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Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2∼Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region (ccRING3) drive MIB1 dimerization and that MIB1 ubiquitin transfer activity relies solely on ccRING3. We report X-ray crystal structures of a UbcH5B-ccRING3 complex and the ANK domain. Directly tethering the MIB1 N-terminal region to ccRING3 forms a minimal MIB1 protein sufficient to induce a Notch response in receiver cells and rescue mib knockout phenotypes in flies. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.

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AIM: To investigate the performance of the 2021 ERC/ESICM-recommended algorithm for predicting poor outcome after cardiac arrest (CA) and potential tools for predicting neurological recovery in patients with indeterminate outcome. METHODS: Prospective, multicenter study on out-of-hospital CA survivors from 28 ICUs of the AfterROSC network. In patients comatose with a Glasgow Coma Scale motor score ≤3 at ≥72 h after resuscitation, we measured: (1) the accuracy of neurological examination, biomarkers (neuron-specific enolase, NSE), electrophysiology (EEG and SSEP) and neuroimaging (brain CT and MRI) for predicting poor outcome (modified Rankin scale score ≥4 at 90 days), and (2) the ability of low or decreasing NSE levels and benign EEG to predict good outcome in patients whose prognosis remained indeterminate. RESULTS: Among 337 included patients, the ERC-ESICM algorithm predicted poor neurological outcome in 175 patients, and the positive predictive value for an unfavourable outcome was 100% [98-100]%. The specificity of individual predictors ranged from 90% for EEG to 100% for clinical examination and SSEP. Among the remaining 162 patients with indeterminate outcome, a combination of 2 favourable signs predicted good outcome with 99[96-100]% specificity and 23[11-38]% sensitivity. CONCLUSION: All comatose resuscitated patients who fulfilled the ERC-ESICM criteria for poor outcome after CA had poor outcome at three months, even if a self-fulfilling prophecy cannot be completely excluded. In patients with indeterminate outcome (half of the population), favourable signs predicted neurological recovery, reducing prognostic uncertainty.

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BACKGROUND: Acute respiratory distress syndrome (ARDS) remains a significant challenge in critical care, with high mortality rates despite advancements in treatment. Venovenous extracorporeal membrane oxygenation (VV-ECMO) is employed as salvage therapy for refractory cases. However, some patients may continue to experience persistent severe hypoxemia despite being treated with VV-ECMO. To achieve this, moderate hypothermia and short-acting selective ß1-blockers have been proposed. METHODS: Using a swine model of severe ARDS treated with VV-ECMO, this study investigated the efficacy of moderate hypothermia or ß-blockade in improving arterial oxygen saturation (SaO2) three hours after VV-ECMO initiation. Primary endpoints included the ratio of VV-ECMO flow to cardiac output and arterial oxygen saturation before VV-ECMO start (H0) and three hours after ECMO start (H3). Secondary safety criteria encompassed hemodynamics and oxygenation parameters. RESULTS: Twenty-two male pigs were randomized into three groups: control (n = 6), hypothermia (n = 9) and ß-blockade (n = 7). At H0, all groups demonstrated similar hemodynamic and respiratory parameters. Both moderate hypothermia and ß-blockade groups exhibited a significant increase in the ratio of VV-ECMO flow to cardiac output at H3, resulting in improved SaO2. At H3, despite a decrease in oxygen delivery and consumption in the intervention groups compared to the control group, oxygen extraction ratios across groups remained unchanged and lactate levels were normal. CONCLUSIONS: In a swine model of severe ARDS treated with VV-ECMO, both moderate hypothermia and ß-blockade led to an increase in the ratio of VV-ECMO flow to cardiac output resulting in improved arterial oxygen saturation without any impact on tissue perfusion.

