The separate axes of TECPR1 and ATG16L1 in CASM.

Conjugation of ATG8 to single membranes (CASM) is a fundamental cellular process that entails the conjugation of mammalian Atg8 homologs, here referred to as ATG8, to phosphatidylethanolamine (PE) and phosphatidylserine (PS) on endolysosomal compartments. Our current research, together with recent reports from the Randow, Wu, and Wileman labs, has uncovered yet another layer to this process. We discovered that, in addition to ATG16L1-containing complexes, TECPR1 (tectonin beta-propeller repeat containing 1)-containing ATG12-ATG5 E3 complexes can facilitate CASM, thereby providing a broader understanding of this pathway.

TECPR1 is activated by damage-induced sphingomyelin exposure to mediate noncanonical autophagy.

Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin-like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1-containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1-containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage-induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1-ATG5-ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1-ATG5-ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure.

Effectiveness of Smartphone-Based Community Case Management on the Urgent Referral, Reconsultation, and Hospitalization of Children Aged Under 5 Years in Malawi: Cluster-Randomized, Stepped-Wedge Trial.

BACKGROUND: Integrated community case management (CCM) has led to reductions in child mortality in Malawi resulting from illnesses such as malaria, pneumonia, and diarrhea. However, adherence to CCM guidelines is often poor, potentially leading to inappropriate clinical decisions and poor outcomes. We determined the impact of an e-CCM app on the referral, reconsultation, and hospitalization rates of children presenting to village clinics in Malawi. OBJECTIVE: We determined the impact of an electronic version of a smartphone-based CCM (e-CCM) app on the referral, reconsultation, and hospitalization rates of children presenting to village clinics in Malawi. METHODS: We used a stepped-wedge, cluster-randomized trial to compare paper-based CCM (control) with and without the use of an e-CCM app on smartphones from November 2016 to February 2017. A total of 102 village clinics from 2 districts in northern Malawi were assigned to 1 of 6 clusters, which were randomized on the sequencing of the crossover from the control phase to the intervention phase as well as the duration of exposure in each phase. Children aged ≥2 months to <5 years who presented with acute illness were enrolled consecutively by health surveillance assistants. The primary outcome of urgent referrals to higher-level facilities was evaluated by using multilevel mixed effects models. A logistic regression model with the random effects of the cluster and the fixed effects for each step was fitted. The adjustment for potential confounders included baseline factors, such as patient age, sex, and the geographical location of the village clinics. Calendar time was adjusted for in the analysis. RESULTS: A total of 6965 children were recruited-49.11% (3421/6965) in the control phase and 50.88% (3544/6965) in the intervention phase. After adjusting for calendar time, children in the intervention phase were more likely to be urgently referred to a higher-level health facility than children in the control phase (odds ratio [OR] 2.02, 95% CI 1.27-3.23; P=.003). Overall, children in the intervention arm had lower odds of attending a repeat health surveillance assistant consultation (OR 0.45, 95% CI 0.34-0.59; P<.001) or being admitted to a hospital (OR 0.75, 95% CI 0.62-0.90; P=.002), but after adjusting for time, these differences were not significant (P=.07 for consultation; P=.30 for hospital admission). CONCLUSIONS: The addition of e-CCM decision support by using smartphones led to a greater proportion of children being referred to higher-level facilities, with no apparent increase in hospital admissions or repeat consultations in village clinics. Our findings provide support for the implementation of e-CCM tools in Malawi and other low- and middle-income countries with a need for ongoing assessments of effectiveness and integration with national digital health strategies. TRIAL REGISTRATION: ClinicalTrials.gov NCT02763345; https://clinicaltrials.gov/ct2/show/NCT02763345.

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine.

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.

Sustainability of 'mHealth' interventions in sub- Saharan Africa: a stakeholder analysis of an electronic community case management project in Malawi.

