The chicken B-cell line DT40 proteome, beadome and interactomes.

In developing a new quantitative AP-MS method for exploring interactomes in the chicken B-cell line DT40, we also surveyed the most abundant proteins in this organism and explored the likely contaminants that bind to a variety of affinity resins that would later be confirmed quantitatively [1]. We present the 'Top 150 abundant DT40 proteins list', the DT40 beadomes as well as protein interaction lists for the Phosphatidyl inositol 5-phosphate 4-kinase 2β and Fanconi anaemia protein complexes.We thanks Prof. R. Irvine for providing the JPR3 cell line, to Dr. E. Rajendra for providing the FANCC cell line, FANC antibodies and helpful discussions, to Dr. M. Deery and J. Howard for assistance with MS. This work was funded by the Biotechnology and Biological Sciences Research Council (UK) Grant BB/H024085/1.This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.dib.2014.12.00

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3-subunit.

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated β-subunits. The β3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length β3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric β3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the β3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the β3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the β3-subunit is not required for this clustering but β3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the β3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the β3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the β3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the β3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.We would like to thank the Gurdon Institute Imaging Facility for use of their microscope and general assistance. This work was supported by a British Heart Foundation grant (PG/14/79/31102) to APJ and CLHH, The Wellcome Trust, award number: 105727/Z/14/Z to CLHH and a Medical Research Council grant (MR/K015591/1) to CLF, RAL, and STFC

Analysis of the expression patterns, subcellular localisations and interaction partners of Drosophila proteins using a pigP protein trap library.

Although we now have a wealth of information on the transcription patterns of all the genes in the Drosophila genome, much less is known about the properties of the encoded proteins. To provide information on the expression patterns and subcellular localisations of many proteins in parallel, we have performed a large-scale protein trap screen using a hybrid piggyBac vector carrying an artificial exon encoding yellow fluorescent protein (YFP) and protein affinity tags. From screening 41 million embryos, we recovered 616 verified independent YFP-positive lines representing protein traps in 374 genes, two-thirds of which had not been tagged in previous P element protein trap screens. Over 20 different research groups then characterized the expression patterns of the tagged proteins in a variety of tissues and at several developmental stages. In parallel, we purified many of the tagged proteins from embryos using the affinity tags and identified co-purifying proteins by mass spectrometry. The fly stocks are publicly available through the Kyoto Drosophila Genetics Resource Center. All our data are available via an open access database (Flannotator), which provides comprehensive information on the expression patterns, subcellular localisations and in vivo interaction partners of the trapped proteins. Our resource substantially increases the number of available protein traps in Drosophila and identifies new markers for cellular organelles and structures.This work was supported by a project grant from the Wellcome Trust [076739], by a Wellcome Trust Principal Research Fellowship to D.StJ. [049818 and 080007], and by core support from the Wellcome Trust [092096] and Cancer Research UK [A14492].This is the final version of the article. It was first available from The Company of Biologists via http://dx.doi.org/10.1242/dev.11105

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3-subunit

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated β-subunits. The β3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length β3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric β3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the β3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the β3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the β3-subunit is not required for this clustering but β3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the β3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the β3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the β3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the β3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.British Heart Foundation, Grant/Award Number: PG/14/79/31102; Wellcome Trust, Grant/Award Number: 105727/Z/14/Z; Medical Research Council (UK), Grant/Award Number: MR/K015591/

Two-point functions in a holographic Kondo model

We develop the formalism of holographic renormalization to compute two-point functions in a holographic Kondo model. The model describes a $(0+1)$-dimensional impurity spin of a gauged $SU(N)$ interacting with a $(1+1)$-dimensional, large-$N$, strongly-coupled Conformal Field Theory (CFT). We describe the impurity using Abrikosov pseudo-fermions, and define an $SU(N)$-invariant scalar operator \mathcalO built from a pseudo-fermion and a CFT fermion. At large $N$ the Kondo interaction is of the form \mathcalO^\dagger \mathcalO, which is marginally relevant, and generates a Renormalization Group (RG) flow at the impurity. A second-order mean-field phase transition occurs in which \mathcalO condenses below a critical temperature, leading to the Kondo effect, including screening of the impurity. Via holography, the phase transition is dual to holographic superconductivity in $(1+1)$-dimensional Anti-de Sitter space. At all temperatures, spectral functions of \mathcalO exhibit a Fano resonance, characteristic of a continuum of states interacting with an isolated resonance. In contrast to Fano resonances observed for example in quantum dots, our continuum and resonance arise from a $(0+1)$-dimensional UV fixed point and RG flow, respectively. In the low-temperature phase, the resonance comes from a pole in the Green's function of the form $-i \langle \cal O \rangle^2$, which is characteristic of a Kondo resonance

Predicting pain and function outcomes in people consulting with shoulder pain: the PANDA-S clinical cohort and qualitative study protocol

Introduction: People presenting with shoulder pain considered to be of musculoskeletal origin is common in primary care but diagnosing the cause of the pain is contentious, leading to uncertainty in management. To inform optimal primary care for patients with shoulder pain, the study aims to (1) to investigate the short-term and long-term outcomes (overall prognosis) of shoulder pain, (2) estimate costs of care, (3) develop a prognostic model for predicting individuals' level and risk of pain and disability at 6 months and (4) investigate experiences and opinions of patients and healthcare professionals regarding diagnosis, prognosis and management of shoulder pain. Methods and analysis: The Prognostic And Diagnostic Assessment of the Shoulder (PANDA-S) study is a longitudinal clinical cohort with linked qualitative study. At least 400 people presenting to general practice and physiotherapy services in the UK will be recruited. Participants will complete questionnaires at baseline, 3, 6, 12, 24 and 36 months. Short-term data will be collected weekly between baseline and 12 weeks via Short Message Serevice (SMS) text or software application. Participants will be offered clinical (physiotherapist) and ultrasound (sonographer) assessments at baseline. Qualitative interviews with ≈15 dyads of patients and their healthcare professional (general practitioner or physiotherapist).Short-term and long-term trajectories of Shoulder Pain and Disability Index (using SPADI) will be described, using latent class growth analysis. Health economic analysis will estimate direct costs of care and indirect costs related to work absence and productivity losses. Multivariable regression analysis will be used to develop a prognostic model predicting future levels of pain and disability at 6 months using penalisation methods to adjust for overfitting. The added predictive value of prespecified physical examination tests and ultrasound findings will be examined. For the qualitative interviews an inductive, exploratory framework will be adopted using thematic analysis to investigate decision making, perspectives of patients and clinicians on the importance of diagnostic and prognostic information when negotiating treatment and referral options. Ethics and dissemination: The PANDA-S study has ethical approval from Yorkshire and The Humber-Sheffield Research Ethics Committee, UK (18/YH/0346, IRAS Number: 242750). Results will be disseminated through peer-reviewed publications, social and mainstream media, professional conferences, and the patient and public involvement and engagement group supporting this study, and through newsletters, leaflets and posters in participating sites

Global economic burden of unmet surgical need for appendicitis

Background: There is a substantial gap in provision of adequate surgical care in many low-and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods: Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results: Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion: For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe