Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion

Intra-endolysosomal Ca(2+) release is required for endolysosomal membrane fusion with intracellular organelles. However, the molecular mechanisms for intra-endolysosomal Ca(2+) release and the downstream Ca(2+) targets involved in the fusion remain elusive. Previously, we demonstrated that endolysosomal P2X4 forms channels activated by luminal adenosine triphosphate in a pH-dependent manner. In this paper, we show that overexpression of P2X4, as well as increasing endolysosomal P2X4 activity by alkalinization of endolysosome lumen, promoted vacuole enlargement in cells and endolysosome fusion in a cell-free assay. These effects were prevented by inhibiting P2X4, expressing a dominant-negative P2X4 mutant, and disrupting the P2X4 gene. We further show that P2X4 and calmodulin (CaM) form a complex at endolysosomal membrane where P2X4 activation recruits CaM to promote fusion and vacuolation in a Ca(2+)-dependent fashion. Moreover, P2X4 activation-triggered fusion and vacuolation were suppressed by inhibiting CaM. Our data thus suggest a new molecular mechanism for endolysosomal membrane fusion involving P2X4-mediated endolysosomal Ca(2+) release and subsequent CaM activation

Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy

Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a β-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization

Stream-dependent development of higher visual cortical areas

Multiple cortical areas contribute to visual processing in mice. However, the functional organization and development of higher visual areas are unclear. Here, we used intrinsic signal optical imaging and 2-photon calcium imaging to map visual responses in adult and developing mice. We found that visually driven activity was well-correlated among higher visual areas within two distinct subnetworks resembling the dorsal and ventral visual streams. Visual response magnitude in dorsal stream areas slowly increased over the first two weeks of visual experience. By contrast, ventral stream areas exhibited strong responses shortly after eye opening. Neurons in a dorsal stream area showed little change in their tuning sharpness to oriented gratings while those in a ventral stream area increased stimulus selectivity and expanded their receptive fields significantly. Together, these findings provide a functional basis for grouping subnetworks of mouse visual areas and revealed stream differences in the development of receptive field properties

Prospective observational cohort study on grading the severity of postoperative complications in global surgery research

Background The Clavien–Dindo classification is perhaps the most widely used approach for reporting postoperative complications in clinical trials. This system classifies complication severity by the treatment provided. However, it is unclear whether the Clavien–Dindo system can be used internationally in studies across differing healthcare systems in high- (HICs) and low- and middle-income countries (LMICs). Methods This was a secondary analysis of the International Surgical Outcomes Study (ISOS), a prospective observational cohort study of elective surgery in adults. Data collection occurred over a 7-day period. Severity of complications was graded using Clavien–Dindo and the simpler ISOS grading (mild, moderate or severe, based on guided investigator judgement). Severity grading was compared using the intraclass correlation coefficient (ICC). Data are presented as frequencies and ICC values (with 95 per cent c.i.). The analysis was stratified by income status of the country, comparing HICs with LMICs. Results A total of 44 814 patients were recruited from 474 hospitals in 27 countries (19 HICs and 8 LMICs). Some 7508 patients (16·8 per cent) experienced at least one postoperative complication, equivalent to 11 664 complications in total. Using the ISOS classification, 5504 of 11 664 complications (47·2 per cent) were graded as mild, 4244 (36·4 per cent) as moderate and 1916 (16·4 per cent) as severe. Using Clavien–Dindo, 6781 of 11 664 complications (58·1 per cent) were graded as I or II, 1740 (14·9 per cent) as III, 2408 (20·6 per cent) as IV and 735 (6·3 per cent) as V. Agreement between classification systems was poor overall (ICC 0·41, 95 per cent c.i. 0·20 to 0·55), and in LMICs (ICC 0·23, 0·05 to 0·38) and HICs (ICC 0·46, 0·25 to 0·59). Conclusion Caution is recommended when using a treatment approach to grade complications in global surgery studies, as this may introduce bias unintentionally

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

© 2017 British Journal of Anaesthesia 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\u3c0.01], 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\u3c0.01; I2=87%] and reduced complication rates [OR 0.73 (0.61–0.88); P\u3c0.01; 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

Critical care admission following elective surgery was not associated with survival benefit: prospective analysis of data from 27 countries

This was an investigator initiated study funded by Nestle Health Sciences through an unrestricted research grant, and by a National Institute for Health Research (UK) Professorship held by RP. The study was sponsored by Queen Mary University of London