Background: The global health community and funding agencies are currently engaged in ensuring that worthwhile research-based programmes are sustainable. Despite its importance, few studies have analysed the sustainability of global health interventions. In this paper, we aim to explore barriers and facilitators for the wider implementation and sustainability of a mobile health (mHealth) intervention (Supporting LIFE Community Case Management programme) in Malawi, Africa. Methods: Between January and March 2017, a qualitative approach was used to carry out and analyse 13 in-depth semi-structured interviews with key stakeholders across all levels of healthcare provision in Malawi to explore their perceptions with regards to the implementation and sustainability of the mHealth programme. Data were analysed thematically by two reviewers. Results: Overall, our analysis found that the programme was successful in achieving its goals. However, there are many challenges to the wider implementation and sustainability of this programme, including the absence of monetary resources, limited visibility outside the healthcare sector, the lack of integration with community-based and nationwide programmes, services and information and communication technologies, and the limited local capacity in relation to the maintenance, further development, and management. Conclusions: Future developments should be aligned with the strategic goals and interests of the Ministry of Health and engage with national and international stakeholders to develop shared goals and strategies for nationwide scale-up. These developments should also focus on building local capacity by educating trainers and ensuring that training methods and guidelines are appropriately accredited based on national policies. Our findings provide a framework for a variety of stakeholders who are engaged in sustaining mHealth programmes in resource-poor settings and can be used to develop an evidence-based policy for the utilization of technology for healthcare delivery across developing countries.

Parental reactions, distress, and sense of coherence after prenatal versus postnatal diagnosis of complex congenital heart disease.

INTRODUCTION: A diagnosis of congenital heart disease (CHD) in offspring triggers psychological distress in parents. Results of previous studies have been inconsistent regarding the psychological impact of a prenatal versus a postnatal diagnosis. The aim of this study was to evaluate the influence of the time of diagnosis on levels of parental distress. METHODS: Pregnant women and their partners with a fetus diagnosed with complex CHD, parents of children with postnatally diagnosed CHD, and pregnant women and their partners with uncomplicated pregnancies were invited to participate. Data were collected during pregnancy and 2-6 months after delivery using the Hospital Anxiety and Depression Scale, sense of coherence, life satisfaction, and Dyadic Adjustment Scale. RESULTS: During pregnancy, the prenatal group scored lower sense of coherence compared to controls (p=0.044). Postnatally the prenatal group scored lower on sense of coherence compared to the postnatal group and controls (p=0.001; p=0.001). Postnatally, the prenatal and postnatal groups had higher levels of anxiety compared to controls (p=0.025; p=0.0003). Life satisfaction was lower in the prenatal group compared to that in the postnatal group and in controls (p=0.000; p=0.0004). CONCLUSION: Parents with a prenatal diagnosis of CHD in offspring report a low sense of coherence already during pregnancy which decreased further at follow-up. The same group reported a lower satisfaction with life compared to parents of a child with postnatal diagnosis of CHD and parents of a healthy child. This motivates further efforts to improve counselling and support during pregnancy and for parents after a prenatal diagnosis.

Toward the function of mammalian ATG12-ATG5-ATG16L1 complex in autophagy and related processes.

The machinery that decorates autophagic membranes with lipid-conjugated LC3/GABARAP is not yet fully understood. We recently reported the purification of the full-length ATG12-ATG5-ATG16L1 complex, and in reconstitution experiments with purified ATG7, ATG3, and LC3/GABARAP in vitro, together with rescue experiments in knockout cells, important aspects of the complete lipidation reaction were revealed. Hitherto unobserved membrane-binding regions in ATG16L1 were found, contributing to properties that explain the crucial role of this protein in membrane targeting and LC3/GABARAP lipidation in macroautophagy/autophagy and other related processes.

People Welcomed This Innovation with Two Hands: A Qualitative Report of an mHealth Intervention for Community Case Management in Malawi.

