Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis.

Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In Caenorhabditis elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. We report that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. We place Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca2+ sensor dSTIM, and the Ca2+-release-activated Ca2+ channel dOrai in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, our results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway.

Children and Adolescents with Severe Tyrannical Behaviour: Profile of Youth and Their Parents.

This study focused on a subtype of child-to-parent violence, severe tyrannical behaviour (STB). The aim was to examine the clinical characteristics of children and adolescents who physically and/or verbally abuse their parents and the sociodemographic characteristics and generalities of those families. Clinical and sociodemographic data from 73 children and adolescents with STB and their parents have been collected from a randomized control trial. Results showed a specific profile of youth with tyrannical behaviour (aggressive behaviour only in-home settings, only-child, previously mental health care), as well as differential characteristics of these families (late parenthood, high socio-economic status and conjugal family). Children with tyrannical behaviour frequently had psychiatric conditions, such as attention deficit hyperactivity disorder, separation anxiety, sleeping disorders and severe irritability was frequently described. These clinical patterns of combined neurodevelopmental, externalized and internalized symptoms suggest that the combination of individual characteristics, parenting style and parent-child relationship play essential roles in children's STB development.

Is an irritable ADHD profile traceable using personality dimensions? Replicability, stability, and predictive value over time of data-driven profiles.

Pediatric attention deficit/hyperactivity disorder (ADHD) is a heterogeneous condition. In particular, children with ADHD display varying profiles of dispositional traits, as assessed through temperament and personality questionnaires. Previous data-driven community detection analyses based on temperament dimensions identified an irritable profile of patients with ADHD, uniquely characterized by elevated emotional dysregulation symptoms. Belonging to this profile increased the risk of developing comorbid disorders. Here, we investigated whether we could replicate this profile in a sample of 178 children with ADHD, using community detection based on personality dimensions. Stability of the identified profiles, of individual classifications, and clinical prediction were longitudinally assessed over a 1-year interval. Three personality profiles were detected: The first two profiles had high levels of neuroticism, with the first displaying higher ADHD severity and lower openness to experience (profile 1; N = 38), and the second lower agreeableness (profile 2; N = 73). The third profile displayed scores closer to the normative range on all five factors (profile 3; N = 67). The identified profiles did only partially replicate the temperament-based profiles previously reported, as higher levels of neuroticism were found in two of the three detected profiles. Nonetheless, despite changes in individual classifications, the profiles themselves were highly stable over time and of clinical predictive value. Whereas children belonging to profiles 1 and 2 benefited from starting medication, children in profile 3 did not. Hence, belonging to an emotionally dysregulated profile at baseline predicted the effect of medication at follow-up over and above initial ADHD symptom severity. This finding suggests that personality profiles could play a role in predicting treatment response in ADHD.

The Drosophila CD36 Homologue croquemort Is Required to Maintain Immune and Gut Homeostasis during Development and Aging.

Phagocytosis is an ancient mechanism central to both tissue homeostasis and immune defense. Both the identity of the receptors that mediate bacterial phagocytosis and the nature of the interactions between phagocytosis and other defense mechanisms remain elusive. Here, we report that Croquemort (Crq), a Drosophila member of the CD36 family of scavenger receptors, is required for microbial phagocytosis and efficient bacterial clearance. Flies mutant for crq are susceptible to environmental microbes during development and succumb to a variety of microbial infections as adults. Crq acts parallel to the Toll and Imd pathways to eliminate bacteria via phagocytosis. crq mutant flies exhibit enhanced and prolonged immune and cytokine induction accompanied by premature gut dysplasia and decreased lifespan. The chronic state of immune activation in crq mutant flies is further regulated by negative regulators of the Imd pathway. Altogether, our data demonstrate that Crq plays a key role in maintaining immune and organismal homeostasis.

A double-blind placebo-controlled randomised trial of omega-3 supplementation in children with moderate ADHD symptoms.

