A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

Mechanosensitive Enteric Neurons in the Myenteric Plexus of the Mouse Intestine

BACKGROUND: Within the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions. METHODOLOGY/PRINCIPAL FINDINGS: We deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon (calretinin). RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low Ca(++)/high Mg(++). Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin. CONCLUSIONS/SIGNIFICANCE: We showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions

Asteroseismology and Interferometry

Asteroseismology provides us with a unique opportunity to improve our understanding of stellar structure and evolution. Recent developments, including the first systematic studies of solar-like pulsators, have boosted the impact of this field of research within Astrophysics and have led to a significant increase in the size of the research community. In the present paper we start by reviewing the basic observational and theoretical properties of classical and solar-like pulsators and present results from some of the most recent and outstanding studies of these stars. We centre our review on those classes of pulsators for which interferometric studies are expected to provide a significant input. We discuss current limitations to asteroseismic studies, including difficulties in mode identification and in the accurate determination of global parameters of pulsating stars, and, after a brief review of those aspects of interferometry that are most relevant in this context, anticipate how interferometric observations may contribute to overcome these limitations. Moreover, we present results of recent pilot studies of pulsating stars involving both asteroseismic and interferometric constraints and look into the future, summarizing ongoing efforts concerning the development of future instruments and satellite missions which are expected to have an impact in this field of research.Comment: Version as published in The Astronomy and Astrophysics Review, Volume 14, Issue 3-4, pp. 217-36

A Naturally Occurring Plant Cysteine Protease Possesses Remarkable Toxicity against Insect Pests and Synergizes Bacillus thuringiensis Toxin

When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitating the movement of other insecticidal proteins in the diet to the midgut microvilli and thereby enhancing their toxicity. To directly determine the toxicity of Mir1-CP, the purified recombinant enzyme was directly tested against four economically significant Lepidopteran pests in bioassays. Mir1-CP LC50 values were 1.8, 3.6, 0.6, and 8.0 ppm for corn earworm, tobacco budworm, fall armyworm and southwestern corn borer, respectively. These values were the same order of magnitude as those determined for the Bacillus thuringiensis toxin Bt-CryIIA. In addition to being directly toxic to the larvae, 60 ppb Mir1-CP synergized sublethal concentrations of Bt-CryIIA in all four species. Permeabilization of the PM by Mir1-CP probably provides ready access to Bt-binding sites on the midgut microvilli and increases its activity. Consequently, Mir1-CP could be used for controlling caterpillar pests in maize using non-transgenic approaches and potentially could be used in other crops either singly or in combination with Bt-toxins

Dementia and Physical Activity (DAPA) - an exercise intervention to improve cognition in people with mild to moderate dementia: Study protocol for a randomized controlled trial

Background: Dementia is more common in older than in younger people, and as a result of the ageing of the population in developed countries, it is becoming more prevalent. Drug treatments for dementia are limited, and the main support offered to people with dementia and their families is generally services to mitigate against loss of function. Physical exercise is a candidate non-pharmacological treatment for dementia. Methods/Design: DAPA is a randomised controlled trial funded by the National Institute for Health Research Health Technology Assessment programme to estimate the effect of a 4-month, moderate- to hard-intensity exercise training programme and subsequent advice to remain active, on cognition (primary outcome) at 12 months in people with mild to moderate dementia. Community-dwelling participants (with their carers where possible), who are able to walk 3 metres without human assistance, able to undertake an exercise programme and do not have any unstable or terminal illness are recruited. Participants are then randomised by an independent statistician using a computerised random number generator to usual care or exercise at a 2:1 ratio in favour of exercise. The exercise intervention comprises 29, 1-hour-long exercise classes, run twice weekly at suitable venues such as leisure centres, which include aerobic exercise (on static bikes) and resistance exercise (using weights). Goals for independent exercise are set while the classes are still running, and supported thereafter with phone calls. The primary outcome is measured using ADAS-cog. Secondary outcome measures include behavioural symptoms, functional ability, quality of life and carer burden. Primary and secondary outcomes will be measured at baseline and at 6 and 12 months after randomisation, by researchers masked to participant randomisation in the participants' own homes. An economic evaluation will be carried out in parallel to the RCT, as will a qualitative study capturing the experiences of participants, carers and staff delivering the intervention. Discussion: The DAPA study will be the first large, randomised trial of the cognitive effects of exercise on people with dementia. The intervention is designed to be capable of being delivered within the constraints of NHS service provision, and the economic evaluation will allow assessment of its cost-effectiveness. Trial registration: DAPA was registered with the ISRCTN database on 29 July 2011, registration number ISRCTN32612072. © 2016 Atherton et al

Can oral corticosteroids reduce the severity or duration of an acute cough, and the associated National Health Service and societal costs, in adults presenting to primary care?: study protocol for a randomised controlled trial

Background: Acute lower respiratory tract infection (LRTI) is one of the most common conditions managed internationally and is costly to health services and patients. Despite good evidence that antibiotics are not effective for improving the symptoms of uncomplicated LRTI, they are widely prescribed, contributing to antimicrobial resistance. Many of the symptoms observed in LRTI are mediated by inflammatory processes also observed in exacerbations of asthma, for which there is strong evidence of corticosteroid effectiveness. The primary aim of the OSAC (Oral Steroids for Acute Cough) Trial is to determine whether oral prednisolone (40 mg daily for 5 days) can reduce the duration of moderately bad (or worse) cough and the severity of all its associated symptoms on days 2 to 4 post-randomisation (day 1 is trial entry) by at least 20% in adults ≥18 years with acute LRTI presenting to primary care. Methods/design: OSAC is a two-arm, multi-centre, placebo-controlled, randomised superiority trial. The target sample size is 436 patients, which allows for a 20% dropout rate. Patients will be recruited from primary care sites (General Practitioner surgeries) across England and followed up until symptom resolution. The two primary clinical outcomes are the duration of moderately bad (or worse) cough, and the severity of all its associated symptoms on days 2 to 4 post-randomisation. Secondary outcomes include: antibiotic consumption; symptom burden; adverse events; participant satisfaction with treatment and intention to consult for future similar illnesses. A parallel economic evaluation will investigate the cost-effectiveness of the intervention. Discussion: Results from the OSAC trial will increase knowledge regarding the clinical and cost-effectiveness of corticosteroids for LRTI, and will establish the potential of a new treatment option that could substantially improve patient health. We have chosen a relatively high ‘efficacy dose’ as this will enable us to decide on the potential for further research into lower dose oral and/or inhaled corticosteroids. This trial will also contribute to a growing body of research investigating the natural course of this very common illness, as well as the effects of steroids on the undesirable inflammatory symptoms associated with infection. Trial registration: Current Controlled Trials ISRCTN57309858 (31 January 2013)

Establishing a primary care audit and feedback implementation laboratory: a consensus study

Background: There is a significant variation among individual primary care providers in prescribing of potentially problematic, low-value medicines which cause avoidable patient harm. Audit and feedback is generally effective at improving prescribing. However, progress has been hindered by research waste, leading to unanswered questions about how to include audit and feedback for specific problems and circumstances. Trials of different ways of providing audit and feedback in implementation laboratories have been proposed as a way of improving population healthcare while generating robust evidence on feedback effects. However, there is limited experience in their design and delivery. Aim: To explore priorities, feasibility, and ethical challenges of establishing a primary care prescribing audit and feedback implementation laboratory. Design and setting: Two-stage Delphi consensus process involving primary care pharmacy leads, audit and feedback researchers, and patient and public. Method: Participants initially scored statements relating to priorities, feasibility, and ethical considerations for an implementation laboratory. These covered current feedback practice, priority topics for feedback, usefulness of feedback in improving prescribing and different types of prescribing data, acceptability and desirability of different organization levels of randomization, options for trial consent, different methods of delivering feedback, and interest in finding out how effective different ways of presenting feedback would be. After receiving collated results, participants then scored the items again. The consensus was defined using the GRADE criteria. The results were analyzed by group and overall score. Results: Fourteen participants reached consensus for 38 out of 55 statements. Addressing antibiotic and opioid prescribing emerged as the highest priorities for action. The panel supported statements around addressing highpriority prescribing issues, taking an “opt-out” approach to practice consent if waiving consent was not permitted, and randomizing at lower rather than higher organizational levels. Participants supported patient-level prescribing data and further research evaluating most of the different feedback methods we presented them with. Conclusions: There is a good level of support for evaluating a wide range of potential enhancements to improve the effects of feedback on prescribing. The successful design and delivery of a primary care audit and feedback implementation laboratory depend on identifying shared priorities and addressing practical and ethical considerations

