CoESPRIT: A Library-Based Construct Screening Method for Identification and Expression of Soluble Protein Complexes

Structural and biophysical studies of protein complexes require multi-milligram quantities of soluble material. Subunits are often unstable when expressed separately so co-expression strategies are commonly employed since in vivo complex formation can provide stabilising effects. Defining constructs for subunit co-expression experiments is difficult if the proteins are poorly understood. Even more problematic is when subunit polypeptide chains co-fold since individually they do not have predictable domains. We have developed CoESPRIT, a modified version of the ESPRIT random library construct screen used previously on single proteins, to express soluble protein complexes. A random library of target constructs is screened against a fixed bait protein to identify stable complexes. In a proof-of-principle study, C-terminal fragments of the influenza polymerase PB2 subunit containing folded domains were isolated using importin alpha as bait. Separately, a C-terminal fragment of the PB1 subunit was used as bait to trap N-terminal fragments of PB2 resulting in co-folded complexes. Subsequent expression of the target protein without the bait indicates whether the target is independently stable, or co-folds with its partner. This highly automated method provides an efficient strategy for obtaining recombinant protein complexes at yields compatible with structural, biophysical and functional studies

Workforce preparation: the Biohealth computing model for Master and PhD students

Abstract The article addresses the strategic role of workforce preparation in the process of adoption of Systems Medicine as a driver of biomedical research in the new health paradigm. It reports on relevant initiatives, like CASyM, fostering Systems Medicine at EU level. The chapter focuses on the BioHealth Computing Program as a reference for multidisciplinary training of future systems-oriented researchers describing the productive interactions with the Synergy-COPD project

Inclusive photon production at forward rapidities in proton-proton collisions at s\sqrt{s} = 0.9, 2.76 and 7 TeV

See paper for full list of authors – 24 pages, 10 captioned figures, 4 tables, authors from page 19, figures at http://aliceinfo.cern.ch/ArtSubmission/node/1024International audienceThe multiplicity and pseudorapidity distributions of inclusive photons have been measured at forward rapidities ($2.3 < \eta < 3.9$) in proton-proton collisions at three center-of-mass energies, $\sqrt{s}=0.9$, 2.76 and 7 TeV using the ALICE detector. It is observed that the increase in the average photon multiplicity as a function of beam energy is compatible with both a logarithmic and a power-law dependence. The relative increase in average photon multiplicity produced in inelastic pp collisions at 2.76 and 7 TeV center-of-mass energies with respect to 0.9 TeV are 37.2% $\pm$ 0.3% (stat) $\pm$ 8.8% (sys) and 61.2% $\pm$ 0.3% (stat) $\pm$ 7.6% (sys), respectively. The photon multiplicity distributions for all center-of-mass energies are well described by negative binomial distributions. The multiplicity distributions are also presented in terms of KNO variables. The results are compared to model predictions, which are found in general to underestimate the data at large photon multiplicities, in particular at the highest center-of-mass energy. Limiting fragmentation behavior of photons has been explored with the data, but is not observed in the measured pseudorapidity range

Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR

<p>Abstract</p> <p>Background</p> <p>This study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for <it>Plasmodium </it>species-specific real-time PCR.</p> <p>Methods</p> <p>First, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag <it>Plasmodium falciparum</it>/Pan test) were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four <it>Plasmodium </it>species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples.</p> <p>Results</p> <p>Best results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of <it>P. falciparum </it>(n = 60), <it>Plasmodium vivax </it>(n = 10), <it>Plasmodium ovale </it>(n = 10) and <it>Plasmodium malariae </it>(n = 10). Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20) gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests.</p> <p>Conclusions</p> <p>RDTs are a reliable source of DNA for <it>Plasmodium </it>real-time PCR. This study demonstrates the best method of RDT fragment sampling for a wide range of RDT brands in combination with a simple and low cost extraction method, allowing RDT quality control.</p

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

The Athena X-ray Integral Field Unit (X-IFU)

The X-ray Integral Field Unit (X-IFU) is the high resolution X-ray spectrometer of the ESA Athena X-ray observatory. Over a field of view of 5' equivalent diameter, it will deliver X-ray spectra from 0.2 to 12 keV with a spectral resolution of 2.5 eV up to 7 keV on similar to 5 '' pixels. The X-IFU is based on a large format array of super-conducting molybdenum-gold Transition Edge Sensors cooled at similar to 90 mK, each coupled with an absorber made of gold and bismuth with a pitch of 249 mu m. A cryogenic anti-coincidence detector located underneath the prime TES array enables the non X-ray background to be reduced. A bath temperature of similar to 50 mK is obtained by a series of mechanical coolers combining 15K Pulse Tubes, 4K and 2K Joule-Thomson coolers which pre-cool a sub Kelvin cooler made of a He-3 sorption cooler coupled with an Adiabatic Demagnetization Refrigerator. Frequency domain multiplexing enables to read out 40 pixels in one single channel. A photon interacting with an absorber leads to a current pulse, amplified by the readout electronics and whose shape is reconstructed on board to recover its energy with high accuracy. The defocusing capability offered by the Athena movable mirror assembly enables the X-IFU to observe the brightest X-ray sources of the sky (up to Crab-like intensities) by spreading the telescope point spread function over hundreds of pixels. Thus the X-IFU delivers low pile-up, high throughput (> 50%), and typically 10 eV spectral resolution at 1 Crab intensities, i.e. a factor of 10 or more better than Silicon based X-ray detectors. In this paper, the current X-IFU baseline is presented, together with an assessment of its anticipated performance in terms of spectral resolution, background, and count rate capability. The X-IFU baseline configuration will be subject to a preliminary requirement review that is scheduled at the end of 2018. The X-IFU will be provided by an international consortium led by France, the Netherlands and Italy, with further ESA member state contributions from Belgium, Czech Republic, Finland, Germany, Ireland, Poland, Spain, Switzerland and contributions from Japan and the United States.Peer reviewe

Impact of Cerebral Microbleeds in Stroke Patients with Atrial Fibrillation

OBJECTIVES: Cerebral microbleeds are associated with the risks of ischemic stroke and intracranial hemorrhage, causing clinical dilemmas for antithrombotic treatment decisions. We aimed to evaluate the risks of intracranial hemorrhage and ischemic stroke associated with microbleeds in patients with atrial fibrillation treated with Vitamin K antagonists, direct oral anticoagulants, antiplatelets, and combination therapy (i.e. concurrent oral anticoagulant and antiplatelet) METHODS: We included patients with documented atrial fibrillation from the pooled individual patient data analysis by the Microbleeds International Collaborative Network. Risks of subsequent intracranial hemorrhage and ischemic stroke were compared between patients with and without microbleeds, stratified by antithrombotic use. RESULTS: A total of 7,839 patients were included. The presence of microbleeds was associated with an increased relative risk of intracranial hemorrhage (aHR 2.74, 95% confidence interval 1.76 - 4.26) and ischemic stroke (aHR 1.29, 95% confidence interval 1.04 - 1.59). For the entire cohort, the absolute incidence of ischemic stroke was higher than intracranial hemorrhage regardless of microbleeds burden. However, for the subgroup of patients taking combination of anticoagulant and antiplatelet therapy, the absolute risk of intracranial hemorrhage exceeded that of ischemic stroke in those with 2-4 microbleeds (25 vs 12 per 1,000 patient-years) and ≥11 microbleeds (94 vs 48 per 1,000 patient-years). INTERPRETATION: Patients with atrial fibrillation and high burden of microbleeds receiving combination therapy have a tendency of higher rate of intracranial hemorrhage than ischemic stroke, with potential for net harm. Further studies are needed to help optimize stroke preventive strategies in this high-risk group. This article is protected by copyright. All rights reserved

The Athena X-ray Integral Field Unit: a consolidated design for the system requirement review of the preliminary definition phase

The Athena X-ray Integral Unit (X-IFU) is the high resolution X-ray spectrometer, studied since 2015 for flying in the mid-30s on the Athena space X-ray Observatory, a versatile observatory designed to address the Hot and Energetic Universe science theme, selected in November 2013 by the Survey Science Committee. Based on a large format array of Transition Edge Sensors (TES), it aims to provide spatially resolved X-ray spectroscopy, with a spectral resolution of 2.5 eV (up to 7 keV) over an hexagonal field of view of 5 arc minutes (equivalent diameter). The X-IFU entered its System Requirement Review (SRR) in June 2022, at about the same time when ESA called for an overall X-IFU redesign (including the X-IFU cryostat and the cooling chain), due to an unanticipated cost overrun of Athena. In this paper, after illustrating the breakthrough capabilities of the X-IFU, we describe the instrument as presented at its SRR, browsing through all the subsystems and associated requirements. We then show the instrument budgets, with a particular emphasis on the anticipated budgets of some of its key performance parameters. Finally we briefly discuss on the ongoing key technology demonstration activities, the calibration and the activities foreseen in the X-IFU Instrument Science Center, and touch on communication and outreach activities, the consortium organisation, and finally on the life cycle assessment of X-IFU aiming at minimising the environmental footprint, associated with the development of the instrument. Thanks to the studies conducted so far on X-IFU, it is expected that along the design-to-cost exercise requested by ESA, the X-IFU will maintain flagship capabilities in spatially resolved high resolution X-ray spectroscopy, enabling most of the original X-IFU related scientific objectives of the Athena mission to be retained. (abridged).Comment: 48 pages, 29 figures, Accepted for publication in Experimental Astronomy with minor editin