OBJECTIVE: Clinical trials and inconclusive meta-analyses have investigated the effects of omega-3 supplements in children with Attention-Deficit Hyperactivity Disorder (ADHD). We performed a randomised placebo-controlled trial to evaluate the efficacy of omega-3 fatty acids. METHODS: Children aged 6-15 years with established diagnosis of ADHD were randomised 1:1 to receive either supplements containing docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) or a placebo for 3 months. Psychotropic or omega-3-containing treatments were not authorised during the study. The primary outcome was the change in the Attention-Deficit Hyperactivity Disorder Rating Scale version 4 (ADHD-RS-IV). Other outcomes included safety, lexical level (Alouette test), attention (Test of Attentional Performance for Children-KiTAP), anxiety (48-item Conners Parent Rating Scale-Revised-CPRS-R), and depression (Children's Depression Inventory-CDI). RESULTS: Between 2009 and 2011, 162 children were included in five French child psychiatry centres. The mean age was 9.90 (SD 2.62) years and 78.4% were boys. The inclusion ADHD-RS-IV at was 37.31 (SD 8.40). The total ADHD-RS-IV score reduction was greater in the placebo group than in the DHA-EPA group: -19 (-26, -12)  % and -9.7 (-16.6, -2.9) %, respectively, p = 0.039. The other components of the Conners score had a similar variation but the differences between groups were not significant. Two patients in the DHA-EPA group and none in the placebo group experienced a severe adverse event (hospitalisation for worsening ADHD symptoms). CONCLUSION: This study did not show any beneficial effect of omega-3 supplement in children with mild ADHD symptoms.

Non-violent resistance parental training versus treatment as usual for children and adolescents with severe tyrannical behavior: a randomized controlled trial.

Objective: This single-blinded, randomized, parallel group superiority trial evaluates whether the Non-Violent Resistance (NVR) program, a 10-session parental-group intervention, was more effective in reducing stress in parents of children aged 6-20 years and displaying severe tyrannical behavior (STB) compared to a treatment as usual (TAU) intervention that provided supportive counseling and psychoeducation. Methods: Eighty two parents of youth aged 6-20 years with STB were enrolled by the Child and Adolescent Psychiatry Department at the University Hospital of Montpellier (France). A random block and stratified by age (6-12 and 13-20 years) randomization, was performed. All participants were interviewed by independent, blinded to group assignments, research assistants, and completed their assessments at baseline and treatment completion (4 months from baseline). Since this program has not been previously evaluated in this population, the study primarily evaluated the efficacy, using the Parenting Stress Index/Short Form (PSI-SF). The primary outcome was the change from baseline to treatment completion of the PSI-SF total score. Results: Seventy three participants completed the study and were available for analysis (36 NVR and 37 TAU). At completion, between-groups comparison of the change (completion minus baseline) in the total score of PSI-SF was not significant (NVR: -4.3 (± 13.9); TAU: -7.6 (± 19.6); two-sample t-test p = 0.43; effect size of -0.19 [-0.67, 0.28]). Conclusion: Contrary to our expectation, NVR was not superior to TAU in reducing parental stress at completion for parents of children with STB. However, NVR showed positive outcomes in the follow-up, pointing to the importance to implement parental strategies and following this population over longer time periods in future projects.Clinical trial registration: Clinicaltrials.gov, identifier NCT05567276.

[Separation anxiety in children]. / Anxiété de séparation de l'enfant.

Separation anxiety disorder can be differentiated from developmental anxiety because of its intensity, persistence and negative impact on adaptive functioning. This disorder is closely linked to other anxiety and mood disorders and can also be associated with externalizing psychopathology in children and adolescents. Severe separation anxiety can result in school refusal and intra-familial violence. Cognitive behavioral therapies have the best evidence-based support for the treatment of separation anxiety disorder in children and adolescents. In addition, it is important to detect factors associated with persistence of anxiety such as systematic avoidance of separation and parental overprotection. The role of pediatricians and general practitioners in recognizing clinical separation anxiety and encouraging appropriate care and positive parental attitudes is essential, as separation anxiety is often associated with a variety of somatic symptoms.

A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways.

Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border, and disruption of intercellular junctions. We confirm toxin-dependent re-distribution of Rab11 to enterocytes' apical surface and observe suppression of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). We also report that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs.

In vivo and in vitro methods for studying apoptotic cell engulfment in Drosophila.

