Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics.

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down

Changes in searching behaviour of CSL transcription complexes in Notch active conditions.

During development cells receive a variety of signals, which are of crucial importance to their fate determination. One such source of signal is the Notch signalling pathway, where Notch activity regulates expression of target genes through the core transcription factor CSL. To understand changes in transcription factor behaviour that lead to transcriptional changes in Notch active cells, we have probed CSL behaviours in real time, using in vivo Single Molecule Localisation Microscopy. Trajectory analysis reveals that Notch-On conditions increase the fraction of bound CSL molecules, but also the proportion of molecules with exploratory behaviours. These properties are shared by the co-activator Mastermind. Furthermore, both CSL and Mastermind, exhibit characteristics of local exploration near a Notch target locus. A similar behaviour is observed for CSL molecules diffusing in the vicinity of other bound CSL clusters. We suggest therefore that CSL acquires an exploratory behaviour when part of the activation complex, favouring local searching and retention close to its target enhancers. This change explains how CSL can efficiently increase its occupancy at target sites in Notch-On conditions

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

Peer reviewed: TrueAcknowledgements: We thank Kat Millen and the Genetics Department Fly Facility for performing DNA injections of fly embryos to generate transgenic stocks. We are grateful to Cambridge Advanced Imaging Centre for advice on imaging and to all members of the Bray Lab for helpful discussions and comments on the manuscript. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. The work was funded by a Wellcome Trust Investigator Award (212207/Z/18) and an MRC Programme Grant (MR/T014156/1) to SJB. CR and SB, were supported by studentships from Wolfson College-University of Cambridge (Dept of Physiology Development and Neuroscience-School of Biological Sciences).Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind.

Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation

RAB6 and dynein drive post‐Golgi apical transport to prevent neuronal progenitor delamination

International audienceRadial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that aRG delamination is regulated by the post-Golgi secretory pathway. Using in situ subcellular live imaging, we show that post-Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A' and RAB6B impairs apical localization of CRB3 and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knockout of the dynein activator LIS1. Overall, our results identify a RAB6-dynein-LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity

Comparison of standard prophylactic, intermediate prophylactic and therapeutic anticoagulation in patients with severe COVID-19: protocol for the ANTICOVID multicentre, parallel-group, open-label, randomised controlled trial

International audienceINTRODUCTION: COVID-19 induces venous, arterial and microvascular thrombosis, involving several pathophysiological processes. In patients with severe COVID-19 without macrovascular thrombosis, escalating into high-dose prophylactic anticoagulation (HD-PA) or therapeutic anticoagulation (TA) could be beneficial in limiting the extension of microvascular thrombosis and forestalling the evolution of lung and multiorgan microcirculatory dysfunction. In the absence of data from randomised trials, clinical practice varies widely. METHODS AND ANALYSIS: This is a French multicentre, parallel-group, open-label, randomised controlled superiority trial to compare the efficacy and safety of three anticoagulation strategies in patients with COVID-19. Patients with oxygen-treated COVID-19 showing no pulmonary artery thrombosis on computed tomography with pulmonary angiogram will be randomised to receive either low-dose PA, HD-PA or TA for 14 days. Patients attaining the extremes of weight and those with severe renal failure will not be included. We will recruit 353 patients. Patients will be randomised on a 1:1:1 basis, and stratified by centre, use of invasive mechanical ventilation, D-dimer levels and body mass index. The primary endpoint is a hierarchical criterion at day 28 including all-cause mortality, followed by the time to clinical improvement defined as the time from randomisation to an improvement of at least two points on the ordinal clinical scale. Secondary outcomes include thrombotic and major bleeding events at day 28, individual components of the primary endpoint, number of oxygen-free, ventilator-free and vasopressor-free days at day 28, D-dimer and sepsis-induced coagulopathy score at day 7, intensive care unit and hospital stay at day 28 and day 90, and all-cause death and quality of life at day 90. ETHICS AND DISSEMINATION: The study has been approved by an ethical committee (Ethics Committee, Ile de France VII, Paris, France; reference 2020-A03531-38). Patients will be included after obtaining their signed informed consent. The results will be submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04808882

Effects of Standard-Dose Prophylactic, High-Dose Prophylactic, and Therapeutic Anticoagulation in Patients With Hypoxemic COVID-19 Pneumonia The ANTICOVID Randomized Clinical Trial

