Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

BACKGROUND: Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. RESULTS: Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. CONCLUSIONS: The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-tRNA synthetases, and may play an important functional role. We suggest a manner in which catalytic efficiency could be improved by LysU operating in an alternating sites mechanism

A novel role for the rat retrosplenial cortex in cognitive control

By virtue of its frontal and hippocampal connections, the retrosplenial cortex is uniquely placed to support cognition. Here, we tested whether the retrosplenial cortex is required for frontal tasks analogous to the Stroop Test, i.e., for the ability to select between conflicting responses and inhibit responding to task-irrelevant cues. Rats first acquired two instrumental conditional discriminations, one auditory and one visual, set in two distinct contexts. As a result, rats were rewarded for pressing either the right or left lever when a particular auditory or visual signal was present. In extinction, rats received compound stimuli that either comprised the auditory and visual elements that signaled the same lever response (congruent) or signaled different lever responses (incongruent) during training. On conflict (incongruent) trials, lever selection by sham-operated animals followed the stimulus element that had previously been trained in that same test context, whereas animals with retrosplenial cortex lesions failed to disambiguate the conflicting response cues. Subsequent experiments demonstrated that this abnormality on conflict trials was not due to a failure in distinguishing the contexts. Rather, these data reveal the selective involvement of the rat retrosplenial cortex in response conflict, and so extend the frontal system underlying cognitive control

A Formalization of the Theorem of Existence of First-Order Most General Unifiers

This work presents a formalization of the theorem of existence of most general unifiers in first-order signatures in the higher-order proof assistant PVS. The distinguishing feature of this formalization is that it remains close to the textbook proofs that are based on proving the correctness of the well-known Robinson's first-order unification algorithm. The formalization was applied inside a PVS development for term rewriting systems that provides a complete formalization of the Knuth-Bendix Critical Pair theorem, among other relevant theorems of the theory of rewriting. In addition, the formalization methodology has been proved of practical use in order to verify the correctness of unification algorithms in the style of the original Robinson's unification algorithm.Comment: In Proceedings LSFA 2011, arXiv:1203.542

An evaluation of a nurse led unit: an action research study

This study is an exemplar of working in a participatory way with members of the public and health and social care practitioners as co-researchers. A Nurse Consultant Older People working in a nurse-led bed, intermediate care facility in a community hospital acted as joint project lead with an academic researcher. From the outset, members of the public were part of a team of 16 individuals who agreed an evaluation focus and were involved in all stages of the research process from design through to dissemination. An extensive evaluation reflecting all these stakeholders’ preferences was undertaken. Methods included research and audit including: patient and carer satisfaction questionnaire surveys, individual interviews with patients, carers and staff, staff surveys, graffiti board, suggestion box, first impressions questionnaire, patient tracking and a bed census. A key aim of the study has been capacity building of the research team members which has also been evaluated. In terms of impact, the co-researchers have developed research skills and knowledge, grown in confidence, developed in ways that have impacted elsewhere in their lives, developed posters, presented at conferences and gained a better understanding of the NHS. The evaluation itself has provided useful information on the processes and outcomes of intermediate care on the ward which was used to further improve the service

Characteristics of young people accessing recently implemented Community Forensic Child and Adolescent Mental Health Services (F:CAMHS) in England: insights from national service activity data

Children and young people in contact with forensic child and adolescent mental health services present with more complex needs than young people in the general population. Recent policy in child and adolescent mental health has led to the implementation of new workstreams and programmes to improve service provision. This research examines the characteristics of children and young people referred to recently commissioned Community Forensic Child and Adolescent Services (F:CAMHS) and service activity during the frst 24 months of service. The study is a national cohort study to describe the population and investigate service provision and access across England. Secondary data on 1311 advice cases and 1406 referrals are included in analysis. Findings show that 71.9% of the sample had accessed mainstream CAMHS before their referral, 50.9% had experienced/witnessed multiple traumatic events and 58.4% of young people presented with multiple difculties. The results of the study highlight the complexity of the cohort and a need for interagency trauma-informed working. This is the frst study to describe the characteristics of children and young people referred to Community F:CAMHS and provides valuable information on pathways and needs to inform service policy and provision

The F-box protein Cdc4/Fbxw7 is a novel regulator of neural crest development in Xenopus laevis.

BACKGROUND: The neural crest is a unique population of cells that arise in the vertebrate ectoderm at the neural plate border after which they migrate extensively throughout the embryo, giving rise to a wide range of derivatives. A number of proteins involved in neural crest development have dynamic expression patterns, and it is becoming clear that ubiquitin-mediated protein degradation is partly responsible for this. RESULTS: Here we demonstrate a novel role for the F-box protein Cdc4/Fbxw7 in neural crest development. Two isoforms of Xenopus laevis Cdc4 were identified, and designated xCdc4alpha and xCdc4beta. These are highly conserved with vertebrate Cdc4 orthologs, and the Xenopus proteins are functionally equivalent in terms of their ability to degrade Cyclin E, an established vertebrate Cdc4 target. Blocking xCdc4 function specifically inhibited neural crest development at an early stage, prior to expression of c-Myc, Snail2 and Snail. CONCLUSIONS: We demonstrate that Cdc4, an ubiquitin E3 ligase subunit previously identified as targeting primarily cell cycle regulators for proteolysis, has additional roles in control of formation of the neural crest. Hence, we identify Cdc4 as a protein with separable but complementary functions in control of cell proliferation and differentiation

Dysgranular retrosplenial cortex lesions in rats disrupt cross-modal object recognition

The retrosplenial cortex supports navigation, with one role thought to be the integration of different spatial cue types. This hypothesis was extended by examining the integration of nonspatial cues. Rats with lesions in either the dysgranular subregion of retrosplenial cortex (area 30) or lesions in both the granular and dysgranular subregions (areas 29 and 30) were tested on cross-modal object recognition (Experiment 1). In these tests, rats used different sensory modalities when exploring and subsequently recognizing the same test objects. The objects were first presented either in the dark, i.e., giving tactile and olfactory cues, or in the light behind a clear Perspex barrier, i.e., giving visual cues. Animals were then tested with either constant combinations of sample and test conditions (light to light, dark to dark), or changed “cross-modal” combinations (light to dark, dark to light). In Experiment 2, visual object recognition was tested without Perspex barriers, but using objects that could not be distinguished in the dark. The dysgranular retrosplenial cortex lesions selectively impaired cross-modal recognition when cue conditions switched from dark to light between initial sampling and subsequent object recognition, but no impairment was seen when the cue conditions remained constant, whether dark or light. The combined (areas 29 and 30) lesioned rats also failed the dark to light cross-modal problem but this impairment was less selective. The present findings suggest a role for the dysgranular retrosplenial cortex in mediating the integration of information across multiple cue types, a role that potentially applies to both spatial and nonspatial domains

Gravity waves in the winter stratosphere over the Southern Ocean: high-resolution satellite observations and 3-D spectral analysis.

Atmospheric gravity waves play a key role in the transfer of energy and momentum between layers of the Earth's atmosphere. However, nearly all Global Circulation Models (GCMs) seriously under-represent the momentum fluxes of gravity waves at latitudes near 60° S. This can result in modelled winter stratospheres that are unrealistically cold – a significant bias known as the "cold-pole problem". There is thus a need for measurements of gravity-wave fluxes near 60S to test and constrain GCMs. Such measurements are notoriously difficult, because they require 3-D observations of wave properties if the fluxes are to be estimated without using significant limiting assumptions. Here we use 3-D satellite measurements of stratospheric gravity waves from NASA's AIRS/Aqua instrument. We present the first extended application of a 3-D Stockwell transform (3DST) method to determine localised gravity-wave amplitudes, wavelengths and directions of propagation around the entire region of the Southern Ocean near 60° S during austral winter 2010. We first validate our method using a synthetic wave field and two case studies of real gravity waves over the Southern Andes and the island of South Georgia. A new technique to overcome wave amplitude attenuation problems in previous methods is also presented. We then characterise large-scale gravity-wave occurrence frequencies, directional momentum fluxes and short-timescale intermittency over the entire Southern Ocean. Our results show that highest wave-occurrence frequencies, amplitudes and momentum fluxes are observed in the stratosphere over the mountains of the Southern Andes and Antarctic Peninsula. However, we find that around 60–80 % of total zonal-mean momentum flux is located over the open Southern Ocean during June–August, where a large "belt" of increased wave-occurrence frequencies, amplitudes and fluxes is observed. Our results also suggest significant short-timescale variability of fluxes from both orographic and non-orographic sources in the region. A particularly striking result is a widespread convergence of gravity-wave momentum fluxes towards latitudes around 60° S from the north and south. We propose that this convergence, which is observed at nearly all longitudes during winter, accounts for a significant part of the under-represented flux in GCMs at these latitudes

Lgr5+ stem and progenitor cells reside at the apex of a heterogeneous embryonic hepatoblast pool.

During mouse embryogenesis, progenitors within the liver known as hepatoblasts give rise to adult hepatocytes and cholangiocytes. Hepatoblasts, which are specified at E8.5-E9.0, have been regarded as a homogeneous progenitor population that initiate differentiation from E13.5. Recently, scRNA-seq analysis has identified sub-populations of transcriptionally distinct hepatoblasts at E11.5. Here, we show that hepatoblasts are not only transcriptionally but also functionally heterogeneous, and that a subpopulation of E9.5-E10.0 hepatoblasts exhibit a previously unidentified early commitment to cholangiocyte fate. Importantly, we also identify a subpopulation constituting 2% of E9.5-E10.0 hepatoblasts that express the adult stem cell marker Lgr5, and generate both hepatocyte and cholangiocyte progeny that persist for the lifespan of the mouse. Combining lineage tracing and scRNA-seq, we show that Lgr5 marks E9.5-E10.0 bipotent liver progenitors residing at the apex of a hepatoblast hierarchy. Furthermore, isolated Lgr5+ hepatoblasts can be clonally expanded in vitro into embryonic liver organoids, which can commit to either hepatocyte or cholangiocyte fates. Our study demonstrates functional heterogeneity within E9.5 hepatoblasts and identifies Lgr5 as a marker for a subpopulation of bipotent liver progenitors.M.H. is a Wellcome Trust Sir Henry Dale Fellow and is jointly funded by the Wellcome Trust and the Royal Society (104151/Z/14/Z); M.H. and N.P. are funded by a Horizon 2020 grant (LSFM4LIFE). C.H. was funded by a Cambridge Stem Cell Institute Seed funding for interdisciplinary research awarded to M.H. and B.D.S., B.D.S acknowledges funding from the Royal Society E.P. Abraham Research Professorship (RP\R1\180165) and Wellcome Trust (098357/Z/12/Z). W.L. and B.G. were supported by programmatic funding from the Wellcome Trust, CRUK and Bloodwise, core infrastructure support from the Wellcome and MRC to the Wellcome & MRC Cambridge Stem Cell Institute, and an MRC Clinical Research Infrastructure grant supporting single cell molecular analysis. S.R. was funded on a Herchel-Smith Fellowship. The authors acknowledge core funding to the Gurdon Institute from the Wellcome Trust (092096) and CRUK (C6946/A14492)