Proper development of all multicellular organisms involves programmed apoptosis. Completion of this process requires removal of the resulting cell corpses through phagocytosis by their neighbors or by macrophages. Studies in C. elegans have been fruitful in the genetic dissection of key pathways, but they lack the professional immune system of higher organisms. Mammalian studies have identified a plethora of factors that are required for engulfment, but redundancy in the pathways has made it difficult to explain the genetic hierarchy of these factors. Thus, Drosophila has proven to be a useful evolutionary intermediate in which to examine this phenomenon. Here we describe methods used for dissecting the mechanisms and pathways involved in the engulfment of apoptotic cells by Drosophila phagocytes. Included are methods to be used for in vivo studies in the early embryo that can be used to examine engulfment of dying cells at various stages of embryogenesis. We also describe in vitro techniques for the use of Drosophila cell culture, including cell engulfment assays, that can be used for general phenotypic analysis, as well as live cell studies. We provide advice on imaging, including the preparation of samples for high-resolution microscopy and quantification of potential engulfment phenotypes for both in vivo and in vitro methods.

Apoptotic Cell Clearance in Drosophila melanogaster.

The swift clearance of apoptotic cells (ACs) (efferocytosis) by phagocytes is a critical event during development of all multicellular organisms. It is achieved through phagocytosis by professional or amateur phagocytes. Failure in this process can lead to the development of inflammatory autoimmune or neurodegenerative diseases. AC clearance has been conserved throughout evolution, although many details in its mechanisms remain to be explored. It has been studied in the context of mammalian macrophages, and in the nematode Caenorhabditis elegans, which lacks "professional" phagocytes such as macrophages, but in which other cell types can engulf apoptotic corpses. In Drosophila melanogaster, ACs are engulfed by macrophages, glial, and epithelial cells. Drosophila macrophages perform similar functions to those of mammalian macrophages. They are professional phagocytes that participate in phagocytosis of ACs and pathogens. Study of AC clearance in Drosophila has identified some key elements, like the receptors Croquemort and Draper, promoting Drosophila as a suitable model to genetically dissect this process. In this review, we survey recent works of AC clearance pathways in Drosophila, and discuss the physiological outcomes and consequences of this process.

Components of the Engulfment Machinery Have Distinct Roles in Corpse Processing.

Billions of cells die in our bodies on a daily basis and are engulfed by phagocytes. Engulfment, or phagocytosis, can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification. In this study, we focus on the last two steps, which can collectively be considered corpse processing, in which the engulfed material is degraded. We use the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. We show that engulfed material is processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, we investigated the role of Draper, integrins, and downstream signaling components in corpse processing. We found that some proteins were required for internalization only, while others had defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. We also performed double mutant analysis and found that combined loss of draper and αPS3 still resulted in a small number of engulfed vesicles. Therefore, we investigated another known engulfment receptor, Crq. We found that loss of all three receptors did not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells.

The Pallbearer E3 ligase promotes actin remodeling via RAC in efferocytosis by degrading the ribosomal protein S6.

Clearance of apoptotic cells (efferocytosis) is achieved through phagocytosis by professional or amateur phagocytes. It is critical for tissue homeostasis and remodeling in all animals. Failure in this process can contribute to the development of inflammatory autoimmune or neurodegenerative diseases. We found previously that the PALL-SCF E3-ubiquitin ligase complex promotes apoptotic cell clearance, but it remained unclear how it did so. Here we show that the F-box protein PALL interacts with phosphorylated ribosomal protein S6 (RpS6) to promote its ubiquitylation and proteasomal degradation. This leads to RAC2 GTPase upregulation and activation and F-actin remodeling that promotes efferocytosis. We further show that the specific role of PALL in efferocytosis is driven by its apoptotic cell-induced nuclear export. Finding a role for RpS6 in the negative regulation of efferocytosis provides the opportunity to develop new strategies to regulate this process.

Phagocytosis of apoptotic cells in mammals, caenorhabditis elegans and Drosophila melanogaster: molecular mechanisms and physiological consequences.

