Tracking autophagy during proliferation and differentiation of trypanosoma brucei

Autophagy is a lysosome-dependent degradation mechanism that sequesters target cargo into autophagosomal vesicles. The Trypanosoma brucei genome contains apparent orthologues of several autophagy-related proteins including an ATG8 family. These ubiquitin-like proteins are required for autophagosome membrane formation, but our studies show that ATG8.3 is atypical. To investigate the function of other ATG proteins, RNAi compatible T. brucei were modified to function as autophagy reporter lines by expressing only either YFP-ATG8.1 or YFP-ATG8.2. In the insect procyclic lifecycle stage, independent RNAi down-regulation of ATG3 or ATG7 generated autophagy-defective mutants and confirmed a pro-survival role for autophagy in the procyclic form nutrient starvation response. Similarly, RNAi depletion of ATG5 or ATG7 in the bloodstream form disrupted autophagy, but did not impede proliferation. Further characterisation showed bloodstream form autophagy mutants retain the capacity to undergo the complex cellular remodelling that occurs during differentiation to the procyclic form and are equally susceptible to dihydroxyacetone-induced cell death as wild type parasites, not supporting a role for autophagy in this cell death mechanism. The RNAi reporter system developed, which also identified TOR1 as a negative regulator controlling YFP-ATG8.2 but not YFP-ATG8.1 autophagosome formation, will enable further targeted analysis of the mechanisms and function of autophagy in the medically relevant bloodstream form of T. brucei

Characterisation of autophagy and a metacaspase in Trypanosoma brucei

This project focuses on the characterisation of two separate aspects of Trypanosoma brucei cell biology; the degradative process of autophagy and a specific cysteine peptidase from the metacaspase family. Autophagy is a widely conserved intracellular mechanism for the degradation of long lived proteins and organelles, that requires the formation of an autophagosome (double membrane bound vesicle) around cargo destined for the lysosome. The molecular machinery involved in autophagy has been well characterised in yeast and bioinformatic screens have identified many of the core components in T. brucei. However, beyond bioinformatics there is limited experimental evidence to support the presence of functional autophagy in T. brucei. A key component of the autophagic pathway is ATG8, a ubiquitin-like protein that is incorporated into the autophagosome membrane. To investigate autophagy in T. brucei the three candidate ATG8 genes (ATG8.1 Tb927.7.5900, ATG8.2 Tb927.7.5910 and ATG8.3 Tb927.7.3320) were fused with yellow fluorescent protein (YFP) and expressed in bloodstream form and procyclic form T. brucei cultured parasites. Fluorescent microscopy was used to monitor the formation of YFP-labelled autophagosomes, which enabled the evaluation of the autophagic response towards a variety of different stimuli. We provide the first direct experimental evidence confirming a functional autophagy pathway in T. brucei and show that it is induced in response to nutrient starvation in the procyclic form and neuropeptide treatment in the bloodstream form and can be blocked by the classical autophagy inhibitor wortmannin. Characterisation of the T. brucei ATG8 family revealed that ATG8.1 and ATG8.2 appear to operate as ‘ATG8-like’ proteins, whereas ATG8.3 behaves atypically, possibly functioning as an ATG12 protein. Furthermore, targeted RNAi downregulation of the predicted T. brucei ATG3 (Tb927.2.1890) caused a reduction in cell growth. The vital role of ATG3 in the autophagy pathway suggested that the process was required for normal procyclic form growth. The second focus of the project was metacaspase 4 (Tb927.10.2440) which belongs to the metacaspases (MCAs), cysteine peptidases of the caspase family found in plants, fungi and protozoa, but absent from mammals. Of the five MCAs possessed by T. brucei, only MCA2, MCA3 and MCA5 contain the conserved histidine cysteine catalytic dyad. MCA1 and MCA4 are predicted to contain key substitutions within their active sites, raising interesting questions regarding potential peptidase activity and functions. The exact role of the T. brucei metacaspases remains largely unknown, with MCA2 , MCA3 and MCA5 appearing to function in association with RAB11 positive endosomes, although independently of the known recycling functions of these endosomes (Helms et al. 2006). To develop our understanding of the MCA family in T. brucei a study into the function of MCA4 was undertaken. An antibody was raised against MCA4 and western blotting of cell lysate revealed that MCA4 expression occurred only in bloodstream form T. brucei. Interestingly MCA4 localised to the flagellar membrane, appearing in a linear array of punctate structures. Dual acylation is known to mediate flagellar membrane association in T. brucei and was implicated in MCA4 targeting following bioinformatic predictions and subsequent experimental confirmation of MCA4 palmitoylation using an acyl-biotin exchange reaction. MCA4 contains a non-canonical active site residue (serine-219) in the position of the predicted conserved active site cysteine. Activity assays using purified recombinant protein revealed that full length MCA4 was unable to autoprocess and was inactive. However, MCA4 peptidase activity could be detected following proteolytic activation with MCA2. Interestingly, removal of the MCA4 active site serine by mutagenesis (MCA4S219G) did not abolish activity of the processed enzyme, revealing an alternative nucleophile was capable of contributing to activity. Furthermore, mutation of the active site serine to cysteine, produced a constitutively active peptidase capable of autolytic processing in a calcium dependent manner. Following these key findings the role of MCA4 in the T. brucei lifecycle was investigated by RNAi and genetic knockout. Rapid depletion of MCA4 by RNAi caused a block in cytokinesis followed by cell death, nevertheless the generation of MCA4 null mutant parasites (∆mca4) was possible. A role for MCA4 in mammalian infection was revealed by monitoring infection progression in mice. Deletion of MCA4 increased host survival and parasite virulence could be restored by the ectopic re-expression of MCA4 in ∆mca4 parasites

Potential excess spend in primary care due to NHS drug tariff variability in vitamin D preparations

Objectives Vitamin D is commonly prescribed in primary care for the prevention and treatment of deficiency and for maintenance after treatment (although supplementation for maintenance and prevention can be bought over-the-counter). There is wide variation in the costs to the NHS in England of oral preparations of vitamin D, even for a single-specific dose and route.1 It is possible that the availability of multiple options for the same intended medicine, the costs of which are unlikely to be known by the prescriber, could result in an inadvertent excess spend. We aimed to estimate the annual cost-saving if only the cheapest vitamin D preparations were prescribed. Design Primary care prescribing data for 2018 were downloaded from NHS Digital (https://digital.nhs.uk).2 Monthly datasets include the number of items, quantity and cost of each drug prescribed and dispensed. Private prescriptions are not recorded. All prescription items relating to the vitamin D preparations colecalciferol and ergocalciferol, regardless of dose, route or manufacturer, were extracted. Data for each specific preparation were aggregated across all practices and all months to give the annual number of items and their cost across England. All liquid and injectable preparations were assumed to be appropriately prescribed and therefore excluded from the analysis. Combined preparations – such as calcium/vitamin D, calcium/alendronate and multivitamins – were also excluded. For each defined dose range of vitamin D, the lowest cost preparation was identified, and the potential cost-savings if only these preparations were prescribed was calculated. Setting Primary care in England. Participants All patients registered with a general practitioner in England in 2018. Main Outcome Measure The difference between actual and potential spend on vitamin D prescriptions, if only the lowest priced preparation were available. Results In 2018, over 4 million vitamin D items were prescribed in primary care, at a cost of over £21 million. If only the cheapest options were prescribed for non-liquid preparations across all dose ranges, and assuming all prescriptions were appropriate, it would have resulted in an approximate £15 million (>70%) cost-saving to the NHS. Maintenance doses of vitamin D (designated as 800 to 2000 international units per day as per NICE guidelines3) accounted for more than half of the spend (∼£12 million). It was noted that all the cheapest options available are suitable for vegetarians. Conclusion The prescribing of more expensive vitamin D preparations in primary care may have significant financial consequences, although our results relate to a single year of prescribing within NHS primary care. Since choice of preparation may intentionally be based on factors other than cost (e.g. dietary requirements, bioavailability, what was initially prescribed by a hospital), work is needed nationally to rationalise available prescribing options. If national guidance were produced, the difficulties of implementation within individual Clinical Commissioning Groups might limit economic benefit. National efficient procurement strategies are an alternative approach, but require careful consideration of legislative frameworks (such as the Public Contract Regulations 20154) with safeguards to prevent horizontal cooperation between suppliers

Guidance impact on primary care prescribing rates of simple analgesia: an interrupted time series analysis in England

Background: In March 2018, NHS England published guidance for Clinical Commissioning Groups (CCGs; NHS bodies that commission health services for local areas) to encourage implementation of policy to reduce primary care prescriptions of over-the-counter medications, including simple analgesia. Aims: To investigate: the impact of guidance publication on prescribing rates of simple analgesia (oral paracetamol, oral ibuprofen and topical non-steroidal anti-inflammatory drugs [NSAIDS]) in primary care; CCG implementation intentions; and whether it has created a health inequality based on socioeconomic status. Design and Setting: Interrupted time series analysis of primary care prescribing data in England. Methods: Practice-level prescribing data from January 2015 to March 2019 were obtained from NHS Digital. Interrupted time series analyses assessed the association of guidance publication with prescribing rates. The association between practice-level prescribing rates and Index of Multiple Deprivation score (a marker of socioeconomic deprivation) before and after publication was quantified using multivariable Poisson regression. Freedom of information requests were submitted to all CCGs. Results: There was a 4% reduction in prescribing of simple analgesia following guidance publication (adjusted incidence rate ratio [aIRR] 0.96, 95% CI 0.92-0.99, p=0.027), adjusting for underlying time trend and seasonality. Practice-level prescribing rates were greater in more deprived areas. There was considerable diversity across CCGs in whether or how they chose to implement the guidance. Conclusion: Guidance publication was associated with a small reduction in the prescribing rates of simple analgesia across England, without evidence of creating an additional health inequality. Careful implementation by CCGs would be required to optimise cost-saving to the NHS

