HaloTag display enables quantitative single-particle characterisation and functionalisation of engineered extracellular vesicles.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

Mitochondria-ER-PM contacts regulate mitochondrial division and PI(4)P distribution.

The mitochondria-ER-cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1's functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1's role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions.

Temporal control of contact site formation reveals a relationship between mitochondrial division and Num1-mediated mitochondrial tethering.

Mitochondrial division is critical for maintenance of mitochondrial morphology and cellular homeostasis. Previous studies have suggested that the mitochondria-ER-cortex anchor (MECA), a tripartite membrane contact site between mitochondria, the ER, and the plasma membrane, is involved in mitochondrial division. However, its role is poorly understood. We developed a system to control MECA formation and depletion, which allowed us to investigate the relationship between MECA-mediated contact sites and mitochondrial division. Num1 is the protein that mediates mitochondria-ER-plasma membrane tethering at MECA sites. Using both rapamycin-inducible dimerization and auxin-inducible degradation components coupled with Num1, we developed systems to temporally control the formation and depletion of the native contact site. Additionally, we designed a regulatable Num1-independant mitochondria-PM tether. We found that mitochondria-PM tethering alone is not sufficient to rescue mitochondrial division and that a specific feature of Num1-mediated tethering is required. This study demonstrates the utility of systems that regulate contact-site formation and depletion in studying the biological functions of membrane contact sites.

Loss of Num1-mediated cortical dynein anchoring negatively impacts respiratory growth.

Num1 is a multifunctional protein that both tethers mitochondria to the plasma membrane and anchors dynein to the cell cortex during nuclear inheritance. Previous work has examined the impact loss of Num1-based mitochondrial tethering has on dynein function in Saccharomyces cerevisiae; here, we elucidate its impact on mitochondrial function. We find that like mitochondria, Num1 is regulated by changes in metabolic state, with the protein levels and cortical distribution of Num1 differing between fermentative and respiratory growth conditions. In cells lacking Num1, we observe a reproducible respiratory growth defect, suggesting a role for Num1 in not only maintaining mitochondrial morphology, but also function. A structure-function approach revealed that, unexpectedly, Num1-mediated cortical dynein anchoring is important for normal growth under respiratory conditions. The severe respiratory growth defect in Δnum1 cells is not specifically due to the canonical functions of dynein in nuclear migration but is dependent on the presence of dynein, as deletion of DYN1 in Δnum1 cells partially rescues respiratory growth. We hypothesize that misregulated dynein present in cells that lack Num1 negatively impacts mitochondrial function resulting in defects in respiratory growth.

The Caenorhabditis elegans and Haemonchus contortus beta-tubulin genes cannot substitute for loss of the Saccharomyces cerevisiae beta-tubulin gene.

To better understand the mechanism of resistance caused by putative interactions between beta-tubulin and benzimidazole compounds, we sought to purify nematode-specific beta-tubulins using heterologous expression after replacement of the single Saccharomyces cerevisiae beta-tubulin gene. However, we found that haploid yeast cells containing nematode-specific beta-tubulin genes were not viable, suggesting that nematode beta-tubulin cannot substitute for the loss of the yeast beta-tubulin gene.

The multifunctional nature of mitochondrial contact site proteins.

Mitochondria make physical contact with nearly every other membrane in the cell, and these contacts have a wide variety of functions that are carried out by proteins that reside at the sites of contact. Over the past decade, tremendous insight into the identity and functions of proteins localized to mitochondrial contact sites has been gained. In doing so, it has become clear that one protein or protein complex can contribute to contact site formation and function in a wide variety of ways. Thus, complex and often surprising relationships between the roles of a mitochondrial contact site and its multifunctional resident proteins continue to be unraveled.

"Auto-anti-IgE": naturally occurring IgG anti-IgE antibodies may inhibit allergen-induced basophil activation.