Phagocytosis is the necessary corollary of apoptosis. It leads to the clearance of apoptotic cells by phagocytes, which can be 'professional' or 'amateur'. I review the known molecular aspects of phagocytosis of apoptotic corpses in mammals, Caenorhabditis elegans and Drosophila melanogaster from the point of view of the phagocyte and the apoptotic corpse. I highlight recent advances made in the field and discuss the physiological outcomes and consequences of this process. Indeed, phagocytosis of apoptotic cells is important in shaping or remodeling tissues to maintain their integrity and specialized functions during development and wound healing. It also contributes to the development of inflammation and/or its resolution after an injury or infection. This perhaps explains why the molecular mechanisms of phagocytosis of apoptotic cells are redundant and complex in mammals and suggests why they appear to have been mostly conserved through evolution. Caenorhabditis elegans has already proven to be useful in genetically dissecting the molecular mechanisms underlying phagocytosis of apoptotic corpses by 'amateur' neighboring cells. Drosophila melanogaster will become the model of choice in genetically dissecting the molecular mechanisms underlying phagocytosis of apoptotic cells by 'professional' phagocytes such as macrophages.

Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila.

During developmental remodeling, neurites destined for pruning often degenerate on-site. Physical injury also induces degeneration of neurites distal to the injury site. Prompt clearance of degenerating neurites is important for maintaining tissue homeostasis and preventing inflammatory responses. Here we show that in both dendrite pruning and dendrite injury of Drosophila sensory neurons, epidermal cells rather than hemocytes are the primary phagocytes in clearing degenerating dendrites. Epidermal cells act via Draper-mediated recognition to facilitate dendrite degeneration and to engulf and degrade degenerating dendrites. Using multiple dendritic membrane markers to trace phagocytosis, we show that two members of the CD36 family, croquemort (crq) and debris buster (dsb), act at distinct stages of phagosome maturation for dendrite clearance. Our finding reveals the physiological importance of coordination between neurons and their surrounding epidermis, for both dendrite fragmentation and clearance.

The Drosophila TRPP cation channel, PKD2 and Dmel/Ced-12 act in genetically distinct pathways during apoptotic cell clearance.

Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway.

Pertinence of the self-report mother-to-infant bonding scale in the neonatal unit of a maternity ward.

OBJECTIVE: To assess the relevance of the self-report Mother-to-Infant Bonding Scale (MIBS) to evaluate mother-infant bonding in the neonatal unit of a maternity ward. MATERIAL AND METHODS: Forty-eight hours after delivery, 78 mothers responded to the MIBS, the Edinburgh Postnatal Depression Scale (EPDS), the Adult Attachment Questionnaire (AAQ), and the Mother's Assessment of the Behavior of her Infant (MABI) questionnaire. They were then interviewed 24h later by a pediatric psychiatrist, who assessed the mother-infant relationship. The neonatology nurses also filled out the MIBS, imagining the mothers' responses, and responded anonymously to questionnaires on the use of the MIBS in their daily practice. RESULTS: MIBS satisfactorily detected difficulties in mother-child bonding: the area under the ROC curve was 0.93, with a sensitivity of 0.9 and a specificity of 0.8 for a threshold score ≥2. MIBS was independent of EPDS (r=0.11, p=0.29) and AAQ (r=0.05, p=0.63). However, it was influenced by the infant's behavioral characteristics (r=0.3, p=0.01). MIBS scores of the mothers and nurses showed low correlation (r=0.31, p=0.004) and the item-by-item responses were rarely concordant. Fully 100% of the nurses stated that the MIBS was helpful in evaluating mother-child bonding and 85% of the mothers found it beneficial. CONCLUSION: New mothers need to express their feelings about their babies, as hospital staff observation of mother-infant interactions is not sufficiently reliable for assessing the attachment process. The self-report MIBS is a useful tool for detecting difficulties in early mother-infant bonding.

Requirement for a Drosophila E3-ubiquitin ligase in phagocytosis of apoptotic cells.

Many cells die by apoptosis during animal development. Apoptotic cells are rapidly removed through phagocytosis by their neighbors or by macrophages. To genetically dissect this process, we performed an in vivo screen for genes required for efficient phagocytosis of apoptotic cells by Drosophila macrophages and identified pallbearer (pall), which encodes an F box protein. F box proteins generally provide substrate specificity to Skp Cullin F box (SCF) complexes, acting as E3 ligases that target phosphorylated proteins to ubiquitylation and degradation via the 26S proteasome. We showed that Pallbearer functions in an SCF-dependent manner and provided direct evidence of a role for ubiquitylation and proteasomal degradation in phagocytosis of apoptotic corpses in vivo. This work might further our understanding of the regulation of apoptotic cell engulfment and thus our understanding of innate immunity as a whole.