Incorporation of cholesterol in sphingomyelin- phosphatidylcholine vesicles has profound effects on detergent-induced phase transitions

Abstract Vesicle ↔ micelle transitions are important phenomena during bile formation and intestinal lipid processing. The hepatocyte canalicular membrane outer leaflet contains appreciable amounts of phosphatidylcholine (PC) and sphingomyelin (SM), and both phospholipids are found in the human diet. Dietary SM enrichment inhibits intestinal cholesterol absorption. We therefore studied detergent-induced vesicle → micelle transitions in SM-PC vesicles. Phase transitions were evaluated by spectrophotometry and cryotransmission electron microscopy (cryo-TEM) after addition of taurocholate (3–7 mM) to SM-PC vesicles (4 mM phospholipid, SM/PC 40%/60%, without or with 1.6 mM cholesterol). After addition of excess (5–7 mM) taurocholate, SM-PC vesicles were more sensitive to micellization than PC vesicles. As shown by sequential cryo-TEM, addition of equimolar (4 mM) taurocholate to SM-PC vesicles induced formation of open vesicles, then (at the absorbance peak) fusion of bilayer fragments into large open structures (around 200 nm diameter) coexisting with some multilamellar or fused vesicles and thread-like micelles and, finally, transformation into an uniform picture with long thread-like micelles. Incorporation of cholesterol in the SM/PC bilayer changed initial vesicular shape from spherical into ellipsoid and profoundly increased detergent resistance. Disk-like micelles and multilamellar vesicles, and then extremely large vesicular structures, were observed by sequential cryo-TEM under these circumstances, with persistently increased absorbance values by spectrophotometry. These findings may be relevant for bile formation and intestinal lipid processing. Inhibition of intestinal cholesterol absorption by dietary SM enrichment may relate to high resistance against bile salt-induced micellization of intestinal lipids in presence of the sphingolipid

Structural Evaluation of EGFR Inhibition Mechanisms for Nanobodies/VHH Domains

SummaryThe epidermal growth factor receptor (EGFR) is implicated in human cancers and is the target of several classes of therapeutic agents, including antibody-based drugs. Here, we describe X-ray crystal structures of the extracellular region of EGFR in complex with three inhibitory nanobodies, the variable domains of heavy chain only antibodies (VHH). VHH domains, the smallest natural antigen-binding modules, are readily engineered for diagnostic and therapeutic applications. All three VHH domains prevent ligand-induced EGFR activation, but use two distinct mechanisms. 7D12 sterically blocks ligand binding to EGFR in a manner similar to that of cetuximab. EgA1 and 9G8 bind an epitope near the EGFR domain II/III junction, preventing receptor conformational changes required for high-affinity ligand binding and dimerization. This epitope is accessible to the convex VHH paratope but inaccessible to the flatter paratope of monoclonal antibodies. Appreciating the modes of binding and inhibition of these VHH domains will aid in developing them for tumor imaging and/or cancer therapy

A small protein probe for correlated microscopy of endogenous proteins

Probes are essential to visualize proteins in their cellular environment, both using light microscopy as well as electron microscopy (EM). Correlated light microscopy and electron microscopy (CLEM) requires probes that can be imaged simultaneously by both optical and electron-dense signals. Existing combinatorial probes often have impaired efficiency, need ectopic expression as a fusion protein, or do not target endogenous proteins. Here, we present FLIPPER-bodies to label endogenous proteins for CLEM. Fluorescent Indicator and Peroxidase for Precipitation with EM Resolution (FLIPPER), the combination of a fluorescent protein and a peroxidase, is fused to a nanobody against a target of interest. The modular nature of these probes allows an easy exchange of components to change its target or color. A general FLIPPER-body targeting GFP highlights histone2B-GFP both in fluorescence and in EM. Similarly, endogenous EGF receptors and HER2 are visualized at nm-scale resolution in ultrastructural context. The small and flexible FLIPPER-body outperforms IgG-based immuno-labeling, likely by better reaching the epitopes. Given the modular domains and possibilities of nanobody generation for other targets, FLIPPER-bodies have high potential to become a universal tool to identify proteins in immuno-CLEM with increased sensitivity compared to current approaches

Spiral Vein Reconstruction of the Infected Abdominal Aorta Using the Greater Saphenous Vein: Preliminary Results of the Tilburg Experience

AbstractObjectivesThe aim of this study was to evaluate patients, who underwent spiral vein reconstruction of the abdominal aorta to repair infected aneurysms or replace infected aortic grafts.MethodsAll spiral vein reconstructions between March 2005 and May 2010 because of vascular infections of the abdominal aorta were retrospectively included. Diagnosis was determined by clinical examination, laboratory results, computed tomography (CT) and positron emission tomography (PET) scan, and microbiological tests. Spiral vein reconstruction consisted of harvesting the greater saphenous vein (GSV) and construction into a spiral graft, aortic reconstruction and a transmesenteric omentumplasty. Primary outcomes were survival and limb salvage. Secondary outcomes included technical, clinical and ongoing success, re-infection, ongoing infection and patency.ResultsAll five patients survived surgery, and there were no in-hospital deaths. Survival and limb salvage were 100% after median follow-up of 13 months (6–67 months). Further, technical, clinical and continuing success was 100%. There were no re-infections or ongoing infections.ConclusionsSpiral vein reconstruction using the GSV showed good short-term survival and limb salvage. It, therefore, might be considered as an attractive treatment method for vascular infections of the abdominal aorta. Still, more follow-up is needed to evaluate long-term results

