Bowlby's five therapeutic tasks: bringing them up to date for children.

Bowlby remained attached to his psychoanalytic roots and conceptualised treatment in terms of one-to-one relationships, albeit acknowledging the need for a family formulation. Bowlby's five therapeutic tasks were never adapted to the current understanding of working with the relationships fostering the development and maintenance of children's attachment strategies. This paper goes through each of Bowlby's five tasks and adapts them to our current understanding of development, with consequences for prioritising family approaches, rather than a secure base alone with a therapist. In doing so I will review the process of achieving security, seeing it as more similar to an allostatic process than a state of homeostasis.

Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy.

Autophagy is an intracellular lysosomal degradation pathway by which cytoplasmic cargoes are removed to maintain cellular homeostasis. Monitoring autophagy flux is crucial to understand the autophagy process and its biological significance. However, assays to measure autophagy flux are either complex, low throughput or not sensitive enough for reliable quantitative results. Recently, ER-phagy has emerged as a physiologically relevant pathway to maintain ER homeostasis but the process is poorly understood, highlighting the need for tools to monitor ER-phagy flux. In this study, we validate the use of the signal-retaining autophagy indicator (SRAI), a fixable fluorescent probe recently generated and described to detect mitophagy, as a versatile, sensitive and convenient probe for monitoring ER-phagy. This includes the study of either general selective degradation of the endoplasmic reticulum (ER-phagy) or individual forms of ER-phagy involving specific cargo receptors (e.g., FAM134B, FAM134C, TEX264 and CCPG1). Crucially, we present a detailed protocol for the quantification of autophagic flux using automated microscopy and high throughput analysis. Overall, this probe provides a reliable and convenient tool for the measurement of ER-phagy.

Assessing autism spectrum disorder in children with a background of maltreatment: challenges and guidance.

There may be some similarities in the presentation of children who have autism spectrum disorder (ASD) and those exposed to maltreatment affecting assessment and diagnosis. Overlapping characteristics include difficulties understanding and maintaining relationships, sensitivity to routine and hyper-reactivity to sensory inputs. Children who have been maltreated are at increased risk of various developmental vulnerabilities with both environmental and genetic factors being relevant. The existing epidemiological evidence has found that looked-after children are more likely to screen positively for neurodevelopmental disorders and there are smaller scale studies in adoptive children finding higher rates of ASD than would be expected in the general population. Other research suggests a predominantly genetic basis for this increased risk in keeping with what is generally understood about the aetiology of autism. Children exposed to profound deprivation in Romanian orphanages were found to be at higher risk of a pattern of traits termed 'quasi autistic' which tended to reduce following adoption, but these findings have not been replicated in children experiencing maltreatment in birth families. Reactive attachment disorder (RAD) has some overlapping criteria with ASD, but its prevalence is unknown and children with RAD should be more socially reciprocal and not have the same repetitive and restricted behaviours and interests. We recommend experienced multidisciplinary assessment that considers both the possibility of maltreatment in children with ASD and neurodevelopmental vulnerabilities in children who have been maltreated and advise on assessment and management strategies.

Toll-like receptor 2 orchestrates a tumor suppressor response in non-small cell lung cancer.

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.

Analysis of Pancreatic Acinar Protein Solubility in Autophagy-Deficient Mice.

Autophagy of the endoplasmic reticulum, or ER-phagy, maintains the homeostasis of the secretory pathway. This is particularly prominent in specialized secretory cells such as the acinar cells of the exocrine pancreas. The role for such a homeostatic pathway during ageing of mammals is modelled best by in vivo genetic or pharmacologic intervention in mice. This is due to the paucity of cellular models that can maintain acinar identity outside of an animal. Here we present methods for isolation of soluble and insoluble protein fractions of ER luminal proteins from the pancreas, alongside RNA. Analysis of these macromolecules allows inference of changes in ER luminal proteostasis upon autophagy-targeted interventions. These methods will likely be more widely applicable, beyond autophagy research.

Diagnosis on the way out - personality on the way in? Priorities in treatment in child and adolescent psychiatry.

The scientific basis for practice in child psychiatry has developed apace. And has thrown up several quandries for an accepted paradigm for good practice anchored to the diagnostic schema developed in adult psychiatry. This paper hopes to stimulate discussion about where alternative paradigms might lead us on a path to precision medicine as applied to child psychiatry.

Emotional abuse and neglect in a clinical setting: challenges for mental health professionals.

This article addresses some of the common uncertainties and dilemmas encountered by both adult and child mental health workers in the course of their clinical practice when dealing with cases of suspected emotional abuse or neglect (EAN) of children. We suggest ways of dealing with these according to current best practice guidelines and our own clinical experience working in the field of child maltreatment.

Prevalence of traumatic psychological stress reactions in children and parents following paediatric surgery: a systematic review and meta-analysis.