International audienceImportance Given the high risk of thrombosis and anticoagulation-related bleeding in patients with hypoxemic COVID-19 pneumonia, identifying the lowest effective dose of anticoagulation therapy for these patients is imperative. OBJECTIVES To determine whether therapeutic anticoagulation (TA) or high-dose prophylactic anticoagulation (HD-PA) decreases mortality and/or disease duration compared with standard-dose prophylactic anticoagulation (SD-PA), and whether TA outperforms HD-PA; and to compare the net clinical outcomes among the 3 strategies. DESIGN, SETTINGS, AND PARTICIPANTS The ANTICOVID randomized clinical open-label trial included patients with hypoxemic COVID-19 pneumonia requiring supplemental oxygen and having no initial thrombosis on chest computer tomography with pulmonary angiogram at 23 health centers in France from April 14 to December 13, 2021. Of 339 patients randomized, 334 were included in the primary analysis-114 patients in the SD-PA group, 110 in the HD-PA, and 110 in the TA. At randomization, 90% of the patients were in the intensive care unit. Data analyses were performed from April 13, 2022, to January 3, 2023. INTERVENTIONS Patients were randomly assigned (1:1:1) to receive either SD-PA, HD-PA, or TA with low-molecular-weight or unfractionated heparin for 14 days. MAIN OUTCOMES AND MEASURES A hierarchical criterion of all-cause mortality followed by time to clinical improvement at day 28. Main secondary outcome was net clinical outcome at day 28 (composite of thrombosis, major bleeding, and all-cause death). RESULTS Among the study population of 334 individuals (mean [SD] age, 58.3 [13.0] years; 226 [67.7%] men and 108 [32.3%] women), use of HD-PA and SD-PA had similar probabilities of favorable outcome (47.3% [95%CI, 39.9% to 54.8%] vs 52.7%[95%CI, 45.2%to 60.1%]; P = .48), as did TA compared with SD-PA (50.9% [95%CI, 43.4%to 58.3%] vs 49.1% [95%CI, 41.7%to 56.6%]; P = .82) and TA compared with HD-PA (53.5%[95%CI 45.8% to 60.9%] vs 46.5% [95%CI, 39.1% to 54.2%]; P = .37). Net clinical outcome was met in 29.8% of patients receiving SD-PA (20.2%thrombosis, 2.6%bleeding, 14.0% death), 16.4% receiving HD-PA (5.5%thrombosis, 3.6%bleeding, 11.8%death), and 20.0% receiving TA (5.5% thrombosis, 3.6% bleeding, 12.7%death). Moreover, HD-PA and TA use significantly reduced thrombosis compared with SD-PA (absolute difference, -14.7 [95%CI -6.2 to -23.2] and -14.7 [95%CI -6.2 to -23.2], respectively). Use of HD-PA significantly reduced net clinical outcome compared with SD-PA (absolute difference, -13.5; 95%CI -2.6 to -24.3). CONCLUSIONS AND RELEVANCE This randomized clinical trial found that compared with SD-PA, neither HD-PAnor TAuse improved the primary hierarchical outcome of all-cause mortality or time to clinical improvement in patients with hypoxemicCOVID-19 pneumonia; however, HD-PA resulted in significantly better net clinical outcome by decreasing the risk of de novo thrombosis

Effects of Standard-Dose Prophylactic, High-Dose Prophylactic, and Therapeutic Anticoagulation in Patients With Hypoxemic COVID-19 Pneumonia The ANTICOVID Randomized Clinical Trial

International audienceIMPORTANCE Given the high risk of thrombosis and anticoagulation-related bleeding in patients with hypoxemic COVID-19 pneumonia, identifying the lowest effective dose of anticoagulation therapy for these patients is imperative. OBJECTIVES To determine whether therapeutic anticoagulation (TA) or high-dose prophylactic anticoagulation (HD-PA) decreases mortality and/or disease duration compared with standard-dose prophylactic anticoagulation (SD-PA), and whether TA outperforms HD-PA; and to compare the net clinical outcomes among the 3 strategies. DESIGN, SETTINGS, AND PARTICIPANTS The ANTICOVID randomized clinical open-label trial included patients with hypoxemic COVID-19 pneumonia requiring supplemental oxygen and having no initial thrombosis on chest computer tomography with pulmonary angiogram at 23 health centers in France from April 14 to December 13, 2021. Of 339 patients randomized, 334 were included in the primary analysis-114 patients in the SD-PA group, 110 in the HD-PA, and 110 in the TA. At randomization, 90% of the patients were in the intensive care unit. Data analyses were performed from April 13, 2022, to January 3, 2023. INTERVENTIONS Patients were randomly assigned (1:1:1) to receive either SD-PA, HD-PA, or TA with low-molecular-weight or unfractionated heparin for 14 days. MAIN OUTCOMES AND MEASURES A hierarchical criterion of all-cause mortality followed by time to clinical improvement at day 28. Main secondary outcome was net clinical outcome at day 28 (composite of thrombosis, major bleeding, and all-cause death). RESULTS Among the study population of 334 individuals (mean [SD] age, 58.3 [13.0] years; 226 [67.7%] men and 108 [32.3%] women), use of HD-PA and SD-PA had similar probabilities of favorable outcome (47.3% [95%CI, 39.9% to 54.8%] vs 52.7%[95%CI, 45.2%to 60.1%]; P = .48), as did TA compared with SD-PA (50.9% [95%CI, 43.4%to 58.3%] vs 49.1% [95%CI, 41.7%to 56.6%]; P = .82) and TA compared with HD-PA (53.5%[95%CI 45.8% to 60.9%] vs 46.5% [95%CI, 39.1% to 54.2%]; P = .37). Net clinical outcome was met in 29.8% of patients receiving SD-PA (20.2%thrombosis, 2.6%bleeding, 14.0% death), 16.4% receiving HD-PA (5.5%thrombosis, 3.6%bleeding, 11.8%death), and 20.0% receiving TA (5.5% thrombosis, 3.6% bleeding, 12.7%death). Moreover, HD-PA and TA use significantly reduced thrombosis compared with SD-PA (absolute difference, -14.7 [95%CI -6.2 to -23.2] and -14.7 [95%CI -6.2 to -23.2], respectively). Use of HD-PA significantly reduced net clinical outcome compared with SD-PA (absolute difference, -13.5; 95%CI -2.6 to -24.3). CONCLUSIONS AND RELEVANCE This randomized clinical trial found that compared with SD-PA, neither HD-PAnor TAuse improved the primary hierarchical outcome of all-cause mortality or time to clinical improvement in patients with hypoxemicCOVID-19 pneumonia; however, HD-PA resulted in significantly better net clinical outcome by decreasing the risk of de novo thrombosis