Protein glycation may occur naturally when reducing sugars and proteins coexist, which is often the case for industrial enzymes. The impact of post-translational modifications on enzyme... Show moreProtein glycation may occur naturally when reducing sugars and proteins coexist, which is often the case for industrial enzymes. The impact of post-translational modifications on enzyme performance (e.g., stability or function) is often not predictable, highlighting the importance of having appropriate analytical methodologies to monitor the influence of glycation on performance. Here, a boronate affinity chromatography method was developed to enrich glycated species followed by mass spectrometry for structural characterization and activity assays for functional assessment. This approach was applied to a (temperature-stressed) lipase used for food applications revealing that storage at -20 °C and 4 °C resulted in minor glycation (below 9%), whereas storage at 25 °C led to a higher glycation level with up to four sugars per lipase molecule. Remarkably, activity measurements revealed that glycation did not reduce lipase activity or stability. Altogether, this novel strategy is a helpful extension to the current analytical toolbox supporting development of enzyme products. Show less
Schaick, G. van; Dominguez Vega, E.; Gstottner, C.; Berg-Verleg, J.H. van den; Schouten, O.; Akeroyd, M.; ... ; Franc, V. 2022
Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and,... Show moreMany industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme.We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied.Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished.We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches. (C) 2020 Elsevier B.V. All rights reserved. Show less
Karthaus, E.G.; Tong, T.M.L.; Vahl, A.; Hamming, J.F.; Akker, L.H. van den; Akker, P.J. van den; ... ; Vos 2019
Objective: The aim of this was to analyze differences between saccularshaped abdominal aortic aneurysms (SaAAAs) and fusiform abdominal aortic aneurysms (FuAAAs) regarding patient characteristics,... Show moreObjective: The aim of this was to analyze differences between saccularshaped abdominal aortic aneurysms (SaAAAs) and fusiform abdominal aortic aneurysms (FuAAAs) regarding patient characteristics, treatment, and outcome, to advise a threshold for intervention for SaAAAs.Background: Based on the assumption that SaAAAs are more prone to rupture, guidelines suggest early elective treatment. However, little is known about the natural history of SaAAAs and the threshold for intervention is not substantiated.Methods: Observational study including primary repairs of degenerative AAAs in the Netherlands between 2016 and 2018 in which the shape was registered, registered in the Dutch Surgical Aneurysm Audit (DSAA). Patients were stratified by urgency of surgery; elective versus acute (symptomatic/ruptured). Patient characteristics, treatment, and outcome were compared between SaAAAs and FuAAAs.Results: A total of 7659 primary AAA-patients were included, 6.1% (n = 471) SaAAAs and 93.9% (n = 7188) FuAAAs. There were 5945 elective patients (6.5% SaAAA) and 1714 acute (4.8% SaAAA). Acute SaAAApatients were more often female (28.9% vs 17.2%, P = 0.007) compared with acute FuAAA-patients. SaAAAs had smaller diameters than FuAAAs, in elective (53.0mm vs 61 mm, P = 0.000) and acute (68mm vs 75 mm, P = 0.002) patients, even after adjusting for sex. In addition, 25.2% of acute SaAAA-patients presented with diameters <55mm and 8.4% <45 mm, versus 8.1% and 0.6% of acute FuAAA-patients (P = 0.000). Postoperative outcomes did not significantly differ between shapes in both elective and acute patients.Conclusions: SaAAAs become acute at smaller diameters than FuAAAs in DSAA patients. This study therefore supports the current idea that SaAAAs should be electively treated at smaller diameters than FuAAAs. The exact diameter threshold for elective treatment of SaAAAs is difficult to determine, but a diameter of 45mm seems to be an acceptable threshold. Show less
Winkel, T.A.; Schouten, O.; Hoeks, S.E.; Voute, M.T.; Chonchol, M.; Goei, D.; ... ; Poldermans, D. 2010
Background: Cardiac troponin T (cTnT) assays with increased sensitivity might increase the number of positive tests. Using the area under the curve (AUC) with serial sampling of cTnT an exact... Show moreBackground: Cardiac troponin T (cTnT) assays with increased sensitivity might increase the number of positive tests. Using the area under the curve (AUC) with serial sampling of cTnT an exact quantification of the myocardial damage size can be made. We compared the prognosis of vascular surgery patients with integrated cTnT-AUC values to continuous and standard 12-lead electrocardiography (ECG) changes. Methods: 513 Patients were monitored. cTnT sampling was performed on postoperative days 1, 3, 7, 30 and/or at discharge or whenever