Vesicle capture by membrane-bound Munc13-1 requires self-assembly into discrete clusters

Munc13-1 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggest that multiple copies of Munc13-1 form nano-assemblies in active zones of neurons. However, it is not known whether such clustering of Munc13-1 is correlated with multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13-1 clustering is also unknown. Here, we report that nano-clustering is an inherent property of Munc13-1 and is indeed required for vesicle binding to bilayers containing Munc13-1. Purified Munc13-1 protein reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ˜ 20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters containing a minimum of 6 copies of Munc13-1 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C2C domain of Munc13-1 is not required for Munc13-1 clustering, but is required for efficient vesicle capture. This capture is largely due to a combination of electrostatic and hydrophobic interactions between the C2C domain and the vesicle membrane

CDK1 controls CHMP7-dependent nuclear envelope reformation

Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope (NE) during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III assembly at the NE occurs transiently during mitotic (M) exit and is initiated when CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, interacts with the Inner Nuclear Membrane (INM) protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during M-exit. Firstly, we show that CHMP7 plays an important role in the dissolution of LEM2 clusters that form at the NE during M-exit. Secondly, we show that CDK1 phosphorylates CHMP7 upon M-entry at Ser3 and Ser441 and that this phosphorylation reduces CHMP7’s interaction with LEM2, limiting its assembly during M-phase. We show that spatiotemporal differences in the dephosphorylation of CHMP7 license its assembly at the NE during telophase, but restrict its assembly on the ER at this time. Without CDK1 phosphorylation, CHMP7 undergoes inappropriate assembly in the peripheral ER during M-exit, capturing LEM2 and downstream ESCRT-III components. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration

Risk factors associated with adverse fetal outcomes in pregnancies affected by Coronavirus disease 2019 (COVID-19): a secondary analysis of the WAPM study on COVID-19.

Objectives To evaluate the strength of association between maternal and pregnancy characteristics and the risk of adverse perinatal outcomes in pregnancies with laboratory confirmed COVID-19. Methods Secondary analysis of a multinational, cohort study on all consecutive pregnant women with laboratory-confirmed COVID-19 from February 1, 2020 to April 30, 2020 from 73 centers from 22 different countries. A confirmed case of COVID-19 was defined as a positive result on real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay of nasal and pharyngeal swab specimens. The primary outcome was a composite adverse fetal outcome, defined as the presence of either abortion (pregnancy loss before 22 weeks of gestations), stillbirth (intrauterine fetal death after 22 weeks of gestation), neonatal death (death of a live-born infant within the first 28 days of life), and perinatal death (either stillbirth or neonatal death). Logistic regression analysis was performed to evaluate parameters independently associated with the primary outcome. Logistic regression was reported as odds ratio (OR) with 95% confidence interval (CI). Results Mean gestational age at diagnosis was 30.6+/-9.5 weeks, with 8.0% of women being diagnosed in the first, 22.2% in the second and 69.8% in the third trimester of pregnancy. There were six miscarriage (2.3%), six intrauterine device (IUD) (2.3) and 5 (2.0%) neonatal deaths, with an overall rate of perinatal death of 4.2% (11/265), thus resulting into 17 cases experiencing and 226 not experiencing composite adverse fetal outcome. Neither stillbirths nor neonatal deaths had congenital anomalies found at antenatal or postnatal evaluation. Furthermore, none of the cases experiencing IUD had signs of impending demise at arterial or venous Doppler. Neonatal deaths were all considered as prematurity-related adverse events. Of the 250 live-born neonates, one (0.4%) was found positive at RT-PCR pharyngeal swabs performed after delivery. The mother was tested positive during the third trimester of pregnancy. The newborn was asymptomatic and had negative RT-PCR test after 14 days of life. At logistic regression analysis, gestational age at diagnosis (OR: 0.85, 95% CI 0.8-0.9 per week increase; pPeer reviewe

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Lipid transfer proteins:the lipid commute via shuttles, bridges and tubes

Lipids are distributed in a highly heterogeneous fashion in different cellular membranes. Only a minority of lipids achieve their final intracellular distribution through transport by vesicles. Instead, the bulk of lipid traffic is mediated by a large group of lipid transfer proteins (LTPs), which move small numbers of lipids at a time using hydrophobic cavities that stabilize lipid molecules outside membranes. Although the first LTPs were discovered almost 50 years ago, most progress in understanding these proteins has been made in the past few years, leading to considerable temporal and spatial refinement of our understanding of the function of these lipid transporters. The number of known LTPs has increased, with exciting discoveries of their multimeric assembly. Structural studies of LTPs have progressed from static crystal structures to dynamic structural approaches that show how conformational changes contribute to lipid handling at a sub-millisecond timescale. A major development has been the finding that many intracellular LTPs localize to two organelles at the same time, forming a shuttle, bridge or tube that links donor and acceptor compartments. The understanding of how different lipids achieve their final destination at the molecular level allows a better explanation of the range of defects that occur in diseases associated with lipid transport and distribution, opening up the possibility of developing therapies that specifically target lipid transfer

The surgical safety checklist and patient outcomes after surgery: a prospective observational cohort study, systematic review and meta-analysis

Background: The surgical safety checklist is widely used to improve the quality of perioperative care. However, clinicians continue to debate the clinical effectiveness of this tool. Methods: Prospective analysis of data from the International Surgical Outcomes Study (ISOS), an international observational study of elective in-patient surgery, accompanied by a systematic review and meta-analysis of published literature. The exposure was surgical safety checklist use. The primary outcome was in-hospital mortality and the secondary outcome was postoperative complications. In the ISOS cohort, a multivariable multi-level generalized linear model was used to test associations. To further contextualise these findings, we included the results from the ISOS cohort in a meta-analysis. Results are reported as odds ratios (OR) with 95% confidence intervals. Results: We included 44 814 patients from 497 hospitals in 27 countries in the ISOS analysis. There were 40 245 (89.8%) patients exposed to the checklist, whilst 7508 (16.8%) sustained ≥1 postoperative complications and 207 (0.5%) died before hospital discharge. Checklist exposure was associated with reduced mortality [odds ratio (OR) 0.49 (0.32-0.77); P<0.01], but no difference in complication rates [OR 1.02 (0.88-1.19); P=0.75]. In a systematic review, we screened 3732 records and identified 11 eligible studies of 453 292 patients including the ISOS cohort. Checklist exposure was associated with both reduced postoperative mortality [OR 0.75 (0.62-0.92); P<0.01; I2=87%] and reduced complication rates [OR 0.73 (0.61-0.88); P<0.01; I2=89%). Conclusions: Patients exposed to a surgical safety checklist experience better postoperative outcomes, but this could simply reflect wider quality of care in hospitals where checklist use is routine