The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains

Overexpression of Mcl-1 exacerbates lymphocyte accumulation and autoimmune kidney disease in lpr mice

Cell death by apoptosis has a critical role during embryonic development and in maintaining tissue homeostasis. In mammals, there are two converging apoptosis pathways: the ‘extrinsic’ pathway, which is triggered by engagement of cell surface ‘death receptors’ such as Fas/APO-1; and the ‘intrinsic’ pathway, which is triggered by diverse cellular stresses, and is regulated by prosurvival and pro-apoptotic members of the Bcl-2 family of proteins. Pro-survival Mcl-1, which can block activation of the proapoptotic proteins, Bax and Bak, appears critical for the survival and maintenance of multiple haemopoietic cell types. To investigate the impact on haemopoiesis of simultaneously inhibiting both apoptosis pathways, we introduced the vavP-Mcl-1 transgene, which causes overexpression of Mcl-1 protein in all haemopoietic lineages, into Faslpr/lpr mice, which lack functional Fas and are prone to autoimmunity. The combined mutations had a modest impact on myelopoiesis, primarily an increase in the macrophage/monocyte population in Mcl-1tg/lpr mice compared with lpr or Mcl-1tg mice. The impact on lymphopoiesis was striking, with a marked elevation in all major lymphoid subsets, including the non-conventional double-negative (DN) T cells (TCRβ+ CD4– CD8– B220+ ) characteristic of Faslpr/lpr mice. Of note, the onset of autoimmunity was markedly accelerated in Mcl-1tg/lpr mice compared with lpr mice, and this was preceded by an increase in immunoglobulin (Ig)-producing cells and circulating autoantibodies. This degree of impact was surprising, given the relatively mild phenotype conferred by the vavP-Mcl-1 transgene by itself: a two- to threefold elevation of peripheral B and T cells, no significant increase in the non-conventional DN T-cell population and no autoimmune disease. Comparison of the phenotype with that of other susceptible mice suggests that the development of autoimmune disease in Mcl-1tg/lpr mice may be influenced not only by Ig-producing cells but also other haemopoietic cell types

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Using OPMs to measure neural activity in standing, mobile participants

Optically pumped magnetometer-based magnetoencephalography (OP-MEG) can be used to measure neuromagnetic fields while participants move in a magnetically shielded room. Head movements in previous OP-MEG studies have been up to 20 cm translation and ∼30° rotation in a sitting position. While this represents a step-change over stationary MEG systems, naturalistic head movement is likely to exceed these limits, particularly when participants are standing up. In this proof-of-concept study, we sought to push the movement limits of OP-MEG even further. Using a 90 channel (45-sensor) whole-head OP-MEG system and concurrent motion capture, we recorded auditory evoked fields while participants were: (i) sitting still, (ii) standing up and still, and (iii) standing up and making large natural head movements continuously throughout the recording - maximum translation 120 cm, maximum rotation 198°. Following pre-processing, movement artefacts were substantially reduced but not eliminated. However, upon utilisation of a beamformer, the M100 event-related field localised to primary auditory regions. Furthermore, the event-related fields from auditory cortex were remarkably consistent across the three conditions. These results suggest that a wide range of movement is possible with current OP-MEG systems. This in turn underscores the exciting potential of OP-MEG for recording neural activity during naturalistic paradigms that involve movement (e.g. navigation), and for scanning populations who are difficult to study with stationary MEG (e.g. young children)

Interference suppression techniques for OPM-based MEG: Opportunities and challenges

One of the primary technical challenges facing magnetoencephalography (MEG) is that the magnitude of neuromagnetic fields is several orders of magnitude lower than interfering signals. Recently, a new type of sensor has been developed – the optically pumped magnetometer (OPM). These sensors can be placed directly on the scalp and move with the head during participant movement, making them wearable. This opens up a range of exciting experimental and clinical opportunities for OPM-based MEG experiments, including paediatric studies, and the incorporation of naturalistic movements into neuroimaging paradigms. However, OPMs face some unique challenges in terms of interference suppression, especially in situations involving mobile participants, and when OPMs are integrated with electrical equipment required for naturalistic paradigms, such as motion capture systems. Here we briefly review various hardware solutions for OPM interference suppression. We then outline several signal processing strategies aimed at increasing the signal from neuromagnetic sources. These include regression-based strategies, temporal filtering and spatial filtering approaches. The focus is on the practical application of these signal processing algorithms to OPM data. In a similar vein, we include two worked-through experiments using OPM data collected from a whole-head sensor array. These tutorial-style examples illustrate how the steps for suppressing external interference can be implemented, including the associated data and code so that researchers can try the pipelines for themselves. With the popularity of OPM-based MEG rising, there will be an increasing need to deal with interference suppression. We hope this practical paper provides a resource for OPM-based MEG researchers to build upon