clinically indicated. If cTnT release occurred, daily measurements of cTnT were performed, until baseline was achieved. CTnT AUC was quantified and divided in tertiles. All-cause mortality and cardiovascular events (cardiac death and myocardial infarction) were noted during follow-up. Results: 81/513 (16%) Patients had cTnT release. After adjustment for gender, cardiac risk factors, and site and type of surgery, those in the highest cTnT-AUC tertile were associated with a significantly worse cardiovascular outcome and long-term mortality (HR 20.2; 95% CI 10.2-40.0 and HR 4.0; 95% CI 2.0-7.8 respectively). Receiver operator analysis showed that the best cut-off value for cTnT-AUC was <0.01 days*ng m for predicting long-term cardiovascular events and all-cause mortality. Conclusion: In vascular surgery patients quantitative assessment of cTnT strongly predicts long-term outcome. (C) 2010 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. Show less
Kuijk, J.P. van; Voute, M.T.; Flu, W.J.; Schouten, O.; Chonchol, M.; Hoeks, S.E.; ... ; Poldermans, D. 2010
Background Major vascular surgery patients are at high risk for developing asymptomatic perioperative myocardial ischemia reflected by a postoperative troponin release without the presence of chest... Show moreBackground Major vascular surgery patients are at high risk for developing asymptomatic perioperative myocardial ischemia reflected by a postoperative troponin release without the presence of chest pain or electrocardiographic abnormalities. Long-term prognosis is severely compromised and characterized by an increased risk of long-term mortality and cardiovascular events. Current guidelines on perioperative care recommend single antiplatelet therapy with aspirin as prophylaxis for cardiovascular events. However, as perioperative surgical stress results in a prolonged hypercoagulable state, the postoperative addition of clopidogrel to aspirin within 7 days after perioperative asymptomatic cardiac ischemia could provide improved effective prevention for cardiovascular events. Study design DECREASE-VII is a phase III, randomized, double-blind, placebo-controlled, multicenter clinical trial designed to evaluate the efficacy and safety of early postoperative dual antiplatelet therapy (aspirin and clopidogrel) for the prevention of cardiovascular events after major vascular surgery. Eligible patients undergoing a major vascular surgery (abdominal aorta or lower extremity vascular surgery) who developed perioperative asymptomatic troponin release are randomized 1: 1 to clopidogrel or placebo (300-mg loading dose, followed by 75mg daily) in addition to standard medical treatment with aspirin. The primary efficacy end point is the composite of cardiovascular death, stroke, or severe ischemia of the coronary or peripheral arterial circulation leading to an intervention. The evaluation of long-term safety includes bleeding defined by TIMI criteria. Recruitment began early 2010. The trial will continue until 750 patients are included and followed for at least 12 months. Summary DECREASE-VII is evaluating whether early postoperative dual antiplatelet therapy for patients developing asymptomatic cardiac ischemia after vascular surgery reduces cardiovascular events with a favorable safety profile. (Am Heart J 2010;160:387-93.) Show less
Background: Atrial fibrillation (AF) is a significant risk factor for cardiovascular (CV) mortality. This study aims to evaluate the prognostic implication of AF in patients with peripheral... Show moreBackground: Atrial fibrillation (AF) is a significant risk factor for cardiovascular (CV) mortality. This study aims to evaluate the prognostic implication of AF in patients with peripheral arterial disease (PAD). Methods: The International Reduction of Atherothrombosis for Continued Health (REACH) Registry included 23,542 outpatients in Europe with established coronary artery disease, cerebrovascular disease (CVD), PAD and/or >= 3 risk factors. Of these, 3753 patients had symptomatic PAD. CV risk factors were determined at baseline. Study end point was a combination of cardiac death, non-fatal myocardial infarction (MI) and stroke (CV events) during 2 years of follow-up. Cox regression analysis adjusted for age, gender and other risk factors (i.e., congestive heart failure, coronary artery re-vascularisation, coronary artery bypass grafting (CABG), MI, hypertension, stroke, current smoking and diabetes) was used. Results: Of 3753 PAD patients, 392 (10%) were known to have AF. Patients with AF were older and had a higher prevalence of CVD, diabetes and hypertension. Long-term CV mortality occurred in 5.6% of patients with AF and in 1.6% of those without AF (p < 0.001). Multivariable analyses showed that AF was an independent predictor of late CV events (hazard ratio (HR): 1.5; 95% confidence interval (CI): 1.09-2.0). Conclusion: AF is common in European patients with symptomatic PAD and is independently associated with a worse 2-year CV outcome. (C) 2010 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. Show less