INTRODUCTION: Community Case Management (CCM) aims to improve health outcomes among children under five with malaria, diarrhea, and pneumonia, but its effectiveness in Malawi is limited by inconsistent standards of delivery characteristic of paper-based interventions. This may lead to negative impacts on child health outcomes and inefficient use of health system resources. This study evaluated the acceptability and impact of the Supporting LIFE Community Case Management App (SL eCCM App) by Health Surveillance Assistants (HSAs) and caregivers in two districts of Northern Malawi. METHODS: Data were collected through semi-structured interviews with HSAs and caregivers as part of a nested study within a larger trial. We used deductive and inductive approaches during data analysis. Relevant constructs were identified from the Consolidated Framework for Implementation Research and combined with emerging concepts from the data. The Framework Method was used to chart and explore data, leading to the development of themes. RESULTS: Seventeen HSAs and 28 caregivers were interviewed. Participants were generally enthusiastic about the SL eCCM App. Nearly all HSAs expressed a preference for the App over routine paper-based CCM. Most HSAs claimed the App was more reliable and less error prone, facilitated more accurate diagnoses and treatment recommendations, and enhanced professional confidence and respect in the community. Some HSAs believed additional features would improve usability of the App, others identified mobile network or electricity shortages as barriers. Not all caregivers understood the purpose of the App, but most welcomed it as a health and technological advancement. CONCLUSION: The SL eCCM App is acceptable to both HSAs and caregivers, and in most cases, preferred, as it was believed to foster improvements in CCM delivery. Our findings suggest that mobile health interventions for CCM, such as the SL eCCM App, may have potential to improve the effectiveness and efficiency of care to children under five.

Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes.

Covalent modification of LC3 and GABARAP proteins to phosphatidylethanolamine in the double-membrane phagophore is a key event in the early phase of macroautophagy, but can also occur on single-membrane structures. In both cases this involves transfer of LC3/GABARAP from ATG3 to phosphatidylethanolamine at the target membrane. Here we have purified the full-length human ATG12-5-ATG16L1 complex and show its essential role in LC3B/GABARAP lipidation in vitro. We have identified two functionally distinct membrane-binding regions in ATG16L1. An N-terminal membrane-binding amphipathic helix is required for LC3B lipidation under all conditions tested. By contrast, the C-terminal membrane-binding region is dispensable for canonical autophagy but essential for VPS34-independent LC3B lipidation at perturbed endosomes. We further show that the ATG16L1 C-terminus can compensate for WIPI2 depletion to sustain lipidation during starvation. This C-terminal membrane-binding region is present only in the ß-isoform of ATG16L1, showing that ATG16L1 isoforms mechanistically distinguish between different LC3B lipidation mechanisms under different cellular conditions.

Evaluation of the Jönköping dental fear coping model: a patient perspective.

OBJECTIVE: This study is a part of a project with the aim to construct and evaluate a structured treatment model (the Jönköping Dental Fear Coping Model, DFCM) for the treatment of dental patients. The aim of the present study was to evaluate the DFCM from a patient perspective. MATERIAL AND METHODS: The study was performed at four Public Dental Clinics, with the same 13 dentists and 14 dental hygienists participating in two treatment periods. In Period I, 1351 patients were included and in Period II, 1417. Standard care was used in Period I, and in Period II the professionals had been trained in and worked according to the DFCM. In the evaluation, the outcome measures were self-rated discomfort, pain and tension, and satisfaction with the professionals. RESULTS: In comparison with standard care, less tension was reported among patients treated according to the DFCM, (p = .041), which was also found among female patients in a subgroup analysis (p = .028). Additional subgroup analyses revealed that patients expecting dental treatment (as opposed to examination only) reported less discomfort (p = .033), pain (p = .016) and tension (p = .012) in Period II than in Period I. Patients with low to moderate dental fear reported less pain in Period II than in Period I (p = .014). CONCLUSIONS: The DFCM has several positive effects on adult patients in routine dental care. In a Swedish context, the differences between standard care and treatment according to the model were small but, in part, statistically significant. However, it is important to evaluate the model in further studies to allow generalization to other settings.