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Members of the LGD/CC2D1 protein family contain repeats of the family-defining DM14 domains. Via this domain, they interact with members of the CHMP family, which are essential for the ESCRT machinery-mediated formation of intraluminal vesicles during endosome maturation. Here, we investigate the requirement of the DM14 domains for the function of Lgd in detail. We found that although both odd-numbered DM14s can act in a functionally redundant manner, the redundancy is not complete and both contribute to the full function of Lgd. Our analysis indicates that some of the AAs that form the KARRxxR motif of the onDM14s are not exchangeable by similarly charged AAs without loss of function, indicating that they not only provide charge, but also fulfil structural roles. Furthermore, we show that the region of Lgd between DM14-4 and the C2 domain as well as its C-terminal region to the C2 domain are important for protein stability/function. Moreover, we analysed the importance of AAs that are conserved in all DM14 domains. Finally, our analysis of the C. elegans ortholog of Lgd revealed that it has only one DM14 domain that is functionally equivalent to the onDM14s. Altogether, the results further the understanding of how Lgd family members regulate the ESCRT machinery.

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OBJECTIVE: Evidence on the optimal "dose" of cognitive behavioral therapy (CBT) for treating major depressive disorder is sparse. This analysis aimed to evaluate the dose-response curve in CBT using a nonlinear approach, whereby "dose" was defined as number of treatment sessions. The dose-response curve of CBT was compared to other psychotherapies and pharmacological treatments for depression. METHOD: A systematic review and metaregression analysis of randomized controlled trials (RCTs) examining the efficacy of CBT in adults with acute depression was conducted. Treatment arms examining other psychosocial or pharmacological interventions were also analyzed. Cubic spline metaregression techniques were used to model nonlinear dose-response curves. RESULTS: Seventy-two studies and 7,377 participants were included. Modeling the dose-response curve between change of depression symptom severity and the number of CBT sessions resulted in a nonlinear curve characterized by a strong improvement in symptom severity from baseline within the first eight sessions. Symptom reduction continues in the further course of the treatment, but at a slower pace. A similar pattern of symptom development was found for other therapies as well, although the prominence of early improvement and overall effect sizes vary across treatment arms. CONCLUSION: Results imply a general tendency for the strongest alleviation of depressive symptom severity in early stages of CBT treatment, thus, if aiming at symptom alleviation, speak for short CBT interventions. However, these findings have to be discussed in the light of the limited data regarding the sustainability of treatment effects in short-term therapies and effects beyond symptomatic changes. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

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Background: Scalable psychological interventions such as the WHO's Self-Help Plus (SH+) have been developed for clinical and non-clinical populations in need of psychological support. SH+ has been successfully implemented to prevent common mental disorders among asylum seekers and refugees who are growing in number due to increasing levels of forced migration. These populations are often exposed to multiple, severe sources of traumatisation, and evidence of the effect of such events on treatment is insufficient, especially for non-clinical populations.Objective: We aim to study the effect of potentially traumatic experiences (PTEs) and the mediating role of symptoms of posttraumatic stress disorder (PTSD) on the improvement following SH+.Method: Participants allocated to SH+ who received at least three sessions (N = 345) were extracted from two large, randomised, European prevention trials involving asylum seekers and refugees. Measures of distress, depression, functional impairment, and post-traumatic stress symptoms were administered at baseline and 6 months post-intervention, together with measures of well-being and quality of life. Adjusted models were constructed to examine the effect of PTEs on post-intervention improvement. The possible mediating role of PTSD symptoms in this relationship was then tested.Results: Increasing numbers of PTEs decreased the beneficial effect of SH+ for all measures. This relationship was mediated by symptoms of PTSD when analysing measures of well-being and quality of life. However, this did not apply for measures of mental health problems.Conclusions: Exposure to PTEs may largely reduce benefits from SH+. PTSD symptomatology plays a specific, mediating role on psychological well-being and quality of life of participants who experienced PTE. Healthcare professionals and researchers should consider the role of PTEs and PTSD symptoms in the treatment of migrants and refugees and explore possible feasible add-on solutions for cases exposed to multiple PTEs.

Increasing numbers of potentially traumatic experiences can decrease the beneficial effect of a manualized group psychotherapeutic intervention in migrants and refugees across multiple countries.In absence of a full threshold diagnosis of post-traumatic stress disorder, post-traumatic stress symptoms still mediate the relation between potentially traumatic experiences and some outcome improvements at follow-up.While the moderating role of number of potentially traumatic experiences applies to all outcomes (depression symptoms, psychological distress, functional impairment, well-being, and quality of life), the mediating role of post-traumatic stress symptoms in this relation only applies to well-being and quality of life.

