Investigating mechanisms of triacylglycerol synthesis induced by nitrogen depletion in the diatom Phaeodactylum tricornutum and characterising its diacylglycerol acyltransferases and phospholipid: diacylglycerol acyl transferase

The ability of the diatom Phaeodactylum tricornutum (P. tricornutum) to accumulate triacylglycerol (TAG) and synthesise the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) has led to interest in their development for biofuels and human nutrition. Nitrogen depletion is a well-studied experimental system for producing high levels of TAG in P. tricornutum. Identication of the mechanisms involved in TAG synthesis holds promise for increasing TAG yields. Numerous studies of nitrogen depletion have been carried out that combine physiological and transcriptomic or proteomic measurements. These studies provide insight into global and local metabolic responses but experimental setups varied. To identify a consistent response, experimental conditions and physiological changes were compiled and transcripts and proteins were functionally grouped in a systematic review. Comprehensive analysis of lipid metabolism and autophagy genes was undertaken. Analysis of regulated genes supports involvement of central pathways and lipid metabolism in TAG accumulation: acetyl-CoA synthesis may increase while FA synthesis, the Kennedy pathway, malonyl-CoA/ACP transacylase, plastidial desaturase and FA transport enzymes were upregulated. Desaturases, elongases, Lands cycle enzymes and long chain acyl-CoA synthases are targets for overexpression to increase EPA production during nitrogen depletion. Several lipidomic experiments have highlighted the potential for TAG synthesis from chloroplast lipids, particularly monogalactosyl diacylglycerol (MGDG), during disassembly of thylakoid membranes under N-depletion. Identifying MGDG derived TAG accumulation may yield enzymes that target EPA to TAG and thereby enable use of developed extraction processes. Some archaeplastida also synthesise TAG from MGDG in nitrogen depletion and freezing stress. A similar response in diatoms would demonstrate this metabolic feature in diverse photosynthetic lineages. In this thesis, the fatty acid synthase inhibitor cerulenin was used to probe TAG accumulation during N-depletion. Neutral lipid staining data support the possibility of an MGDG to TAG route but were not conclusive. Concurrently, candidate enzymes were identied based on conserved sequence domains and experimentally characterised genes from Chlamydomonas reinhardtii (C. reinhardtii ) and Arabidopsis thaliana (A. thaliana). P. tricornutum was subsequently transformed with acyl hydrolase:YFP constructs. Confocal microscopy supported locations of two acyl hydrolases: in the endoplasmic reticulum (ER) and chloroplast ER for Phatr3 J44028 and in the ER or cytoplasm for Phatr3 J41624. P. tricornutum encodes multiple isoforms of the TAG synthesis enzymes diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). Previous studies supported functionality of multiple PtDGATs and dierential regulation and predicted locations suggest non-redundancy. To investigate their role, overexpression of multiple DGATs and an N-terminal truncated PDAT was induced under nitrogen depletion. Total lipid extraction was carried out and species and FA composition of TAG was measured using tandem mass spectrometry. Total TAG was not altered. Overexpression of DGAT2B decreased the proportion of the 48:2 (carbon atoms:double bonds) TAG species and increased 50:2 and 50:3 while palmitic and oleic acid formed an increased and decreased proportion of TAG respectively. Overexpression of PDAT decreased the 48:1 and 48:2 TAG species, decreased the proportion of TAG composed of palmitic acid and increased the amount composed of EPA. Knowledge of typical TAG species composition in P. tricornutum supported respective preferences for incorporation of 18 carbon acyl-CoAs and EPA by DGAT2B and PDAT and their use of C16/C16 diacylglycerols.Sponsored as part of a BBSRC sLOLA gran

System-wide approaches to antimicrobial therapy and antimicrobial resistance in the UK:the AMR-X framework

Antimicrobial resistance (AMR) threatens human, animal, and environmental health. Acknowledging the urgency of addressing AMR, an opportunity exists to extend AMR action-focused research beyond the confines of an isolated biomedical paradigm. An AMR learning system, AMR-X, envisions a national network of health systems creating and applying optimal use of antimicrobials on the basis of their data collected from the delivery of routine clinical care. AMR-X integrates traditional AMR discovery, experimental research, and applied research with continuous analysis of pathogens, antimicrobial uses, and clinical outcomes that are routinely disseminated to practitioners, policy makers, patients, and the public to drive changes in practice and outcomes. AMR-X uses connected data-to-action systems to underpin an evaluation framework embedded in routine care, continuously driving implementation of improvements in patient and population health, targeting investment, and incentivising innovation. All stakeholders co-create AMR-X, protecting the public from AMR by adapting to continuously evolving AMR threats and generating the information needed for precision patient and population care.</p

