Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit-Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.

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BackgroundAdmission neutrophil-lymphocyte ratio (NLR) is significantly correlated to clinical outcomes in acute ischemic stroke (AIS). We investigated follow-up NLR and temporal changes in NLR after endovascular thrombectomy (EVT) with respect to successful revascularization, clinical outcomes, symptomatic intracranial hemorrhage (sICH) and mortality.MethodsRetrospective analysis of EVT for anterior circulation emergent LVO was performed with both admission (NLR1) and 3–7 day follow-up NLR (NLR2) laboratory data. Patient demographics, National Institutes of Health Stroke Scale (NIHSS) presentations, reperfusion efficacy (modified Thrombolysis in Cerebral Infarction (mTICI) score), sICH, and clinical outcomes (modified Rankin Scale (mRS)) at 90 days were studied. Univariate analyses correlated NLR1, NLR2, and temporal change in NLR (NLR2-NLR1) with successful reperfusion (mTICI ≥2b), favorable outcomes (mRS ≤2), sICH, and mortality. Multivariable logistic regression model evaluated the independent effects of NLR2 on favorable outcomes.Results142 AIS patients with median NIHSS 17 underwent EVT within 24 hours, and met NLR laboratory inclusion criteria. Lower follow-up NLR2 and less temporal change in NLR over 3–7 days, but not admission NLR1, inversely correlated with successful reperfusion (p<0.05) and favorable clinical outcomes (p<0.001). Higher follow-up NLR2 and greater temporal change in NLR was significantly associated with sICH and mortality (p≤0.05). In multivariable logistic regression, lower follow-up NLR2 remained a predictor of favorable outcomes (OR 0.785, p=0.001), independent of age or successful reperfusion.ConclusionsFollow-up NLR is a readily available and modifiable biomarker that correlates with the degree of reperfusion after mechanical stroke thrombectomy. Lower follow-up NLR2 at 3–7 days is associated with successful reperfusion and an independent predictor of favorable clinical outcomes, with reduced risk for sICH and mortality.

Summary3β-Azidocholest-5-ene (3) and (3β)-3-(prop-2-yn-1-yloxy)cholest-5-ene (10) were prepared as substrates to synthesize a variety of three-motif pharmacophoric conjugates through CuAAC. Basically, these conjugates included cholesterol and 1,2,3-triazole moieties, while the third, the pharmacophore, was either a chalcone, a lipophilic residue or a carbohydrate tag. These compounds were successfully prepared in good yields and characterized by NMR, MS and IR spectroscopic techniques. Chalcone conjugate 6c showed the best antimicrobial activity, while the lactoside conjugate 27 showed the best cytotoxic effect in vitro.

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