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Curcumin, the fat-soluble active ingredient and major compound of curcuminoids contained in the curcuma root, is known for its physiological low absorption and bioavailability. Various formulations and galenic technologies are currently available on the market. In this study, the product tested was provided as a soft gelatin capsule containing curcuminoids in an oily matrix mixed with phospholipids (oil/phospholipids [PL]-based, no new technologies applied or artificial excipients added). This was intended to improve bioavailability of curcuminoids as well as to mimic the natural digestion process of fat-soluble substances. In particular, the oral bioavailability of curcuminoids in the oil/PL-based formulation was compared with the pure curcuminoids extract alone (reference product), in a randomized, cross-over, single oral dose study design. Twelve healthy subjects were administered 200 mg curcuminoids under fasting conditions. Pharmacokinetic parameters were analyzed from individual concentration-time curves of total curcuminoids, as well as the curcumin metabolite tetrahydrocurcumin (THC). Results showed significantly higher AUC0-8h levels after the intake of the oil/PL-based formulation for total curcuminoids (205.60 vs. 112.50 ng/mL*h, P = .0001) as well as for THC (347.30 vs. 118.90 ng/mL*h, P < .0001) in comparison to the pure curcuminoids extract. Cmax was also significantly higher for both parameters analyzed (total curcuminoids: 47.54 vs. 21.16 ng/mL, P = .0001; THC: 96.69 vs. 29.83 ng/mL, P < .0001). In addition, the uptake kinetic of total curcuminoids was significantly fastened with the oil/PL-based curcuminoids formulation compared with the pure curcuminoids extract (P = .0446). These data suggest an improved impact on curcuminoids uptake of the oil/PL-based formulation and confirms its good tolerability.

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Acral burning pain triggered by fever, thermal hyposensitivity and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide. To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via gene editing. We subjected induced pluripotent stem cells to targeted peripheral neuronal differentiation and observed intra-lysosomal globotriaosylceramide accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in isogenic control neurons compared with healthy control neurons (P < 0.001). Moreover, we demonstrate a drastic increase in Fabry sensory neuron calcium levels at 39°C mimicking clinical fever (P < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons differentiated from induced pluripotent stem cells derived from Fabry patients compared with healthy control cells (P < 0.001). Linear-nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed mitochondrial aggregation at sphingolipid accumulations within Fabry sensory neurites utilizing a click chemistry approach together with mitochondrial dysmorphism compared with healthy control cells. We pioneer pilot insights into the cellular mechanisms contributing to pain, thermal hyposensitivity and denervation in Fabry small fibre neuropathy and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.

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Fabry disease (FD) is a lysosomal storage disorder of X-linked inheritance. Mutations in the α-galactosidase A gene lead to cellular globotriaosylceramide (Gb3) depositions and triggerable acral burning pain in both sexes as an early FD symptom of unknown pathophysiology. We aimed at elucidating the link between skin cells and nociceptor sensitization contributing to FD pain in a sex-associated manner. We used cultured keratinocytes and fibroblasts of 27 adult FD patients and 20 healthy controls. Epidermal keratinocytes and dermal fibroblasts were cultured and immunoreacted to evaluate Gb3 load. Gene expression analysis of pain-related ion channels and pro-inflammatory cytokines was performed in dermal fibroblasts. We further investigated electrophysiological properties of induced pluripotent stem cell (iPSC) derived sensory-like neurons of a man with FD and a healthy man and incubated the cells with interleukin 8 (IL-8) or fibroblast supernatant as an in vitro model system. Keratinocytes displayed no intracellular, but membrane-bound Gb3 deposits. In contrast, fibroblasts showed intracellular Gb3 and revealed higher gene expression of potassium intermediate/small conductance calcium-activated potassium channel 3.1 (KCa 3.1, KCNN4) in both, men and women with FD compared to controls. Additionally, cytokine expression analysis showed increased IL-8 RNA levels only in female FD fibroblasts. Patch-clamp studies revealed reduced rheobase currents for both iPSC neuron cell lines incubated with IL-8 or fibroblast supernatant of women with FD. We conclude that Gb3 deposition in female FD patient skin fibroblasts may lead to increased KCa3.1 activity and IL-8 secretion. This may result in cutaneous nociceptor sensitization as a potential mechanism contributing to a sex-associated FD pain phenotype.