Background: Children undergoing surgery and their parents are at risk of developing post-traumatic stress reactions. We systematically reviewed the literature to understand the prevalence of this issue, as well as potential risk factors. Methods: We conducted a systematic review and meta-analysis, using PubMed, PsycInfo, Web of Science and Google Scholar, with searches conducted in February 2021. Papers were included if they measured post-traumatic stress in children and/or parents following paediatric surgery and were excluded if they did not use a validated measure of post-traumatic stress. Data were extracted from published reports. Findings: Our search yielded a total of 1672 papers, of which 16 met our inclusion criteria. In meta-analysis, pooled studies of children estimated an overall prevalence of 16% meeting criteria for post-traumatic stress disorder post surgery (N=187, 95% CI 5% to 31%, I2=80%). After pooling studies of parents, overall prevalence was estimated at 23% (N=1444, 95% CI 16% to 31%, I2=91%). Prevalence rates were higher than those reported in the general population. Risk factors reported within studies included length of stay, level of social support and parental mental health. Interpretation: There is consistent evidence of traumatic stress following surgery in childhood which warrants further investigation. Those delivering surgical care to children would benefit from a raised awareness of the potential for post-traumatic stress in their patients and their families, including offering screening and support.

The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276.

BACKGROUND: The links between the p53/MDM2 pathway and the expression of pro-oncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1. METHODS: Proximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype. RESULTS: CD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control. CONCLUSION: Genetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis.

While studies of the autophagy-related (ATG) genes in knockout models have led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP family proteins (human ATG8 equivalents) are still poorly understood. A major drawback in understanding their roles is that the available interactome data has largely been acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was employed to map endogenous GABARAPL2 protein complexes using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions, whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Furthermore, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together these data allow us to define ACSL3 as a novel regulator of the enigmatic UFM1 conjugation pathway.

Selective Autophagy Conceals the Enemy: Why Cytotoxic T Cells Don't (MH)C Pancreatic Cancer.

Cell surface MHC-I can present tumor antigens to CD8+ T cells. In pancreatic cancer, selective autophagy instead reroutes MHC-I to lysosomes, using the ubiquitin-binding receptor NBR1, precluding T cell recognition. Accordingly, immune clearance of tumors can be facilitated by blocking autophagy.

Development of a Gaussian Process - feature selection model to characterise (poly)dimethylsiloxane (Silastic® ) membrane permeation.

OBJECTIVES: The current study aims to determine the effect of physicochemical descriptor selection on models of polydimethylsiloxane permeation. METHODS: A total of 2942 descriptors were calculated for a data set of 77 chemicals. Data were processed to remove redundancy, single values, imbalanced and highly correlated data, yielding 1363 relevant descriptors. For four independent test sets, feature selection methods were applied and modelled via a variety of Machine Learning methods. KEY FINDINGS: Two sets of molecular descriptors which can provide improved predictions, compared to existing models, have been identified. Best permeation predictions were found with Gaussian Process methods. The molecular descriptors describe lipophilicity, partial charge and hydrogen bonding as key determinants of PDMS permeation. CONCLUSIONS: This study highlights important considerations in the development of relevant models and in the construction and use of the data sets used in such studies, particularly that highly correlated descriptors should be removed from data sets. Predictive models are improved by the methodology adopted in this study, notably the systematic evaluation of descriptors, rather than simply using any and all available descriptors, often based empirically on in vitro experiments. Such findings also have clear relevance to a number of other fields.

Child psychiatry: a model for specific goals for in-patient treatment linked to resources and limitations in out-patient treatment.

SUMMARY: I present a rationale for two different types of in-patient child psychiatric unit: 24/7 intensive units and 24/5 child and family units. Intensive units address safety requirements. The developing personality of young people is at the centre of in-patient approaches on the child and family units. This requires attachment-informed practice. Families must always be involved and placement of units must facilitate their participation. The primary skill characterising these units is use of the milieu for therapy and combining this with family therapy. In other words, nurses and allied professionals need to be the dominant force in unit development, under the reflective guidance of consultants and clinical psychologists.

Emerging Principles of Selective ER Autophagy.

The endoplasmic reticulum (ER) is a fundamental organelle in cellular metabolism and signal transduction. It is subject to complex, dynamic sculpting of morphology and composition. Degradation of ER content has an important role to play here. Indeed, a major emerging player in ER turnover is ER-phagy, the degradation of ER fragments by selective autophagy, particularly macroautophagy. This article proposes a number of unifying principles of ER-phagy mechanism and compares these with other selective autophagy pathways. A perspective on the likely roles of ER-phagy in determining cell fate is provided. Emerging related forms of intracellular catabolism of the ER or contents, including ER-phagy by microautophagy and selective ER protein removal via the lysosome, are outlined for comparison. Unresolved questions regarding the mechanism of ER-phagy and its significance in cellular and organismal health are put forward. This review concludes with a perspective on how this fundamental knowledge might inform future clinical developments.

The influence of diffusion cell type and experimental temperature on machine learning models of skin permeability.