System-wide approaches to antimicrobial therapy and antimicrobial resistance in the UK:the AMR-X framework

Antimicrobial resistance (AMR) threatens human, animal, and environmental health. Acknowledging the urgency of addressing AMR, an opportunity exists to extend AMR action-focused research beyond the confines of an isolated biomedical paradigm. An AMR learning system, AMR-X, envisions a national network of health systems creating and applying optimal use of antimicrobials on the basis of their data collected from the delivery of routine clinical care. AMR-X integrates traditional AMR discovery, experimental research, and applied research with continuous analysis of pathogens, antimicrobial uses, and clinical outcomes that are routinely disseminated to practitioners, policy makers, patients, and the public to drive changes in practice and outcomes. AMR-X uses connected data-to-action systems to underpin an evaluation framework embedded in routine care, continuously driving implementation of improvements in patient and population health, targeting investment, and incentivising innovation. All stakeholders co-create AMR-X, protecting the public from AMR by adapting to continuously evolving AMR threats and generating the information needed for precision patient and population care.</p

Rječnik hrvatskoga ili srpskoga jezika dovršen

Simple protocols have been devised for peroxyoxalate chemiluminescence demonstrations that use urea–hydrogen peroxide or sodium percarbonate as oxidants in place of aqueous solutions of hydrogen peroxide. The procedures described are compatible with well-known peroxyoxalates and fluorescers and have been found to be convenient for use for outreach activities in venues with limited laboratory facilities.PostprintPeer reviewe

Improving care for older people with long-term conditions and social care needs in Salford : the CLASSIC mixed-methods study, including RCT

BackgroundThe Salford Integrated Care Programme (SICP) was a large-scale transformation project to improve care for older people with long-term conditions and social care needs. We report an evaluation of the ability of the SICP to deliver an enhanced experience of care, improved quality of life, reduced costs of care and improved cost-effectiveness.ObjectivesTo explore the process of implementation of the SICP and the impact on patient outcomes and costs.DesignQualitative methods (interviews and observations) to explore implementation, a cohort multiple randomised controlled trial to assess patient outcomes through quasi-experiments and a formal trial, and an analysis of routine data sets and appropriate comparators using non-randomised methodologies.SettingSalford in the north-west of England.ParticipantsOlder people aged ≥ 65 years, carers, and health and social care professionals.InterventionsA large-scale integrated care project with three core mechanisms of integration (community assets, multidisciplinary groups and an ‘integrated contact centre’).Main outcome measuresPatient self-management, care experience and quality of life, and health-care utilisation and costs.Data sourcesProfessional and patient interviews, patient self-report measures, and routine quantitative data on service utilisation.ResultsThe SICP and subsequent developments have been sustained by strong partnerships between organisations. The SICP achieved ‘functional integration’ through the pooling of health and social care budgets, the development of the Alliance Agreement between four organisations and the development of the shared care record. ‘Service-level’ integration was slow and engagement with general practice was a challenge. We saw only minor changes in patient experience measures over the period of the evaluation (both improvements and reductions), with some increase in the use of community assets and care plans. Compared with other sites, the difference in the rates of admissions showed an increase in emergency admissions. Patient experience of health coaching was largely positive, although the effects of health coaching on activation and depression were not statistically significant. Economic analyses suggested that coaching was likely to be cost-effective, generating improvements in quality of life [mean incremental quality-adjusted life-year gain of 0.019, 95% confidence interval (CI) –0.006 to 0.043] at increased cost (mean incremental total cost increase of £150.58, 95% CI –£470.611 to £711.776).LimitationsThe Comprehensive Longitudinal Assessment of Salford Integrated Care study represents a single site evaluation, with consequent limits on external validity. Patient response rates to the cohort survey were ConclusionsThe SICP has been implemented in a way that is consistent with the original vision. However, there has been more rapid success in establishing new integrated structures (such as a formal integrated care organisation), rather than in delivering mechanisms of integration at sufficient scale to have a large impact on patient outcomes.Future workFurther research could focus on each of the mechanisms of integration. The multidisciplinary groups may require improved targeting of patients or disease subgroups to demonstrate effectiveness. Development of a proven model of health coaching that can be implemented at scale is required, especially one that would provide cost savings for commissioners or providers. Similarly, further exploration is required to assess the longer-term benefits of community assets and whether or not health impacts translate to reductions in care use.Trial registrationCurrent Controlled Trials ISRCTN12286422.FundingThis project was funded by the NIHR Health Services and Delivery Research programme and will be published in full in Health Services and Delivery Research; Vol. 6, No. 31. See the NIHR Journals Library website for further project information

