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Contains fulltext : 287508.pdf (Publisher’s version ) (Closed access) BACKGROUND: Pooled analyses of previous randomised studies have suggested that very early treatment with glyceryl trinitrate (also known as nitroglycerin) improves functional outcome in patients with acute ischaemic stroke or intracerebral haemorrhage, but this finding was not confirmed in a more recent trial (RIGHT-2). We aimed to assess whether patients with presumed acute stroke benefit from glyceryl tr initrate started within 3 h after symptom onset. METHODS: MR ASAP was a phase 3, randomised, open-label, blinded endpoint trial done at six ambulance services serving 18 hospitals in the Netherlands. Eligible participants (aged ≥18 years) had a probable diagnosis of acute stroke (as assessed by a paramedic), a face-arm-speech-time test score of 2 or 3, systolic blood pressure of at least 140 mm Hg, and could start treatment within 3 h of symptom onset. Participants were randomly assigned (1:1) by ambulance personnel, using a secure web-based electronic application with random block sizes stratified by ambulance service, to receive either transdermal glyceryl trinitrate 5 mg/day for 24 h plus standard care (glyceryl trinitrate group) or to standard care alone (control group) in the prehospital setting. Informed consent was deferred until after arrival at the hospital. The primary outcome was functional outcome assessed with the modified Rankin Scale (mRS) at 90 days. Safety outcomes included death within 7 days, death within 90 days, and serious adverse events. Analyses were based on modified intention to treat, and treatment effects were expressed as odds ratios (ORs) or common ORs, with adjustment for baseline prognostic factors. We separately analysed the total population and the target population (ie, patients with intracerebral haemorrhage, ischaemic stroke, or transient ischaemic attack). The target sample size was 1400 patients. The trial is registered as ISRCTN99503308. FINDINGS: On June 24, 2021, the MR ASAP trial was prematurely terminated on the advice of the data and safety monitoring board, with recruitment stopped because of safety concerns in patients with intracerebral haemorrhage. Between April 4, 2018, and Feb 12, 2021, 380 patients were randomly allocated to a study group. 325 provided informed consent or died before consent could be obtained, of whom 170 were assigned to the glyceryl trinitrate group and 155 to the control group. These patients were included in the total population. 201 patients (62%) had ischaemic stroke, 34 (10%) transient ischaemic attack, 56 (17%) intracerebral haemorrhage, and 34 (10%) a stroke-mimicking condition. In the total population (n=325), the median mRS score at 90 days was 2 (IQR 1-4) in both the glyceryl trinitrate and control groups (adjusted common OR 0·97 [95% CI 0·65-1·47]). In the target population (n=291), the 90-day mRS score was 2 (2-4) in the glyceryl trinitrate group and 3 (1-4) in the control group (0·92 [0·59-1·43]). In the total population, there were no differences between the two study groups with respect to death within 90 days (adjusted OR 1·07 [0·53-2·14]) or serious adverse events (unadjusted OR 1·23 [0·76-1·99]). In patients with intracerebral haemorrhage, 12 (34%) of 35 patients allocated to glyceryl trinitrate versus two (10%) of 21 allocated to the control group died within 7 days (adjusted OR 5·91 [0·78-44·81]); death within 90 days occurred in 16 (46%) of 35 in the glyceryl trinitrate group and 11 (55%) of 20 in the control group (adjusted OR 0·87 [0·18-4·17]). INTERPRETATION: We found no sign of benefit of transdermal glyceryl trinitrate started within 3 h of symptom onset in the prehospital setting in patients with presumed acute stroke. The signal of potential early harm of glyceryl trinitrate in patients with intracerebral haemorrhage suggests that glyceryl trinitrate should be avoided in this setting. FUNDING: The Collaboration for New Treatments of Acute Stroke consortium, the Brain Foundation Netherlands, the Ministry of Economic Affairs, Stryker, Medtronic, Cerenovus, and the Dutch Heart Foundation.