OBJECTIVES: The aim of this study was to use Gaussian process regression (GPR) methods to quantify the effect of experimental temperature (Texp ) and choice of diffusion cell on model quality and performance. METHODS: Data were collated from the literature. Static and flow-through diffusion cell data were separated, and a series of GPR experiments was conducted. The effect of Texp was assessed by comparing a range of datasets where Texp either remained constant or was varied from 22 to 45 °C. KEY FINDINGS: Using data from flow-through diffusion cells results in poor model performance. Data from static diffusion cells resulted in significantly greater performance. Inclusion of data from flow-through cell experiments reduces overall model quality. Consideration of Texp improves model quality when the dataset used exhibits a wide range of experimental temperatures. CONCLUSIONS: This study highlights the problem of collating literature data into datasets from which models are constructed without consideration of the nature of those data. In order to optimise model quality data from only static, Franz-type, experiments should be used to construct the model and Texp should either be incorporated as a descriptor in the model if data are collated from a range of studies conducted at different temperatures.

Picky Eating at the ER-phagy Buffet.

The degradation of the endoplasmic reticulum (ER) by autophagy (ER-phagy) regulates proteostasis. Two studies (An et al., Mol. Cell, 2019; Chino et al., Mol. Cell, 2019) have uncovered a new ER-phagy molecule, TEX264, yielding insight into how ER is packaged for degradation, and have illuminated the extent of redundancy between different ER-phagy 'pathways' in remodelling the ER proteome.

Endocytosis and Lack of Cytotoxicity of Alkyl-Capped Silicon Quantum Dots Prepared from Porous Silicon.

Freely-dissolved silicon quantum dots were prepared by thermal hydrosilation of 1-undecene at high-porosity porous silicon under reflux in toluene. This reaction produces a suspension of alkyl-capped silicon quantum dots (alkyl SiQDs) with bright orange luminescence, a core Si nanocrystal diameter of about 2.5 nm and a total particle diameter of about 5 nm. Previous work has shown that these particles are rapidly endocytosed by malignant cell lines and have little or no acute toxicity as judged by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for viability and the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis. We have extended this work to the CACO-2 cell line, an established model for the human small intestinal mucosa, and demonstrate that neither acute nor chronic (14 days) toxicity is observed as judged by cell morphology, viability, ATP production, ROS production and DNA damage (single cell gel electrophoresis) at doses of 50-200 µ g mL - 1 . Quantitative assessment of the extent of uptake of alkyl SiQDs by CACO-2, HeLa, HepG2, and Huh7 cell lines by flow cytometry showed a wide variation. The liver cell lines (HepG2 and Huh7) were the most active and HeLa and CACO-2 showed comparable activity. Previous work has reported a cholesterol-sensitivity of the endocytosis (HeLa), which suggests a caveolin-mediated pathway. However, gene expression analysis by quantitative real-time polymerase chain reaction (RT-PCR) indicates very low levels of caveolins 1 and 2 in HepG2 and much higher levels in HeLa. The data suggest that the mechanism of endocytosis of the alkyl SiQDs is cell-line dependent.

ER-phagy: shaping up and destressing the endoplasmic reticulum.

The endoplasmic reticulum (ER) network has central roles in metabolism and cellular organization. The ER undergoes dynamic alterations in morphology, molecular composition and functional specification. Remodelling of the network under fluctuating conditions enables the continual performance of ER functions and minimizes stress. Recent data have revealed that selective autophagy-mediated degradation of ER fragments, or ER-phagy, fundamentally contributes to this remodelling. This review provides a perspective on established views of selective autophagy, comparing these with emerging mechanisms of ER-phagy and related processes. The text discusses the impact of ER-phagy on the function of the ER- and the cell, both in normal physiology and when dysregulated within disease settings. Finally, unanswered questions regarding the mechanisms and significance of ER-phagy are highlighted.

Assessment of hepatotoxicity and dermal toxicity of butyl paraben and methyl paraben using HepG2 and HDFn in vitro models.

Parabens, esters of parahydroxybenzoic acid, are widely used in cosmetic, food and pharmaceutical industries mainly for their antibacterial and fungicidal properties. Methyl paraben has shown very low toxicity in a wide range of in vitro and animal tests. However, butyl paraben and derivatives, such as isobutyl parabens, are classified as allergens and have been shown to induce toxic effects. In the present study the effects of exposure to methyl or butyl paraben (5-1000 µM) on cytotoxicity, oxidative stress, mitochondrial dysfunction and genotoxicity were measured in a hepatocarcinoma cell line (HepG2) and human dermal fibroblasts neonatal (HDFn). Butyl paraben caused a concentration dependent decrease (above 400 µM) in cell viability for both cell lines. Toxicity of butyl paraben observed appeared to be mediated via ATP depletion as seen from luminescence assays. Depletion of glutathione was also observed for higher concentrations of butyl paraben, which may indicate the involvement of oxidative stress. Methyl paraben, however, did not show any significant decrease in cell viability, reduction in ATP or glutathione levels in HepG2 and HDFn cell lines at the concentrations tested. In vitro studies based on human cell lines can provide information in the early stages of multitier paraben toxicity studies and can be combined with in vivo and ex vivo studies to build more comprehensive, scientifically sound strategies for paraben safety testing. The results obtained in this study could supplement existing in vivo toxicity data for defining more robust limits for human exposure.