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It has been suggested that the novel selective phosphodiesterase 9 (PDE9) inhibitor may improve cardiac and renal function by blocking 3',5'-cyclic guanosine monophosphate (cGMP) degradation. 5/6 nephrectomized (5/6Nx) rats were used to investigate the effects of the PDE9 inhibitor (BAY 73-6691) on the heart and kidney. Two doses of BAY 73-6691 (1 mg/kg/day and 5 mg/kg/day) were given for 95 days. The 5/6Nx rats developed albuminuria, a decrease in serum creatinine clearance (Ccr), and elevated serum troponin T levels. Echocardiographic data showed that 5/6 nephrectomy resulted in increased fractional shortening (FS), stroke volume (SV), and left ventricular ejection fraction (EF). However, 95 days of PDE9 inhibitor treatment did not improve any cardiac and renal functional parameter. Histopathologically, 5/6 nephrectomy resulted in severe kidney and heart damage, such as renal interstitial fibrosis, glomerulosclerosis, and enlarged cardiomyocytes. Telmisartan attenuated renal interstitial fibrosis and glomerulosclerosis as well as improved cardiomyocyte size. However, except for cardiomyocyte size and renal perivascular fibrosis, BAY 73-6691 had no effect on other cardiac and renal histologic parameters. Pathway enrichment analysis using RNA sequencing data of kidney and heart tissue identified chronic kidney disease pathways, such as phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, complement and coagulation cascades, and nuclear factor kappa B (NF-κB) signaling pathway. PDE9i did not affect any of these disease-related pathways. Two dosages of the PDE9 inhibitor BAY 73-6691 known to be effective in other rat models have only limited cardio-renal protective effects in 5/6 nephrectomized rats.

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Type-2 diabetes (T2D) worsens stroke recovery, amplifying post-stroke disabilities. Currently, there are no therapies targeting this important clinical problem. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are potent anti-diabetic drugs that also efficiently reduce cardiovascular death and heart failure. In addition, SGLT2i facilitate several processes implicated in stroke recovery. However, the potential efficacy of SGLT2i to improve stroke recovery in T2D has not been investigated. Therefore, we determined whether a post-stroke intervention with the SGLT2i Empagliflozin could improve stroke recovery in T2D mice. T2D was induced in C57BL6J mice by 8 months of high-fat diet feeding. Hereafter, animals were subjected to transient middle cerebral artery occlusion and treated with vehicle or the SGLTi Empagliflozin (10 mg/kg/day) starting from 3 days after stroke. A similar study in non diabetic mice was also conducted. Stroke recovery was assessed using the forepaw grip strength test. To identify potential mechanisms involved in the Empagliflozin-mediated effects, several metabolic parameters were assessed. Additionally, neuronal survival, neuroinflammation, neurogenesis and cerebral vascularization were analyzed using immunohistochemistry/quantitative microscopy. Empagliflozin significantly improved stroke recovery in T2D but not in non-diabetic mice. Improvement of functional recovery was associated with lowered glycemia, increased serum levels of fibroblast growth factor-21 (FGF-21), and the normalization of T2D-induced aberration of parenchymal pericyte density. The global T2D-epidemic and the fact that T2D is a major risk factor for stroke are drastically increasing the number of people in need of efficacious therapies to improve stroke recovery. Our data provide a strong incentive for the potential use of SGLT2i for the treatment of post-stroke sequelae in T2D.

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Digital Mental Health Technologies (DMHTs) have the potential to close treatment gaps in settings where mental healthcare is scarce or even inaccessible. For this, DMHTs need to be affordable, evidence-based, justice-oriented, user-friendly, and embedded in a functioning digital infrastructure. This viewpoint discusses areas crucial for future developments of DMHTs. Drawing back on interdisciplinary scholarship, questions of health equity, consumer-, patient- and developer-oriented legislation, and requirements for successful implementation of technologies across the globe are discussed. Economic considerations and policy implications complement these aspects. We discuss the need for cultural adaptation specific to the context of use and point to several benefits as well as pitfalls of DMHTs for research and healthcare provision. Nonetheless, to circumvent technology-driven solutionism, the development and implementation of DMHTs require a holistic, multi-sectoral, and participatory approach.