The Athena X-ray Integral Field Unit: a consolidated design for the system requirement review of the preliminary definition phase

The Athena X-ray Integral Unit (X-IFU) is the high resolution X-ray spectrometer studied since 2015 for flying in the mid-30s on the Athena space X-ray Observatory. Athena is a versatile observatory designed to address the Hot and Energetic Universe science theme, as selected in November 2013 by the Survey Science Committee. Based on a large format array of Transition Edge Sensors (TES), X-IFU aims to provide spatially resolved X-ray spectroscopy, with a spectral resolution of 2.5 eV (up to 7 keV) over a hexagonal field of view of 5 arc minutes (equivalent diameter). The X-IFU entered its System Requirement Review (SRR) in June 2022, at about the same time when ESA called for an overall X-IFU redesign (including the X-IFU cryostat and the cooling chain), due to an unanticipated cost overrun of Athena. In this paper, after illustrating the breakthrough capabilities of the X-IFU, we describe the instrument as presented at its SRR (i.e. in the course of its preliminary definition phase, so-called B1), browsing through all the subsystems and associated requirements. We then show the instrument budgets, with a particular emphasis on the anticipated budgets of some of its key performance parameters, such as the instrument efficiency, spectral resolution, energy scale knowledge, count rate capability, non X-ray background and target of opportunity efficiency. Finally, we briefly discuss the ongoing key technology demonstration activities, the calibration and the activities foreseen in the X-IFU Instrument Science Center, touch on communication and outreach activities, the consortium organisation and the life cycle assessment of X-IFU aiming at minimising the environmental footprint, associated with the development of the instrument. Thanks to the studies conducted so far on X-IFU, it is expected that along the design-to-cost exercise requested by ESA, the X-IFU will maintain flagship capabilities in spatially resolved high resolution X-ray spectroscopy, enabling most of the original X-IFU related scientific objectives of the Athena mission to be retained. The X-IFU will be provided by an international consortium led by France, The Netherlands and Italy, with ESA member state contributions from Belgium, Czech Republic, Finland, Germany, Poland, Spain, Switzerland, with additional contributions from the United States and Japan.The French contribution to X-IFU is funded by CNES, CNRS and CEA. This work has been also supported by ASI (Italian Space Agency) through the Contract 2019-27-HH.0, and by the ESA (European Space Agency) Core Technology Program (CTP) Contract No. 4000114932/15/NL/BW and the AREMBES - ESA CTP No.4000116655/16/NL/BW. This publication is part of grant RTI2018-096686-B-C21 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. This publication is part of grant RTI2018-096686-B-C21 and PID2020-115325GB-C31 funded by MCIN/AEI/10.13039/501100011033

Twenty-three unsolved problems in hydrology (UPH) – a community perspective

This paper is the outcome of a community initiative to identify major unsolved scientific problems in hydrology motivated by a need for stronger harmonisation of research efforts. The procedure involved a public consultation through on-line media, followed by two workshops through which a large number of potential science questions were collated, prioritised, and synthesised. In spite of the diversity of the participants (230 scientists in total), the process revealed much about community priorities and the state of our science: a preference for continuity in research questions rather than radical departures or redirections from past and current work. Questions remain focussed on process-based understanding of hydrological variability and causality at all space and time scales. Increased attention to environmental change drives a new emphasis on understanding how change propagates across interfaces within the hydrological system and across disciplinary boundaries. In particular, the expansion of the human footprint raises a new set of questions related to human interactions with nature and water cycle feedbacks in the context of complex water management problems. We hope that this reflection and synthesis of the 23 unsolved problems in hydrology will help guide research efforts for some years to come