Perceptions of a mobile health intervention for Community Case Management in Malawi: Opportunities and challenges for Health Surveillance Assistants in a community setting.

Background: Improved availability of mobile phones in low- and middle-income countries (LMICs) offer an opportunity to improve delivery of Community Case Management (CCM). Despite enthusiasm for introducing mHealth into healthcare across LMICs, end-user attitudes towards mHealth solutions for CCM are limited. We aimed to explore Health Surveillance Assistants' (HSAs) perceptions of the Supporting LIFE electronic CCM Application (SL eCCM App) and their experiences incorporating it as part of their clinical practice. Methods: This exploratory qualitative study was part of a mixed methods feasibility study investigating whether children under-5 presenting to village clinics could be followed-up to collect patient outcome data. The convenience sample of 12 HSAs enrolled into the feasibility study participated in semi-structured interviews, which were conducted at village clinics after HSAs had field-tested the SL eCCM App over a 10-day period. Interviews explored HSAs perceptions of the SL eCCM App and their experiences in using the App in addition to paper CCM to assess and treat acutely unwell children. Open coding was used to label emerging concepts, which were iteratively defined and developed into six key themes. Results: HSAs' perceived enhanced clinical decision-making, quality of CCM delivery, and work efficiency as opportunities associated with using the SL eCCM App. HSAs believed the inability to retrieve patient records,, cumbersome duplicate assessments/data entry study procedures, and inconsistencies between the SL eCCM App and paper-based CCM guidelines as challenges to implementation. Adding features to the App, such as, permitting communication between colleagues/supervisors, drug stock-out reporting, and community assessments, were identified as potentially supporting HSAs' many roles in the community. Conclusion: This study identified opportunities and challenges associated with using the SL eCCM App in Malawi. This information can be used to inform future development and evaluation of the SL eCCM App, and similar mHealth solutions for CCM in Malawi and other developing countries.

Development and evaluation of the Jönköping Dental Fear Coping Model: a health professional perspective.

OBJECTIVE: The purpose of this study was to design a structured treatment model focusing on all levels of adult's dental fear, the Jönköping Dental Fear Coping Model (DFCM). The aim was to study the DFCM from a dental health professional perspective. MATERIAL AND METHODS: The DFCM was studied by means of quantitative and qualitative analyses. Nine dental clinics participated in Period I (pre-intervention/standard care), and 133 dental health professionals (dentists, dental hygienists, dental assistants) and 3088 patients were included. After completion of Period I, four of the clinics were randomized to Period II (intervention), beginning with the professionals undergoing DFCM training. Following that, 51 dental health professionals treated 1417 patients according to the DFCM. The other five clinics served as controls. RESULTS: Half or more of the dental health professionals assessed the model as better than standard care, regarding anamnesis and diagnostics, communication and contact, and understanding of patients and dental fear. The dental health professionals reported higher tension in their fearful patients in Period II compared with Period I, possibly due to their increased awareness of dental fear. CONCLUSIONS: The qualitative data suggest that dental health professionals find the DFCM beneficial in routine dental care. The model promotes a holistic approach to the treatment of adult patients. However, stress among the professionals was not reduced when measured, neither quantitatively nor qualitatively. It is important to evaluate the model in further studies to make it possible to draw generalizable conclusions.

SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin-2.

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18-induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin-2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1- and WIPI2-positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin-2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.

The added value of a mobile application of Community Case Management on referral, re-consultation and hospitalization rates of children aged under 5 years in two districts in Northern Malawi: study protocol for a pragmatic, stepped-wedge cluster-randomized controlled trial.