BACKGROUND: Naturally occurring IgE-specific IgG autoantibodies have been identified in patients with asthma and other diseases, but their spectrum of functions is poorly understood. OBJECTIVE: Address the hypothesis that: (i) IgG anti-IgE autoantibodies are detectable in the serum of all subjects but elevated in asthmatic patients regardless of atopic status as compared with controls; (ii) some activate IgE-sensitized basophils; and (iii) some inhibit allergen-induced basophil activation. METHODS: IgE-specific IgG autoantibodies were detected and quantified in sera using ELISA. Sera were examined for their ability to activate IgE-sensitized human blood basophils in the presence and absence of allergen using a basophil activation test, and to inhibit allergen binding to specific IgE on a rat basophilic cell line stably expressing human FcεRI. RESULTS: IgG autoantibodies binding to both free and FcεRI-bound IgE were detected in patients with atopic and non-atopic asthma, as well as controls. While some were able to activate IgE-sensitised basophils, others inhibited allergen-induced basophil activation, at least partly by inhibiting binding of IgE to specific allergen. CONCLUSION: Naturally occurring IgG anti-IgE autoantibodies may inhibit, as well as induce, basophil activation. They act in a manner distinct from therapeutic IgG anti-IgE antibodies such as omalizumab. They may at least partly explain why atopic subjects who make allergen-specific IgE never develop clinical symptoms, and why omalizumab therapy is of variable clinical benefit in severe atopic asthma.

The impact of the food-based and nutrient-based standards on lunchtime food and drink provision and consumption in secondary schools in England.

OBJECTIVES: To assess lunchtime provision of food and drink in English secondary schools and the choices and consumption of food and drink by pupils having school lunches, and to compare provision in 2011 with that in 2004. DESIGN: Cross-sectional data collected between October 2010 and April 2011. In each school, food and drink provision, including portion weights and number of portions of each item served at lunchtime, were recorded over five consecutive days. Caterers provided recipe information. SETTING: England. SUBJECTS: A random selection of 5969 pupils having school lunches in a nationally representative sample of eighty secondary schools in England. RESULTS: Compared with 2004, significantly more schools in 2011 provided main dishes, vegetables and salads, water, fruit juice and other drinks on 4 or 5 d/week (P < 0.005). The number of schools offering items not permitted under the food-based standards for school food on 4 or 5 d/week fell significantly over time (P < 0.005), while the number not offering these items on any day increased significantly (P < 0.005). Meals eaten by pupils were well-balanced in relation to macronutrients. CONCLUSIONS: Lunchtime food provision and consumption in secondary schools have improved considerably since 2004, following the introduction of new compulsory standards for school food in 2009. To maximise their energy and nutrient intake at lunchtime, pupils should be encouraged to select a full meal, and to take and eat more fruit and vegetables. Schools also need continued support to increase the micronutrient content of menus and recipes.

Nutrient-based standards for school lunches complement food-based standards and improve pupils' nutrient intake profile.

Following concerns about the nutritional content of school lunches and the increased prevalence of overweight and obesity in the UK, changes to the standards of school meals were made. From September 2008, all primary schools in England were required, by law, to be fully compliant with the new food-based standards (FBS) and nutrient-based standards (NBS) for school lunches. The aim of the present survey was to evaluate the introduction of the NBS for school lunches on the nutritional profile of food and drink items provided by schools and chosen by pupils at lunchtime. A nationally representative sample of 6696 pupils from 136 primary schools in England aged 3-12 years and having school lunches was recruited. Data were collected on lunchtime food and drink provision at each school and on pupil food and drink choices at lunchtime. Caterers also provided planned menus, recipes and other cooking information. Compliance with both the FBS and NBS was then assessed. Results show that even when the FBS was met, many schools did not provide a school lunch that met the NBS as well. The average school lunch eaten was significantly lower in fat, saturated fat and Na in schools that met both the FBS and NBS for school lunches compared with schools that met only the FBS. Change in school lunch policy has contributed to improvements in pupils' choices and the nutritional profile of foods selected at lunchtime.

Short communicationKey differences between school lunches and packed lunches in primary schools in England in 2009.

OBJECTIVE: To compare the key differences between school lunches and packed lunches as consumed in a nationally representative sample of primary schools, 6-8 months after the nutrient-based standards for school lunch became mandatory. DESIGN: Data on 6580 pupils' school lunches and 3422 pupils' packed lunches were collected between February and April 2009 from pupils attending primary schools in England. Fieldwork was conducted over five consecutive school days. Fieldworkers randomly selected ten pupils taking a school lunch and five pupils bringing a packed lunch each day at each school, and recorded and weighed all food and drink items consumed, as well as any leftovers. SETTING: A nationally representative sample of 136 state-maintained primary schools in England. SUBJECTS: A total of 10 002 pupils aged 4-12 years. RESULTS: Mean intakes of protein, fat, saturated fat and vitamin C from both types of lunch met the nutrient-based standards. Pupils taking school lunches on average consumed significantly more protein, NSP, vitamin A, folate and Zn and less fat, saturated fat, non-milk extrinsic sugars (NMES), Na, Ca, vitamin C and Fe than pupils taking packed lunches. Energy intakes were low in both groups. CONCLUSIONS: Packed lunches were less likely to accord with food-based or nutrient-based standards than school lunches. Higher levels of Na, NMES, fat and percentage energy from saturated fat emphasise the difficulties associated with optimising nutrient intakes from packed lunches.

The impact of the food-based and nutrient-based standards on lunchtime food and drink provision and consumption in primary schools in England.

OBJECTIVE: To assess lunchtime provision of food and drink in English primary schools and to assess both choices and consumption of food and drink by pupils having school lunches. These findings were compared with similar data collected in 2005. DESIGN: Cross-sectional data collected between February and April 2009. In each school, food and drink provision, including portion weights and number of portions of each item served at lunchtime, were recorded over five consecutive days. Caterers provided school lunchtime menus and recipes. SETTING: England. SUBJECTS: A random selection of 6696 pupils having school lunches in a nationally representative sample of 136 primary schools in England. RESULTS: Compared with 2005, schools in 2009 provided significantly more fruit, fruit-based desserts, vegetables and salad, water and fruit juice, and less ketchup, sauces and gravy, starchy foods cooked in fat, snacks and confectionery (P < 0·01). Pupils were also making healthier choices, choosing an average of 2·2 portions of fruit and vegetables from their 'five a day', but about one-third to two-fifths of these were wasted. CONCLUSIONS: Lunchtime food provision and consumption in primary schools have improved substantially since 2005, following the introduction of new standards for school food in 2008. However, improvements still need to be made to increase the Fe and Zn content and to decrease the Na content of recipes, and in encouraging pupils to eat more of the fruits and vegetables taken at lunchtime.

Systematic care for asthma in Australian general practice: a randomised controlled trial.

OBJECTIVE: To evaluate whether systematic asthma care involving a register-recall system, postcard prompts for review, and education for general practitioners and staff in Australian general practice improves the quality of care and health outcomes for adult patients with moderate to severe asthma. DESIGN AND SETTING: Cluster randomised controlled trial in 40 general practices in urban and rural South Australia and New South Wales over the 2 years 2004 and 2005; practices were randomly allocated to the intervention or control group. PARTICIPANTS: 565 adult patients of these randomly allocated practices who had doctor-diagnosed moderate to severe asthma and were taking inhaled corticosteroids. MAIN OUTCOME MEASURES: Clinical asthma indicators, quality of care, acceptability of the intervention to patients, quality of life, and asthma self-management skills at baseline, 6 months and 12 months. RESULTS: Although 46% of patients in the intervention group practices responded to the postcard prompts, only 32% actually attended for their asthma review. At 12 months, there was a statistically significant difference in provision of written asthma action plans (rate ratio, 1.9; 95% CI, 1.0-3.5; P = 0.04) for intervention group patients compared with control group patients; there was no significant difference in other indicators. CONCLUSION: We found little objective evidence of improvement in patient management and outcomes resulting from a systematic model of asthma care. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12605000091606.