Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure

Understanding what works, why and in what circumstances in Hospice at Home Services for End of Life Care: applying a realist logic of analysis to a systematically searched literature review

Background: We have undertaken a systematically searched literature review using a realist logic of analysis to help synthesise the diverse range of literature available on hospice at home services. Aim: To find out in the existing literature what features of hospice at home models work best, for whom and under what circumstances. Design: A realist logic of analysis was applied to synthesise the evidence focusing on mechanisms by which an intervention worked (or did not work). An initial programme theory was developed using the National Association for Hospice at Home standards, Normalisation Process Theory and through refinement using stakeholder engagement. Data sources: PubMed, Science Direct, AMED, BNI, CINAHL, EMBASE, Health Business Elite, HMIC, Medline, PsychINFO, SCOPUS, Web of Science, DARE, Google Scholar, NHS Evidence, NIHR CRN portfolio database, NIHR journal library of funded studies, including searches on websites of relevant professional bodies [August 2014, June 2017, June 2019]. Results: Forty-nine papers were reviewed, of which 34 wereincluded contributing evidence to at least one of eight theory areas: marketing and referral, sustainable funding model, service responsiveness and availability, criteria for service admission, knowledge and skills of care providers, integration and co-ordination, anticipatory care, support directed at carers. Conclusions: Our literature review showed how it was possible to develop a coherent framework and test it against 34 published papers and abstracts. Central to this review was theory building, and as further evidence emerges, our programme theories can be refined and tested against any new empirical evidence

A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods

The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds, and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation may control its homeostasis and regulation of protein turnover is separable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final 2 positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCE The interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon-stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell and interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity

The mechanisms and microstructures of passive atmospheric CO2 mineralisation with slag at ambient conditions

Removal of CO2 already in the Earth's atmosphere through CO2 mineralisation with alkaline waste materials such as steel slag is one approach to mitigate the effects of anthropogenically-induced climate change. However, the microstructures produced during passive carbonation of slag are not well known. Here we use Scanning Electron Microscopy imaging and chemical mapping, X-Ray diffraction and stable isotopes (δ13C and δ18O) to show that ingassed and hydroxylated atmospheric CO2 reacts with Ca leached from slag to precipitate calcite directly on the slag surface. Precipitated calcite crystal morphologies vary, ranging from bladed and acicular crystals to layered deposits of micron-scale equant crystals. The variable morphology and extent of calcite precipitation documented is linked to a combination of internal (i.e. microstructural properties of the slag itself) and external (environmental conditions) factors. This work shows that atmospheric CO2 can be drawn down and mineralised passively by the slag at ambient conditions as part of the slag valorisation and reutilisation process

Attachment style moderates partner presence effects on pain : A laser-evoked potentials study

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly citedSocial support is crucial for psychological and physical well-being. Yet, in experimental and clinical pain research, the presence of others has been found to both attenuate and intensify pain. To investigate the factors underlying these mixed effects, we administered noxious laser stimuli to 39 healthy women while their romantic partner was present or absent, and measured pain ratings and laser-evoked potentials to assess the effects of partner presence on subjective pain experience and underlying neural processes. Further, we examined whether individual differences in adult attachment style, alone or in interaction with the partner's level of attentional focus (manipulated to be either on or away from the participant) might modulate these effects. We found that the effects of partner presence versus absence on pain-related measures depended on adult attachment style but not partner attentional focus. The higher participants' attachment avoidance, the higher pain ratings and N2 and P2 local peak amplitudes were in the presence compared to the absence of the romantic partner. As laser-evoked potentials are thought to reflect activity relating to the salience of events, our data suggest that partner presence may influence the perceived salience of events threatening the body, particularly in individuals who tend to mistrust others.Peer reviewedFinal Published versio