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Traditionally, peripheral sensory neurons are assumed as the exclusive transducers of external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nerve fiber interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of afferents and adjacent connexin 43 contacts in native skin and have applied a pipeline based on expansion microscopy to quantify these parameter in skin sections of healthy participants versus patients with small fiber neuropathy. We further derived a fully human co-culture system, visualizing ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and potential interaction sites advances research at the neuro-cutaneous unit. These findings are crucial on the way to decipher the mechanisms of cutaneous nociception.

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Magnesium alloys play an essential role in metallic lightweight construction for modern mobility applications due to their low density, excellent specific strength, and very good castability. For some years now, degradable implants have also been made from magnesium alloys, which, thanks to this special functionality, save patients a second surgery for explantation. New additive manufacturing processes, which are divided into powder-based and wire-based processes depending on the feedstock used, can be utilized for these applications. Therefore, magnesium alloys should also be used here, but this is hardly ever implemented, and few literature reports exist on this subject. This is attributable to the high affinity of magnesium to oxygen, which makes the use of powders difficult. Therefore, magnesium wires are likely to be used. In this paper, a magnesium-based nanocomposite wire is made from an AM60 (Mg-6Al-0.4Mn) (reinforced with 1 wt% AlN nanoparticles and containing calcium to reduce flammability), using a high-shear process and then extruded into wires. These wires are then used as feedstock to build up samples by wire-arc directed energy deposition, and their mechanical properties and microstructure are examined. Our results show that although the ductility is reduced by adding calcium and nanoparticles, the yield strength in the welding direction and perpendicular to it is increased to 131 MPa.

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This systematic review examines moderators and mediators tested in evaluations of stress management interventions for hospital employees to determine their significance for intervention outcomes. To be included, studies had to comprise a moderator or mediator analysis and a quantitative assessment of stress or mental well-being, and to be published in English or German language. Five databases (APA PsycInfo, APA PsycArticles, Embase, Medline, and Web of Science) were searched. Moderators and mediators were categorised thematically and examined using effect direction plots. Study quality was assessed using RoB 2 and ROBINS-I. In fifteen included studies, 22 moderators and ten mediators were identified. Moderators and mediators were categorised into individual psychological factors (14), socio-economic status (6), work situation (5), intervention (3), and duration of employment (3). Two moderators (perceived stressfulness of residency, job control) had a positive, two a negative impact (spirituality, socially desirable responding). One moderator (years of professional experience) had a positive and negative impact. Three moderators measured on categorical scales (gender, profession, and shiftwork) also had effects, favouring women, physicians and night-shift employees. Five mediators (adherence to intervention, mindfulness, non-reactivity to inner experience, total observing, and self-compassion) had a positive impact, while three (isolation, over-identification, psychological inflexibility) had a negative impact. In conclusion, effects of interventions were predominantly driven by individual psychological factors, while the role of other variables seems to be limited. Interventions focussing on primary or tertiary prevention were rare. Also processes through which organisational-level interventions can be most effective have been hardly investigated. Larger and methodologically robust studies are needed to better understand causal pathways and optimise matching of interventions to target groups.

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Glucagon-like peptide-1 receptor (GLP-1R) agonists are approved treatments for Type 2 diabetes mellitus, with liraglutide and semaglutide also approved for the treatment of obesity. The natural gut hormone oxyntomodulin is a weak dual agonist of the glucagon receptor (GCGR) and GLP-1R. Development of poly-agonists mimicking oxyntomodulin, such as the novel dual GCGR/GLP-1R agonist survodutide, represents an important step towards a more effective treatment for people with Type 2 diabetes mellitus and obesity. Survodutide is a 29-amino acid peptide derived from glucagon, with the incorporation of potent GLP-1 activities. It contains a C18 diacid which mediates binding to albumin, thereby prolonging the half-life to enable once-weekly subcutaneous dosing. The utilisation of GCGR agonism aims to enhance body weight-lowering effects by increasing energy expenditure in addition to the anorectic action of GLP-1R agonists. Glucose-lowering efficacy of survodutide has been demonstrated in a Phase II trial in patients with Type 2 diabetes mellitus and obesity and was associated with clinically meaningful body weight loss. These data highlight the potential of dual GCGR/GLP-1R agonism for reducing glycated haemoglobin and body weight in patients with Type 2 diabetes mellitus, and for greater therapeutic efficacy compared with GLP-1R agonism alone.

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BACKGROUND: Recent advances in hardware and software enabled the use of artificial intelligence (AI) algorithms for analysis of complex data in a wide range of daily-life use cases. We aim to explore the benefits of applying AI to a specific use case in transplant nephrology: risk prediction for severe posttransplant events. For the first time, we combine multinational real-world transplant data, which require specific legal and technical protection measures. OBJECTIVE: The German-Canadian NephroCAGE consortium aims to develop and evaluate specific processes, software tools, and methods to (1) combine transplant data of more than 8000 cases over the past decades from leading transplant centers in Germany and Canada, (2) implement specific measures to protect sensitive transplant data, and (3) use multinational data as a foundation for developing high-quality prognostic AI models. METHODS: To protect sensitive transplant data addressing the first and second objectives, we aim to implement a decentralized NephroCAGE federated learning infrastructure upon a private blockchain. Our NephroCAGE federated learning infrastructure enables a switch of paradigms: instead of pooling sensitive data into a central database for analysis, it enables the transfer of clinical prediction models (CPMs) to clinical sites for local data analyses. Thus, sensitive transplant data reside protected in their original sites while the comparable small algorithms are exchanged instead. For our third objective, we will compare the performance of selected AI algorithms, for example, random forest and extreme gradient boosting, as foundation for CPMs to predict severe short- and long-term posttransplant risks, for example, graft failure or mortality. The CPMs will be trained on donor and recipient data from retrospective cohorts of kidney transplant patients. RESULTS: We have received initial funding for NephroCAGE in February 2021. All clinical partners have applied for and received ethics approval as of 2022. The process of exploration of clinical transplant database for variable extraction has started at all the centers in 2022. In total, 8120 patient records have been retrieved as of August 2023. The development and validation of CPMs is ongoing as of 2023. CONCLUSIONS: For the first time, we will (1) combine kidney transplant data from nephrology centers in Germany and Canada, (2) implement federated learning as a foundation to use such real-world transplant data as a basis for the training of CPMs in a privacy-preserving way, and (3) develop a learning software system to investigate population specifics, for example, to understand population heterogeneity, treatment specificities, and individual impact on selected posttransplant outcomes. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/48892.

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The execution of a Notch signal at the plasma membrane relies on the mechanical force exerted onto Notch by its ligand. It has been appreciated that the DSL ligands need to collaborate with a ubiquitin (Ub) ligase, either Neuralized or Mindbomb1, in order to exert this pulling force, but the role of ubiquitylation per se is uncertain. Regarding the Delta-Neur pair, it is documented that neither the Neur catalytic domain nor the Delta intracellular lysines (putative Ub acceptors) are needed for activity. Here, we present a dissection of the Delta activity using the Delta-Notch-dependent expression of Hey in newborn Drosophila neurons as a sensitive in vivo assay. We show that the Delta-Neur interaction per se, rather than ubiquitylation, is needed for activity, pointing to the existence of a Delta-Neur signaling complex. The Neur catalytic domain, although not strictly needed, greatly improves Delta-Neur complex functionality when the Delta lysines are mutated, suggesting that the ubiquitylation of some component of the complex, other than Delta, can enhance signaling. Since Hey expression is sensitive to the perturbation of endocytosis, we propose that the Delta-Neur complex triggers a force-generating endocytosis event that activates Notch in the adjacent cell.

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BACKGROUND: Ubiquitylation (ubi) of the intracellular domain of the Notch ligand Delta (Dl) by the E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) on lysines (Ks) is thought to be essential for the its signalling activity. Nevertheless, we have previously shown that DlK2R-HA, a Dl variant where all Ks in its intracellular domain (ICD) are replaced by the structurally similar arginine (R), still possess weak activity if over-expressed. This suggests that ubi is not absolutely required for Dl signalling. However, it is not known whether the residual activity of DlK2R-HA is an effect of over-expression and, if not, whether DlK2R can provide sufficient activity for the whole development of Drosophila. RESULTS: To clarify these issues, we generated and analysed DlattP-DlK2R-HA, a knock-in allele into the Dl locus. Our analysis of this allele reveals that the sole presence of one copy of DlattP-DlK2R-HA can provide sufficient activity for completion of development. It further indicates that while ubi is required for the full activity of Dl in Mib1-dependent processes, it is not essential for Neur-controlled neural development. We identify three modes of Dl signalling that are either dependent or independent of ubi. Importantly, all modes depend on the presence of the endocytic adapter Epsin. During activation of Dl, direct binding of Epsin appears not to be an essential requirement. In addition, our analysis further reveals that the Ks are required to tune down the cis-inhibitory interaction of Dl with Notch. CONCLUSIONS: Our results indicate that Dl can activate the Notch pathway without ubi of its ICD. It signals via three modes. Ubi is specifically required for the Mib1-dependent processes and the adjustment of cis-inhibition. In contrast to Mib1, Neur can efficiently activate Dl without ubi. Neur probably acts as an endocytic co-adapter in addition to its role as E3 ligase. Endocytosis, regulated in a ubi-dependent or ubi-independent manner is required for signalling and also suppression of cis-inhibition. The findings clarify the role of ubi of the ligands during Notch signalling.

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BACKGROUND: Out-of-hospital cardiac arrest (OHCA) is a heterogeneous entity with multiple origins and prognoses. An early, reliable assessment of the prognosis is useful to adapt therapeutic strategy, tailor intensity of care, and inform relatives. We aimed primarily to undertake a prospective multicentric study to evaluate predictive performance of the Cardiac Arrest Prognosis (CAHP) Score as compare to historical dataset systematically collected after OHCA (Utstein style criteria). Our secondary aim was to evaluate other dedicated scores for predicting outcome after OHCA and to compare them to Utstein style criteria. METHODS: We prospectively collected data from 24 French and Belgium Intensive Care Units (ICUs) between August 2020 and June 2022. All cases of non-traumatic OHCA (cardiac and non-cardiac causes) patients with stable return of spontaneous circulation (ROSC) and comatose at ICU admission (defined by Glasgow coma score ≤ 8) on ICU admission were included. The primary outcome was the modified Rankin scale (mRS) at day 90 after cardiac arrest, assessed by phone interviews. A wide range of developed scores (CAHP, OHCA, CREST, C-Graph, TTM, CAST, NULL-PLEASE, and MIRACLE2) were included, and their accuracies in predicting poor outcome at 90 days after OHCA (defined as mRS ≥ 4) were determined using the area under the receiving operating characteristic curve (AUROC) and the calibration belt. RESULTS: During the study period, 907 patients were screened, and 658 were included in the study. Patients were predominantly male (72%), with a mean age of 61 ± 15, most having collapsed from a supposed cardiac cause (64%). The mortality rate at day 90 was 63% and unfavorable neurological outcomes were observed in 66%. The performance (AUROC) of Utstein criteria for poor outcome prediction was moderate at 0.79 [0.76-0.83], whereas AUROCs from other scores varied from 0.79 [0.75-0.83] to 0.88 [0.86-0.91]. For each score, the proportion of patients for whom individual values could not be calculated varied from 1.4% to 17.4%. CONCLUSIONS: In patients admitted to ICUs after a successfully resuscitated OHCA, most of the scores available for the evaluation of the subsequent prognosis are more efficient than the usual Utstein criteria but calibration is unacceptable for some of them. Our results show that some scores (CAHP, sCAHP, mCAHP, OHCA, rCAST) have superior performance, and that their ease and speed of determination should encourage their use. Trial registration https://clinicaltrials.gov/ct2/show/NCT04167891.