A systematic review of the burden of hypertension, access to services and patient views of hypertension in humanitarian crisis settings

Introduction Globally, a record number of people are affected by humanitarian crises caused by conflict and natural disasters. Many such populations live in settings where epidemiological transition is underway. Following the United Nations high level meeting on non-communicable diseases, the global commitment to Universal Health Coverage and needs expressed by humanitarian agencies, there is increasing effort to develop guidelines for the management of hypertension in humanitarian settings. The objective was to investigate the prevalence and incidence of hypertension in populations directly affected by humanitarian crises; the cascade of care in these populations and patient knowledge of and attitude to hypertension. Methods A literature search was carried out in five databases. Grey literature was searched. The population of interest was adult, non-pregnant, civilians living in any country who were directly exposed to a crisis since 1999. Eligibility assessment, data extraction and quality appraisal were carried out in duplicate. Results Sixty-one studies were included in the narrative synthesis. They reported on a range of crises including the wars in Syria and Iraq, the Great East Japan Earthquake, Hurricane Katrina and Palestinian refugees. There were few studies from Africa or Asia (excluding Japan). The studies predominantly assessed prevalence of hypertension. This varied with geography and age of the population. Access to care, patient understanding and patient views on hypertension were poorly examined. Most of the studies had a high risk of bias due to methods used in the diagnosis of hypertension and in the selection of study populations. Conclusion Hypertension is seen in a range of humanitarian settings and the burden can be considerable. Further studies are needed to accurately estimate prevalence of hypertension in crisis-affected populations throughout the world. An appreciation of patient knowledge and understanding of hypertension as well as the cascade of care would be invaluable in informing service provision

A systematic review of the burden of, access to services for and perceptions of patients with overweight and obesity, in humanitarian crisis settings

Introduction: Excess body weight causes 4 million deaths annually across the world. The number of people affected by humanitarian crises stands at a record high level with 1 in 95 people being forcibly displaced. These epidemics overlap. Addressing obesity is a post-acute phase activity in non-communicable disease management in humanitarian settings. Information is needed to inform guidelines and timing of interventions. The objective of this review was to explore the prevalence of overweight and obesity in populations directly affected by humanitarian crises; the cascade of care in these populations and perceptions of patients with overweight and obesity. Methods: Literature searches were carried out in five databases. Grey literature was identified. The population of interest was non-pregnant, civilian adults who had experience of humanitarian crises (armed conflict, complex emergencies and natural disasters). All study types published from January 1st, 2011, were included. Screening, data extraction and quality appraisal were carried out in duplicate. A narrative synthesis is presented. Results: Fifty-six reports from forty-five studies were included. Prevalence estimates varied widely across the studies and by subgroups. Estimates of overweight and obesity combined ranged from 6.4% to 82.8%. Studies were heterogenous. Global distribution was skewed. Increasing adiposity was seen over time, in older adults and in women. Only six studies were at low risk of bias. Body mass index was the predominant measure used. There were no studies reporting cascade of care. No qualitative studies were identified. Conclusion: Overweight and obesity varied in crisis affected populations but were rarely absent. Improved reporting of existing data could provide more accurate estimates. Worsening obesity may be prevented by acting earlier in long-term crises and targeting risk groups. The use of waist circumference would provide useful additional information. Gaps remain in understanding the existing cascade of care. Cultural norms around diet and ideal body size vary

Author Correction: Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21491-y</jats:p

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand

Abstract: Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum

De la poésie nationale au prisme du roman d’exilMadre piccola de Cristina Ali Farah

L’article se propose d’explorer le rapport paradoxal que l’auteure italo-somalienne Cristina Ali Farah établit avec la poésie nationale somalienne dans son premier roman Madre piccola, écrit en italien. L’étude analysera notamment le prologue de cet ouvrage où la narratrice déclare son appartenance somalienne en utilisant les mots du poème Soomaali baan ahay / Je suis un Somali, du poète Cabdulqaadir Xirsi Siyaad ‘Yamyam’, tout en revisitant la valeur de cette poésie nationaliste à partir de sa position de femme métisse, et à partir de sa condition particulière en diaspora