The mechanisms and drivers of lithification in slag‐dominated artificial ground

Unconsolidated artificial ground is an ever-increasing feature on the Earth's surface but it poses various challenges such as pollutant release and ground instability. The process of lithification could be an important factor in changing the properties of artificial ground and ameliorating these challenges. In this study, a lithified deposit of a furnace slag associated with a former iron and steel works in Scotland was analysed to determine the mechanisms and drivers of lithification. Scanning Electron Microscope analysis showed that Ca leached from around the edges of clasts of slag through reaction of the chemically unstable slag with water from an adjacent water body. Dissolution of Ca (and OH-) from the slag caused the water in contact with the slag to become hyperalkaline, facilitating ingassing and hydroxylation of CO2 from the atmosphere (fingerprinted through carbon isotope analysis). Reaction of the dissolved Ca and CO2 led to precipitation of calcite. Scanning Electron Microscope analysis showed the calcite is distributed between slag clasts, forming rims around clasts and cementing clasts together into a solid rock-like mass. Understanding the mechanisms and drivers of lithification in artificial ground will be important, given its widespread nature particularly in urban areas where artificial ground is the substrate of most development

Framework and guidelines for implementing the proposed IUCN Environmental Impact Classification for Alien Taxa (EICAT)

Recently, Blackburn et al. (2014) developed a simple, objective and transparent method for classifying alien taxa in terms of the magnitude of their detrimental environmental impacts in recipient areas. Here, we present a comprehensive framework and guidelines for implementing this method, which we term the Environmental Impact Classification for Alien Taxa, or EICAT. We detail criteria for applying the EICAT scheme in a consistent and comparable fashion, prescribe the supporting information that should be supplied along with classifications, and describe the process for implementing the method. This comment aims to draw the attention of interested parties to the framework and guidelines, and to present them in their entirety in a location where they are freely accessible to any potential users.Peer Reviewe

Mapping the Transcriptome Underpinning Acute Corticosteroid Action within the Cortical Collecting Duct

Funding: British Heart Foundation (BHF): Research Excellence Award RE/13/3/30183; Kidney Research UK: Innovation Grant IN_001_201703 Postdoctoral Fellowship PDF_008_20151127; Scottish Funding Council (SFC): St Andrews Restarting Research Funding Scheme; Society for Endocrinology (SFE): Early Career Grant.We report the transcriptomes associated with acute corticosteroid regulation of ENaC activity in polarised mCCDcl1 collecting duct cells. 9 genes were regulated by aldosterone (ALDO), 0 with corticosterone alone and 151 with corticosterone when 11βHSD2 activity was inhibited. We validated 3 novel ALDO-induced genes: Rasd1, Sult1d1 and Gm43305 in primary cells isolated from a novel collecting duct reporter mouse. Background Corticosteroids regulate distal nephron and collecting duct Na+ reabsorption, contributing to fluid-volume and blood pressure homeostasis. The transcriptional landscape underpinning the acute stimulation of the epithelial sodium channel (ENaC) by physiological concentrations of corticosteroids remains unclear. Methods Transcriptomic profiles underlying corticosteroid-stimulated ENaC activity in polarised mCCDcl1 cells were generated by coupling electrophysiological measurements of amiloride-sensitive currents with RNAseq. Generation of a collecting-duct specific reporter mouse line, mT/mG-Aqp2Cre, enabled isolation of primary collecting duct cells by FACS and ENaC activity was measured in cultured primary cells following acute application of corticosteroids. Expression of target genes was assessed by qRT-PCR in cultured cells or freshly isolated cells following acute elevation of steroid hormones in mT/mG-Aqp2Cre mice. Results Physiological relevance of the mCCDcl1 model was confirmed with aldosterone-specific stimulation of SGK1 and ENaC activity. Corticosterone only modulated these responses at supraphysiological concentrations or when 11βHSD2 was inhibited. When 11βHSD2 protection was intact, corticosterone caused no significant change in transcripts. We identified a small number of aldosterone-induced transcripts associated with stimulated ENaC activity in mCCDcl1 cells and a much larger number with corticosterone in the absence of 11βHSD2 activity. Cells isolated from mT/mG-Aqp2Cre mice were validated as collecting duct-specific and assessment of identified aldosterone-induced genes revealed that Sgk1, Zbtbt16, Sult1d1, Rasd1 and Gm43305 are acutely upregulated by corticosteroids both in vitro and in vivo. Conclusions This study reports the transcriptome of mCCDcl1 collecting duct cells and identifies a small number of aldosterone-induced genes associated with acute stimulation of ENaC, including 3 previously undescribed genes.PostprintPeer reviewe