The Dutch multicenter experience of the Endo-Sponge treatment for anastomotic leakage after colorectal surgery

Anastomotic leakage is a feared complication following colorectal surgery and is associated with early and long-term morbidity and mortality. The presacral cavity as the result of leakage can be treated with an endo-sponge (B-Braun Medical). The aim of this study was to assess the effectiveness of endo-sponge treatment of the presacral cavity as the result of anastomotic leakage in the Netherlands. Between July 2006 and April 2008, 16 patients (M/F = 9:7) with median age 64 years (range 19-78 years) who underwent surgery for rectal cancer (n = 13) or ulcerative colitis (n = 3) were treated with the endo-sponge treatment after anastomotic leakage. Of the 16 patients, eight patients started with the endo-sponge treatment within 6 weeks after the initial surgery. In these patients the endo-sponge was placed after a median of 24 days (range 13-39 days) following surgery. In the remaining eight patients the endo-sponge treatment was started later than 6 weeks after the initial surgery. In this group there was a median of 74 days (range 43-1,602 days) between surgery and the start of endo-sponge placement. There was closure in six out of eight patients (75%) in the group that started with the endo-sponge treatment within 6 weeks of surgery compared with three out of eight patients (38%) in the group that started later (p = 0.315). Closure was achieved in a median of 40 (range 28-90) days with a median number of 13 sponge replacements (range 8-17). Endo-sponge placement can be helpful in the treatment for anastomotic leakage after colorectal surgery and might prevent a chronic presacral sinus. However, it is not yet clear if this new treatment modality results in quicker healin

Difurocoumarins, Psoralen Analogs: Synthesis and DNA Photobinding

A new tetracyclic derivative, difurocoumarin, was synthesized and studied in order to ascertain its possible use as a photochemotherapeutic agent alternative to psoralens. The compound proved able to photobind monofunctionally to DNA on irradiation with UV-A. A photocycloadduct with thymine was isolated and characterized spectroscopically

The impact of performing gastric cancer surgery during holiday periods. A population-based study using Dutch upper gastrointestinal cancer audit (DUCA) data

Existing literature suggests inferior quality of oncologic surgery during holiday periods. This study aimed to investigate the impact of holiday periods on surgical treatment of gastric cancer in the Netherlands. This nationwide study included all gastric cancer patients undergoing potentially curative surgery registered in the Dutch Upper Gastrointestinal Cancer Audit (DUCA). For each patient it was established whether they underwent surgery during or outside the 11 Dutch holiday weeks, based on date and region of surgery. Separate, single-day holidays were not included. Baseline and treatment characteristics were compared using descriptive statistics. Time from diagnosis to treatment and short-term surgical outcomes were compared using multilevel multivariable logistic regression analyses. To prevent bias from recent advancements, analyses were repeated in a recent cohort of patients (2015-2018). Between 2011-2018, 3440 patients were included in the DUCA. Some 555 (16.1%) patients underwent surgery during 11 holiday weeks. There were no differences in patient, tumor and treatment characteristics and time to treatment between holidays and non-holidays. Tumor-positive resection margins (R1/R2 vs R0) occurred more frequent during holidays (aOR:1.47, 95%CI:1.07-2.04). Subgroup analyses in a recent cohort of patients also found higher tumor-positive resection margins (aOR:1.59, 95%CI:1.01-2.43) and higher failure-to-rescue rates (aOR:2.55, 95%CI:1.18-5.49) during holidays. Even though time to treatment and patient, tumor and treatment characteristics were comparable between holidays and non-holidays, tumor-positive resection margin and failure to-rescue rates were higher during holidays. This suggests that steps must be taken to keep specialized and dedicated gastric cancer expertise up to standard during holiday periods. (C)& nbsp;2022 The Author(s). Published by Elsevier Inc.& nbsp;& nbsp

Postoperative intensive care unit stay after minimally invasive esophagectomy shows large hospital variation:Results from the Dutch Upper Gastrointestinal Cancer Audit

Item does not contain fulltextINTRODUCTION: The value of routine intensive care unit (ICU) admission after minimally invasive esophagectomy (MIE) has been questioned. This study aimed to investigate Dutch hospital variation regarding length of direct postoperative ICU stay, and the impact of this hospital variation on short-term surgical outcomes. MATERIALS AND METHODS: Patients registered in the Dutch Upper Gastrointestinal Cancer Audit (DUCA) undergoing curative MIE were included. Length of direct postoperative ICU stay was dichotomized around the national median into short ICU stay ( ≤ 1 day) and long ICU stay ( > 1 day). A case-mix corrected funnel plot based on multivariable logistic regression analyses investigated hospital variation. The impact of this hospital variation on short-term surgical outcomes was investigated using multilevel multivariable logistic regression analyses. RESULTS: Between 2017 and 2019, 2110 patients from 16 hospitals were included. Median length of postoperative ICU stay was 1 day [hospital variation: 0-4]. The percentage of short ICU stay ranged from 0 to 91% among hospitals. Corrected for case-mix, 7 hospitals had statistically significantly higher short ICU stay rates and 6 hospitals had lower rates. ICU readmission, in-hospital/30-day mortality, failure to rescue, postoperative pneumonia, cardiac complications and anastomotic leakage were not associated with hospital variation in length of ICU stay. Total length of hospital stay was significantly shorter in hospitals with relatively short ICU stay. CONCLUSION: This study showed significant hospital variation in postoperative length of ICU stay after MIE. Short ICU stay was associated with shorter overall hospital admission and did not negatively impact short-term surgical outcomes. More selected use of ICU resources could result in a national significant cost reduction