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic

First Sagittarius A* Event Horizon Telescope results. II. EHT and multiwavelength observations, data processing, and calibration

We present Event Horizon Telescope (EHT) 1.3 mm measurements of the radio source located at the position of the supermassive black hole Sagittarius A* (Sgr A*), collected during the 2017 April 5–11 campaign. The observations were carried out with eight facilities at six locations across the globe. Novel calibration methods are employed to account for Sgr A*'s flux variability. The majority of the 1.3 mm emission arises from horizon scales, where intrinsic structural source variability is detected on timescales of minutes to hours. The effects of interstellar scattering on the image and its variability are found to be subdominant to intrinsic source structure. The calibrated visibility amplitudes, particularly the locations of the visibility minima, are broadly consistent with a blurred ring with a diameter of ∼50 μas, as determined in later works in this series. Contemporaneous multiwavelength monitoring of Sgr A* was performed at 22, 43, and 86 GHz and at near-infrared and X-ray wavelengths. Several X-ray flares from Sgr A* are detected by Chandra, one at low significance jointly with Swift on 2017 April 7 and the other at higher significance jointly with NuSTAR on 2017 April 11. The brighter April 11 flare is not observed simultaneously by the EHT but is followed by a significant increase in millimeter flux variability immediately after the X-ray outburst, indicating a likely connection in the emission physics near the event horizon. We compare Sgr A*'s broadband flux during the EHT campaign to its historical spectral energy distribution and find that both the quiescent emission and flare emission are consistent with its long-term behavior.http://iopscience.iop.org/2041-8205Physic

First Sagittarius A* Event Horizon Telescope Results. II. EHT and Multiwavelength Observations, Data Processing, and Calibration

We present Event Horizon Telescope (EHT) 1.3 mm measurements of the radio source located at the position of the supermassive black hole Sagittarius A* (Sgr A*), collected during the 2017 April 5–11 campaign. The observations were carried out with eight facilities at six locations across the globe. Novel calibration methods are employed to account for Sgr A*'s flux variability. The majority of the 1.3 mm emission arises from horizon scales, where intrinsic structural source variability is detected on timescales of minutes to hours. The effects of interstellar scattering on the image and its variability are found to be subdominant to intrinsic source structure. The calibrated visibility amplitudes, particularly the locations of the visibility minima, are broadly consistent with a blurred ring with a diameter of ∼50 μas, as determined in later works in this series. Contemporaneous multiwavelength monitoring of Sgr A* was performed at 22, 43, and 86 GHz and at near-infrared and X-ray wavelengths. Several X-ray flares from Sgr A* are detected by Chandra, one at low significance jointly with Swift on 2017 April 7 and the other at higher significance jointly with NuSTAR on 2017 April 11. The brighter April 11 flare is not observed simultaneously by the EHT but is followed by a significant increase in millimeter flux variability immediately after the X-ray outburst, indicating a likely connection in the emission physics near the event horizon. We compare Sgr A*’s broadband flux during the EHT campaign to its historical spectral energy distribution and find that both the quiescent emission and flare emission are consistent with its long-term behavior

Alternative hydrogen peroxide sources for peroxyoxalate “glowstick” chemiluminescence demonstrations

Simple protocols have been devised for peroxyoxalate chemiluminescence demonstrations that use urea–hydrogen peroxide or sodium percarbonate as oxidants in place of aqueous solutions of hydrogen peroxide. The procedures described are compatible with well-known peroxyoxalates and fluorescers and have been found to be convenient for use for outreach activities in venues with limited laboratory facilities