Modelling optically pumped magnetometer interference in MEG as a spatially homogeneous magnetic field

Here we propose that much of the magnetic interference observed when using optically pumped magnetometers for MEG experiments can be modeled as a spatially homogeneous magnetic field. We show that this approximation reduces sensor level variance and substantially improves statistical power. This model does not require knowledge of the underlying neuroanatomy nor the sensor positions. It only needs information about the sensor orientation. Due to the model's low rank there is little risk of removing substantial neural signal. However, we provide a framework to assess this risk for any sensor number, design or subject neuroanatomy. We find that the risk of unintentionally removing neural signal is reduced when multi-axis recordings are performed. We validated the method using a binaural auditory evoked response paradigm and demonstrated that removing the homogeneous magnetic field increases sensor level SNR by a factor of 3. Considering the model's simplicity and efficacy, we suggest that this homogeneous field correction can be a powerful preprocessing step for arrays of optically pumped magnetometers

Markers of serotonergic function in the orbitofrontal cortex and dorsal raphé nucleus predict individual variation in spatial-discrimination serial reversal learning.

Dysfunction of the orbitofrontal cortex (OFC) impairs the ability of individuals to flexibly adapt behavior to changing stimulus-reward (S-R) contingencies. Impaired flexibility also results from interventions that alter serotonin (5-HT) and dopamine (DA) transmission in the OFC and dorsomedial striatum (DMS). However, it is unclear whether similar mechanisms underpin naturally occurring variations in behavioral flexibility. In the present study, we used a spatial-discrimination serial reversal procedure to investigate interindividual variability in behavioral flexibility in rats. We show that flexibility on this task is improved following systemic administration of the 5-HT reuptake inhibitor citalopram and by low doses of the DA reuptake inhibitor GBR12909. Rats in the upper quintile of the distribution of perseverative responses during repeated S-R reversals showed significantly reduced levels of the 5-HT metabolite, 5-hydroxy-indoleacetic acid, in the OFC. Additionally, 5-HT2A receptor binding in the OFC of mid- and high-quintile rats was significantly reduced compared with rats in the low-quintile group. These perturbations were accompanied by an increase in the expression of monoamine oxidase-A (MAO-A) and MAO-B in the lateral OFC and by a decrease in the expression of MAO-A, MAO-B, and tryptophan hydroxylase in the dorsal raphé nucleus of highly perseverative rats. We found no evidence of significant differences in markers of DA and 5-HT function in the DMS or MAO expression in the ventral tegmental area of low- vs high-perseverative rats. These findings indicate that diminished serotonergic tone in the OFC may be an endophenotype that predisposes to behavioral inflexibility and other forms of compulsive behavior.This work was supported by Medical Research Council Grants (G0701500; G0802729), a 503 Wellcome Trust Programme Grant (grant number 089589/Z/09/Z), and by a Core Award 504 from the Medical Research Council and the Wellcome Trust to the Behavioural and Clinical 505 21 Neuroscience Institute (MRC Ref G1000183; WT Ref 093875/Z/10/Z). RLB was supported 506 by a studentship from the Medical Research Council. JA was supported by a Fellowship from 507 the Swedish Research Council (350-2012-230). BJ was supported by Fellowships from the 508 AXA Research Fund and the National Health and Medical Research Council of Australia. 509 Financial support from the Fredrik and Ingrid Thuring Foundation is also acknowledged.This is the accepted manuscript. The final version is available from Nature Publishing at http://www.nature.com/npp/journal/vaop/ncurrent/full/npp2014335a.html

2017 HRS/EHRA/ECAS/APHRS/SOLAECE expert consensus statement on catheter and surgical ablation of atrial fibrillation: executive summary.

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