The effect of local anaesthetic wound infiltration on chronic pain after lower limb joint replacement: A protocol for a double-blind randomised controlled trial

<p>Abstract</p> <p>Background</p> <p>For the majority of patients with osteoarthritis (OA), joint replacement is a successful intervention for relieving chronic joint pain. However, between 10-30% of patients continue to experience chronic pain after joint replacement. Evidence suggests that a risk factor for chronic pain after joint replacement is the severity of acute post-operative pain. The aim of this randomised controlled trial (RCT) is to determine if intra-operative local anaesthethic wound infiltration additional to a standard anaethesia regimen can reduce the severity of joint pain at 12-months after total knee replacement (TKR) and total hip replacement (THR) for OA.</p> <p>Methods</p> <p>300 TKR patients and 300 THR patients are being recruited into this single-centre double-blind RCT. Participants are recruited before surgery and randomised to either the standard care group or the intervention group. Participants and outcome assessors are blind to treatment allocation throughout the study. The intervention consists of an intra-operative local anaesthetic wound infiltration, consisting of 60 mls of 0.25% bupivacaine with 1 in 200,000 adrenaline. Participants are assessed on the first 5 days post-operative, and then at 3-months, 6-months and 12-months. The primary outcome is the WOMAC Pain Scale, a validated measure of joint pain at 12-months. Secondary outcomes include pain severity during the in-patient stay, post-operative nausea and vomiting, satisfaction with pain relief, length of hospital stay, joint pain and disability, pain sensitivity, complications and cost-effectiveness. A nested qualitative study within the RCT will examine the acceptability and feasibility of the intervention for both patients and healthcare professionals.</p> <p>Discussion</p> <p>Large-scale RCTs assessing the effectiveness of a surgical intervention are uncommon, particulary in orthopaedics. The results from this trial will inform evidence-based recommendations for both short-term and long-term pain management after lower limb joint replacement. If a local anaesthetic wound infiltration is found to be an effective and cost-effective intervention, implementation into clinical practice could improve long-term pain outcomes for patients undergoing lower limb joint replacement.</p> <p>Trial registration</p> <p>Current Controlled Trials <a href="http://www.controlled-trials.com/ISRCTN96095682">ISRCTN96095682</a></p

Multiple Neural Oscillators and Muscle Feedback Are Required for the Intestinal Fed State Motor Program

After a meal, the gastrointestinal tract exhibits a set of behaviours known as the fed state. A major feature of the fed state is a little understood motor pattern known as segmentation, which is essential for digestion and nutrient absorption. Segmentation manifests as rhythmic local constrictions that do not propagate along the intestine. In guinea-pig jejunum in vitro segmentation constrictions occur in short bursts together with other motor patterns in episodes of activity lasting 40–60 s and separated by quiescent episodes lasting 40–200 s. This activity is induced by luminal nutrients and abolished by blocking activity in the enteric nervous system (ENS). We investigated the enteric circuits that regulate segmentation focusing on a central feature of the ENS: a recurrent excitatory network of intrinsic sensory neurons (ISNs) which are characterized by prolonged after-hyperpolarizing potentials (AHPs) following their action potentials. We first examined the effects of depressing AHPs with blockers of the underlying channels (TRAM-34 and clotrimazole) on motor patterns induced in guinea-pig jejunum, in vitro, by luminal decanoic acid. Contractile episode durations increased markedly, but the frequency and number of constrictions within segmenting bursts and quiescent period durations were unaffected. We used these observations to develop a computational model of activity in ISNs, excitatory and inhibitory motor neurons and the muscle. The model predicted that: i) feedback to ISNs from contractions in the circular muscle is required to produce alternating activity and quiescence with the right durations; ii) transmission from ISNs to excitatory motor neurons is via fast excitatory synaptic potentials (EPSPs) and to inhibitory motor neurons via slow EPSPs. We conclude that two rhythm generators regulate segmentation: one drives contractions within segmentation bursts, the other the occurrence of bursts. The latter depends on AHPs in ISNs and feedback to these neurons from contraction of the circular muscle