Background The pathophysiology of new-onset cardiac arrhythmias is complex and may bring about severe cardiovascular complications. The relevance of perioperative arrhythmias during vascular... Show moreBackground The pathophysiology of new-onset cardiac arrhythmias is complex and may bring about severe cardiovascular complications. The relevance of perioperative arrhythmias during vascular surgery has not been investigated. The aim of this study was to assess risk factors and prognosis of new-onset arrhythmias during vascular surgery. Methods A total of 513 vascular surgery patients, without a history of arrhythmias, were included. Cardiac risk factors, inflammatory status, and left ventricular function (LVF; N-terminal pro-B-type natriuretic peptide and echocardiography) were assessed. Continuous electrocardiography (ECG) recordings for 72 hours were used to identify ischemia and new-onset arrhythmias: atrial fibrillation, sustained ventricular tachycardia, supraventricular tachycardia, and ventricular fibrillation. Logistic regression analysis was applied to identify preoperative risk factors for arrhythmias. Cox regression analysis assessed the impact of arrhythmias on cardiovascular event-free survival during 1.7 years. Results New-onset arrhythmias occurred in 55 (11%) of 513 patients: atrial fibrillation, ventricular tachycardia, supraventricular tachycardia, and ventricular fibrillation occurred in 4%, 7%, 1%, and 0.2%, respectively. Continuous ECG showed myocardial ischemia and arrhythmias in 17 (3%) of 513 patients. Arrhythmia was preceded by ischemia in 10 of 55 cases. Increased age and reduced LVF were risk factors for the development of arrhythmias. Multivariate analysis showed that perioperative arrhythmias were associated with long-term cardiovascular events, irrespective of the presence of perioperative ischemia (hazard ratio 2.2, 95% CI 1.3-3.8, P=.004). Conclusion New-onset perioperative arrhythmias are common after vascular surgery. The elderly and patients with reduced LVF show arrhythmias. Perioperative continuous ECG monitoring helps to identify this high-risk group at increased risk of cardiovascular events and death. (Am Heart J 2010;159:1108-15.) Show less
Despite recent advancements in perioperative care and guideline recommendations, patients undergoing vascular surgery remain at risk for perioperative cardiovascular complications. In this review,... Show moreDespite recent advancements in perioperative care and guideline recommendations, patients undergoing vascular surgery remain at risk for perioperative cardiovascular complications. In this review, the results are summarized of the most recent studies on the effectiveness and safety of perioperative statin use for the prevention of these perioperative cardiovascular complications. Perioperative statin therapy was associated with an improvement in postoperative cardiovascular outcome and a reduction in serum lipid levels and levels of inflammation markers. Show less
Lier, F. van; Schouten, O.; Hoeks, S.E.; Ven, L. van de; Stolker, R.J.; Bax, J.J.; Poldermans, D. 2010
Blockers are widely used to improve the postoperative cardiac outcome in patients with coronary artery disease scheduled for noncardiac surgery. However, recently serious concerns regarding the... Show moreBlockers are widely used to improve the postoperative cardiac outcome in patients with coronary artery disease scheduled for noncardiac surgery. However, recently serious concerns regarding the safety of perioperative beta blockers have emerged. To assess the incidence, risk factors, and beta-blocker use associated with postoperative stroke in the Dutch Echocardiographic Cardiac Risk Evaluation Applying Stress Echocardiography (DECREASE) trials, we evaluated all 3,884 patients of the DECREASE trials for postoperative stroke. All cardiac risk factors and medication use were assessed. The incidence of stroke within 30 days after surgery was recorded. The incidence of postoperative stroke in the DECREASE trials was 0.46% (18 of 3,884). For the p-blocker users, the incidence was 0.5%. All the strokes had an ischemic origin. A history of stroke was associated with a greater incidence of postoperative stroke (odds ratio [OR] 3.79, 95% confidence interval [CI] 1.2 to 11.6). Statins and anticoagulants were not associated with postoperative stroke (OR 0.85, 95% Cl 0.3 to 2.4; and OR 1.27, 95% CI 0.4 to 4.6, respectively). No association with bisoprolol therapy was found (OR 1.16, 95% CI 0.4 to 3.4). In conclusion, with a low-dose bisoprolol regimen started >= 30 days before surgery, no association was observed between beta-blocker use and postoperative stroke. (C) 2010 Elsevier Inc. All rights reserved. (Am J Cardiol 2010;105:43-47) Show less