BACKGROUND: There is evidence to suggest that frontline community health workers in Malawi are under-referring children to higher-level facilities. Integrating a digitized version of paper-based methods of Community Case Management (CCM) could strengthen delivery, increasing urgent referral rates and preventing unnecessary re-consultations and hospital admissions. This trial aims to evaluate the added value of the Supporting LIFE electronic Community Case Management Application (SL eCCM App) compared to paper-based CCM on urgent referral, re-consultation and hospitalization rates, in two districts in Northern Malawi. METHODS/DESIGN: This is a pragmatic, stepped-wedge cluster-randomized trial assessing the added value of the SL eCCM App on urgent referral, re-consultation and hospitalization rates of children aged 2 months and older to up to 5 years, within 7 days of the index visit. One hundred and two health surveillance assistants (HSAs) were stratified into six clusters based on geographical location, and clusters randomized to the timing of crossover to the intervention using simple, computer-generated randomization. Training workshops were conducted prior to the control (paper-CCM) and intervention (paper-CCM + SL eCCM App) in assigned clusters. Neither participants nor study personnel were blinded to allocation. Outcome measures were determined by abstraction of clinical data from patient records 2 weeks after recruitment. A nested qualitative study explored perceptions of adherence to urgent referral recommendations and a cost evaluation determined the financial and time-related costs to caregivers of subsequent health care utilization. The trial was conducted between July 2016 and February 2017. DISCUSSION: This is the first large-scale trial evaluating the value of adding a mobile application of CCM to the assessment of children aged under 5 years. The trial will generate evidence on the potential use of mobile health for CCM in Malawi, and more widely in other low- and middle-income countries. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT02763345 . Registered on 3 May 2016.

A multi-facet pain survey of psychosocial complaints among patients with long-standing non-malignant pain.

BACKGROUND AND AIMS: Psychometric inventories and scales intended to measure cognitive, emotional and behavioural concomitants of pain are typically constructed by deducting items from theoretically derived concepts related to pain states, e.g. social support, perceived control, depressiveness, and catastrophizing. The aim of this study was to design a clinically useful, generic pain distress inventory - The Multi-Facet Pain Survey (MFPS) - inductively derived from psychological and social complaints reported by a study group of individuals with severe chronic nonmalignant pain. METHODS: Extensive clinical interviews with hospitalized chronic pain patients were made by clinical psychologists. The purpose was to highlight the patients' pain histories and their beliefs and feelings about the pain, and to determine factors possibly influencing their rehabilitation potential. The types of distress reported were sorted into categories with a procedure similar to content analysis. Distress reports were converted to statements, forming items in a questionnaire, the Multi-Facet Pain Survey. RESULTS: Our analyses supported a distress structure including 15 categories, or "facets", comprising in all 190 types of psychosocial distress. Ten of the facets denote beliefs about the present condition and aspects of distress experienced in daily life (e.g. cognitive problems); three facets reflect the illness history, and two the patient's views on future prospects. To improve the clinical utility, we shortened the scale into a 53 items inventory. A factor analysis of these 53 items revealed four clinically meaningful factors: (1) stress-related exhaustion; (2) impact of pain on daily life; (3) self-inefficacy in regard to future prospects; and (4) negative experiences of health care. While the second factor represents distress directly related to the pain, the first factor reflects long-term exhaustion effects of the pain condition similar to those seen in individuals exposed to long periods of stress. Items loading in the third factor reflect a pessimistic outlook on the future. The content validity of the scale was explored by predicting and testing correlations between the 15 MFPS facets, and the Symptom Checklist (SCL-90) and the West Haven Yale Multidimensional Pain Inventory (MPI). Some of the MFPS facets showed little or no agreement with any of the subscales of the comparison measures. The homogeneity was satisfactory both for facets and factors. CONCLUSIONS: The Multi-Facet Pain Survey (MFPS) facets cover a broad array of experienced psychosocial distress in patients with severe, longstanding pain. Some facets of psychosocial impact of longstanding pain states shown in the qualitatively derived distress facets, or by the latent factors found in the factor analysis, may complement our understanding of the long-term impact of pain. Consequently, MFPS may improve the assessment of psychological and social complaints and complications in patients with chronic pain. IMPLICATIONS: The MFPS will hopefully be an assessment tool supporting the psychological contribution to a biopsychosocial evaluation of patients with severe, longstanding pain. By exposing a broad range of suffering, MFPS may contribute to alternative treatment options and a better prognosis of future rehabilitation.

HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.

Translation and psychometric properties of the Swedish version of the Index of Dental Anxiety and Fear (IDAF-4C⁺).

Dental anxiety (DA) is a common condition, with significant medical, psychological, and social consequences. High-quality psychometric tools for the assessment of dental anxiety are necessary for clinical and research purposes. The aim of this study was to adapt the Index of Dental Anxiety and Fear (IDAF-4C(+) ) to the Swedish language and to explore the psychometric properties of the translated version. The study included a clinical sample (n = 414; 17-91 yr of age) and a non-clinical sample (n = 51; 19-47 yr of age). The scales used were the IDAF-4C(+) , the Single-Question Assessment of Dental Anxiety (SQDA), the Dental Fear Survey (DFS), and the Internal Health Locus of Control (IHLOC). The Swedish IDAF anxiety module showed a clear, one-dimensional structure, good internal consistency (Cronbach's alpha = 0.95), and adequate validity, as evidenced by strong correlations with the other DA measures (SQDA and DFS) and weak correlations with the IHLOC. In addition, the IDAF phobia module and the IDAF stimulus module were strongly correlated with the other DA measures. To conclude, the study shows promising findings for the reliability and validity of the Swedish translation of the IDAF-4C(+) , as a useful measure of dental anxiety in research and clinical practice.

[The substance addiction team: a bio-psycho-social melting pot. Physicians, psychologists and social workers do not cooperate fully--even though they want to]. / Beroendeteamet: en bio-psyko-social smältdegel - Läkare, psykologer och socionomer samverkar inte fullt ut - trots att de vill.

Medical doctors, psychologists and social workers are often collaborating in health care units for substance addiction. In a survey answered by 141 persons with any of these professions, they communicated somewhat disparate views on the causes of addiction, and how our knowledge of addiction could be best promoted. At the same time, they expressed a demand for intensified forms of inter-professional team collaboration. There seems to be a need to improve the understanding and acceptance between the different professions.

Membrane dynamics in autophagosome biogenesis.

Bilayered phospholipid membranes are vital to the organization of the living cell. Based on fundamental principles of polarity, membranes create borders allowing defined spaces to be encapsulated. This compartmentalization is a prerequisite for the complex functional design of the eukaryotic cell, yielding localities that can differ in composition and operation. During macroautophagy, cytoplasmic components become enclosed by a growing double bilayered membrane, which upon closure creates a separate compartment, the autophagosome. The autophagosome is then primed for fusion with endosomal and lysosomal compartments, leading to degradation of the captured material. A large number of proteins have been found to be essential for autophagy, but little is known about the specific lipids that constitute the autophagic membranes and the membrane modeling events that are responsible for regulation of autophagosome shape and size. In this Commentary, we review the recent progress in our understanding of the membrane shaping and remodeling events that are required at different steps of the autophagy pathway. This article is part of a Focus on Autophagosome biogenesis. For further reading, please see related articles: 'ERES: sites for autophagosome biogenesis and maturation?' by Jana Sanchez-Wandelmer et al. (J. Cell Sci. 128, 185-192) and 'WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome' by Tassula Proikas-Cezanne et al. (J. Cell Sci. 128, 207-217).

Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains.

BACKGROUND: Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric ß-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied. METHODOLOGY/PRINCIPAL FINDINGS: OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor. CONCLUSION/SIGNIFICANCE: Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.