Homogeneous batch micro-crystallization of proteins from ammonium sulfate

The emergence of X-ray free-electron lasers has led to the development of serial macromolecular crystallography techniques, making it possible to study smaller and more challenging crystal systems and to perform time-resolved studies on fast time scales. For most of these studies the desired crystal size is limited to a few micrometres, and the generation of large amounts of nanocrystals or microcrystals of defined size has become a bottleneck for the wider implementation of these techniques. Despite this, methods to reliably generate microcrystals and fine-tune their size have been poorly explored. Working with three different enzymes, L-aspartate alpha-decarboxylase, copper nitrite reductase and copper amine oxidase, the precipitating properties of ammonium sulfate were exploited to quickly transition from known vapour-diffusion conditions to reproducible, large-scale batch crystallization, circumventing the tedious determination of phase diagrams. Furthermore, the specific ammonium sulfate concentration was used to fine-tune the crystal size and size distribution. Ammonium sulfate is a common precipitant in protein crystallography, making these findings applicable to many crystallization systems to facilitate the production of large amounts of microcrystals for serial macromolecular crystallography experiments.Peer reviewe

Relative affinities of protein–cholesterol interactions from equilibrium molecular dynamics simulations

Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-electron microscopy (cryo-EM) densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities using experimental or simulation methods. Here, we develop a method for determining protein–lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in, e.g., cryo-EM derived membrane protein structures surrounded by an array of ancillary lipid densities

Collaborative action for person-centred coordinated care (P3C): an approach to support the development of a comprehensive system-wide solution to fragmented care

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: Fragmented care results in poor outcomes for individuals with complexity of need. Person-centred coordinated care (P3C) is perceived to be a potential solution, but an absence of accessible evidence and the lack of a scalable 'blue print' mean that services are 'experimenting' with new models of care with little guidance and support. This paper presents an approach to the implementation of P3C using collaborative action, providing examples of early developments across this programme of work, the core aim of which is to accelerate the spread and adoption of P3C in United Kingdom primary care settings. METHODS: Two centrally funded United Kingdom organisations (South West Collaboration for Leadership in Applied Health Research and Care and South West Academic Health Science Network) are leading this initiative to narrow the gap between research and practice in this urgent area of improvement through a programme of service change, evaluation and research. Multi-stakeholder engagement and co-design are core to the approach. A whole system measurement framework combines outcomes of importance to patients, practitioners and health organisations. Iterative and multi-level feedback helps to shape service change while collecting practice-based data to generate implementation knowledge for the delivery of P3C. The role of the research team is proving vital to support informed change and challenge organisational practice. The bidirectional flow of knowledge and evidence relies on the transitional positioning of researchers and research organisations. RESULTS: Extensive engagement and embedded researchers have led to strong collaborations across the region. Practice is beginning to show signs of change and data flow and exchange is taking place. However, working in this way is not without its challenges; progress has been slow in the development of a linked data set to allow us to assess impact innovations from a cost perspective. Trust is vital, takes time to establish and is dependent on the exchange of services and interactions. If collaborative action can foster P3C it will require sustained commitment from both research and practice. This approach is a radical departure from how policy, research and practice traditionally work, but one that we argue is now necessary to deal with the most complex health and social problems.This research was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care South West Peninsula at the Royal Devon and Exeter NHS Foundation Trust. TS’s current post at the University of Oxford is funded by the NIHR Collaboration for Leadership in Applied Health Research and Care Oxford at Oxford Health NHS Foundation Trust. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. This research was also funded by the South West Academic health Science Network (AHSN). The views expressed are those of the authors and not necessarily those of the AHSN.The NIHR CLAHRC Programme fund the substantive posts of HML, RB, NB, HW, TPS and MP as members of the South West Peninsula CLARHC. All other co-authors are members of the wider collaboration and are substantially employed by their respective organisations

From Duty to Right: The Role of Public Education in the Transition to Aging Societies

This paper argues that the introduction of compulsory schooling in early industrialization promoted the growth process that eventually led to a vicious cycle of population aging and negative pressure on education policy. In the early phases of industrialization, public education was undesirable for the young poor who relied on child labor. Compulsory schooling therefore discouraged childbirth, while the accompanying industrialization stimulated their demand for education. The subsequent rise in the share of the old population, however, limited government resources for education, placing heavier financial burdens on the young. This induced further fertility decline and population aging, and the resulting cycle may have delayed the growth of advanced economies in the last few decades

Neutral sphingomyelinase-3 mediates TNF-stimulated oxidant activity in skeletal muscle

Aims: Sphingolipid and oxidant signaling affect glucose uptake, atrophy, and force production of skeletal muscle similarly and both are stimulated by tumor necrosis factor (TNF), suggesting a connection between systems. Sphingolipid signaling is initiated by neutral sphingomyelinase (nSMase), a family of agonist-activated effector enzymes. Northern blot analyses suggest that nSMase3 may be a striated muscle-specific nSMase. The present study tested the hypothesis that nSMase3 protein is expressed in skeletal muscle and functions to regulate TNF-stimulated oxidant production. Results: We demonstrate constitutive nSMase activity in skeletal muscles of healthy mice and humans and in differentiated C2C12 myotubes. nSMase3 (Smpd4 gene) mRNA is highly expressed in muscle. An nSMase3 protein doublet (88 and 85 kD) is derived from alternative mRNA splicing of exon 11. The proteins partition differently. The full-length 88 kD isoform (nSMase3a) fractionates with membrane proteins that are resistant to detergent extraction; the 85 kD isoform lacking exon 11 (nSMase3b) is more readily extracted and fractionates with detergent soluble membrane proteins; neither variant is detected in the cytosol. By immunofluorescence microscopy, nSMase3 resides in both internal and sarcolemmal membranes. Finally, myotube nSMase activity and cytosolic oxidant activity are stimulated by TNF. Both if these responses are inhibited by nSMase3 knockdown. Innovation: These findings identify nSMase3 as an intermediate that links TNF receptor activation, sphingolipid signaling, and skeletal muscle oxidant production. Conclusion: Our data show that nSMase3 acts as a signaling nSMase in skeletal muscle that is essential for TNF-stimulated oxidant activity

PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets1. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P2 binding. Endogenous lipids were also observed bound directly to the trimeric Gαsβγ protein complex of the adenosine A2A receptor (A2AR) in the gas phase. Using engineered Gα subunits (mini-Gαs, mini-Gαi and mini-Gα12)2, we demonstrate that the complex of mini-Gαs with the β1 adrenergic receptor (β1AR) is stabilized by the binding of two PtdIns(4,5)P2 molecules. By contrast, PtdIns(4,5)P2 does not stabilize coupling between β1AR and other Gα subunits (mini-Gαi or mini-Gα12) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P2. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαiβγ complex in the presence of PtdIns(4,5)P2 provide further evidence for a specific effect of PtdIns(4,5)P2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs