQY1 gene was deleted from a wild-type yeast and cells were cultured in vitro with cycled hypo-osmolar or hyper-osmolar stresses, the AQY1 null yeast showed significantly improved viability when compared with the parental wild-type strain. We conclude that Saccharomyces cerevisiae contains at least one aquaporin gene, but it is not functional in laboratory strains due to apparent negative selection pressures resulting from in vitro methods