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.

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.

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.

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.

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.

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.

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).

Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules.

Sorting nexins (SNXs) are regulators of endosomal sorting. For the SNX-BAR subgroup, a Bin/Amphiphysin/Rvs (BAR) domain is vital for formation/stabilization of tubular subdomains that mediate cargo recycling. Here, by analysing the in vitro membrane remodelling properties of all 12 human SNX-BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX-BARs display a restricted pattern of BAR domain-mediated dimerization, and by resolving a 2.8 Å structure of a SNX1-BAR domain homodimer, establish that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective 'tip-loop' interactions. Overall, the restricted and selective nature of these interactions provide a molecular explanation for how distinct SNX-BAR-decorated tubules are nucleated from the same endosomal vacuole, as observed in living cells. Our data provide insight into the molecular mechanism that generates and organizes the tubular endosomal network.

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 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.

On the dynamics of dental fear: dental or mental?

This study explored possible predictors of change over time in dental fear. In a population-based sample of 664 women, 38-54 yr of age at first assessment, a self-rating of dental fear was recorded on two occasions, 6 yr apart. Whilst the majority of the women maintained their level of fear, two subgroups reported changes at follow up. Of the 99 subjects indicating high fear at baseline, 54 recorded no or minimal fear at follow up and, among the 565 recording minimal fear at baseline, 27 reported increased fear at follow up. We used psychological and odontological findings at baseline to compare the four subgroups with altered or maintained fear ratings at follow up. The women reporting reduced fear at follow up differed significantly in the psychological variables from those reporting unchanged fear; there were no significant differences between these groups in any of the odontological variables. The group indicating increased fear at follow up did not differ from those with a maintained fearlessness in any of the baseline measurements. The results are discussed in terms of a systems-oriented understanding of the development of dental fear.

Dental anxiety - a joint interest for dentists and psychologists.

This introductory article to the Symposium on Behavioral and Community Dentistry aims to describe the development of research and clinical work on dental anxiety, and includes a discussion of the historical background and the evolution since the 1970s. In view of its pioneering activities in this regard, special focus is placed on research and development at the Institute of Odontology of the University of Gothenburg, Sweden.

Dentists' skills with fearful patients: education and treatment.

The aims were to explore dentists' skills in dental fear, current strategies when treating fearful adult patients, and the possible need for additional education among dentists working in Sweden. A sample of 1,293 members of the Association of Public Health Dentists in Sweden were asked to respond to a Web survey concerning dental fear. The response rate was 69% (n = 889); 91% trained in Sweden and 9% trained in another country. The most frequently used pharmacological anxiety-reducing techniques were medication with a midazolame mixture (72%) and benzodiazepine tablets (77%), and the most commonly used psychological techniques were relaxation (68%), distraction (66%), and Tell-Show-Do (86%). A larger proportion of dentists trained in Sweden, compared with dentists who were trained in other countries, reported that they had received undergraduate training in dental fear. Dentists trained in Sweden more often reported competence in pharmacological and psychological anxiety-reducing techniques, compared with dentists who were trained in other countries. Higher levels of self-rated efficacy in treating fearful patients accompanied additional education in dental fear after graduation. In conclusion, Swedish dentists use a variety of techniques to meet the needs of fearful dental patients. Competence in anxiety-reducing techniques is associated with self-efficacy and the site of education.

Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis.

Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.

Dentists' views on fearful patients. Problems and promises.

UNLABELLED: A large number of patients treated in the general dental health service in Western countries report dental fear to some degree. Dentists' views of treating these fearful patients are not well described in the literature.Therefore, the aims of the study were to explore dentists' attitudes towards, experience of, and feelings about treating fearful patients. The sample consisted of 1293 members of the Association of Public Health Dentists in Sweden who were asked to respond to a web survey concerning dental fear. The response rate was 69% (n = 889).The majority of the responding dentists stated that dental fear is a problem in routine dental care,treating patients with dental fear is a positive challenge and they felt they were making a contribution.They also reported that treating patients with dental fear is associated with hard work, poor revenues, and little appreciation by employers. Female dentists reported a greater proportion of patients with dental fear and greater self-efficacy regarding the treatment of these patients, compared with their male colleagues. Dentists trained in other EU countries reported stress more often and less perceived contribution when treating fearful patients, compared with colleagues trained in Sweden. CONCLUSION: Dentists' views of treating fearful patients are mainly positive; however, it is problematic that dentists feel stress and that dentists who treat many fearful patients feel their employers do not appreciate their efforts.

Biochemical and functional characterization of Helicobacter pylori vesicles.

Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigour of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl-Lewis x/a (sLex/a) antigens. Similar to most Gram-negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence-associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and nuclear magnetic resonance. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle-host interactions and found that the vesicles carry effector-promoting properties that are important to disease development.

The PX-BAR membrane-remodeling unit of sorting nexin 9.

Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism.

Psychosocial and dental factors in the maintenance of severe dental fear.

The aim was to study the relationship between psychosocial factors and dental status in adult subjects with severe dental fear (DF). A consecutive sample of 148 adults (mean age 36.1 yrs, range 21-69 yrs) referred for dental fear treatment was investigated using an intake questionnaire on dental attendance and history, psychometric questionnaires on dental fear, general anxiety and depression and a radiographic examination. The subjects had a mean DFMT (Decayed, Filled, Missed Teeth) score of 18.6 (SD = 5.6). A deterioration in dental status defined as the presence of root remnants was present in 57% of the subjects and was related to the negative consequences of dental fear, general anxiety and depression. Most subjects (84%) reported clinical levels of general anxiety and 46% reported clinical levels of depression. In conclusion, subjects with severe DF often suffer from psychosocial consequences and distress.This is even more marked if their dental status has deteriorated.The findings support a biopsychosocial vicious circle understanding of the maintenance of DF.

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.