Background: We aimed to assess whether the effect of intravenous alteplase treatment (IVT) before endovascular treatment (EVT) on outcome is modified by first-line technique during EVT in IVT eligible patients. Methods: This was a post hoc analysis from MR CLEAN-NO IV (Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands - Intravenous Treatment Followed by Intra-Arterial Treatment Versus Direct Intra-Arterial Treatment for Acute Ischemic Stroke Caused by a Proximal Intracranial Occlusion), a randomized trial of IVT followed by EVT versus EVT alone in patients presenting directly to EVT-capable centers. We included data from all patients who underwent EVT with a thrombectomy attempt. We compared patients treated with stent retriever (with or without aspiration) to aspiration alone as first-line EVT technique and assessed the interaction of first-line EVT technique with IVT treatment. Primary outcome was the 90-day modified Rankin Scale score, analyzed with mixed model ordinal regression for a shift towards better outcome. Secondary outcomes included successful reperfusion (extended Thrombolysis in Cerebral Infarction score 2b–3). Results: Of 473 included patients, 102 (21.6%) were treated with aspiration alone as first-line technique. In the full population, functional outcome was similar for patients treated with stent retriever versus aspiration only (adjusted common odds ratio [acOR]‚ 1.07 [95% CI, 0.69–1.66]). We observed a significant interaction between IVT and first-line EVT technique ( P =0.03). In the aspiration-only group, patients treated with EVT alone had worse functional outcome compared to those treated with IVT and EVT (acOR, 0.44 [95% CI, 0.21–0.90]). In the stent retriever group, functional outcome did not differ between patients treated with or without IVT (acOR, 1.08 [95% CI, 0.74–1.57]). There was no statistically significant interaction for successful reperfusion. Conclusions: In MR CLEAN-NO IV, the treatment effect of IVT was modified by first-line EVT technique. Patients treated with aspiration only as first-line technique had worse clinical outcomes if they did not receive IVT. No such difference was observed in patients treated with stent retrievers. Confirmation by pooling with results from other trials is needed to confirm these findings.

Background Insight into outcome variation between hospitals could help to improve quality of care. We aimed to assess the validity of early outcomes as quality indicators for acute ischemic stroke care for patients treated with endovascular therapy (EVT). Methods and Results We used data from the MR CLEAN (Multicenter Randomized Controlled Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands) Registry, a large multicenter prospective cohort study including 3279 patients with acute ischemic stroke undergoing EVT. Random effect linear and proportional odds regression were used to analyze the effect of case mix on between‐hospital differences in 2 early outcomes: the National Institutes of Health Stroke Scale (NIHSS) score at 24 to 48 hours and the expanded thrombolysis in cerebral infarction score. Between‐hospital variation in outcomes was assessed using the variance of random hospital effects (tau 2 ). In addition, we estimated the correlation between hospitals' EVT‐patient volume and (case‐mix–adjusted) outcomes. Both early outcomes and case‐mix characteristics varied significantly across hospitals. Between‐hospital variation in the expanded thrombolysis in cerebral infarction score was not influenced by case‐mix adjustment (tau 2 =0.17 in both models). In contrast, for the NIHSS score at 24 to 48 hours, case‐mix adjustment led to a decrease in variation between hospitals (tau 2 decreases from 0.19 to 0.17). Hospitals' EVT‐patient volume was strongly correlated with higher expanded thrombolysis in cerebral infarction scores ( r =0.48) and weakly with lower NIHSS score at 24 to 48 hours ( r =0.15). Conclusions Between‐hospital variation in NIHSS score at 24 to 48 hours is significantly influenced by case‐mix but not by patient volume. In contrast, between‐hospital variation in expanded thrombolysis in cerebral infarction score is strongly influenced by EVT‐patient volume but not by case‐mix. Both outcomes may be suitable for comparing hospitals on quality of care, provided that adequate adjustment for case‐mix is applied for NIHSS score.

Background: A larger thrombus in patients with acute ischemic stroke might result in more complex endovascular treatment procedures, resulting in poorer patient outcomes. Current evidence on thrombus volume and length related to procedural and functional outcomes remains contradicting. This study aimed to assess the prognostic value of thrombus volume and thrombus length and whether this relationship differs between first-line stent retrievers and aspiration devices for endovascular treatment. Methods: In this multicenter retrospective cohort study, 670 of 3279 patients from the MR CLEAN Registry (Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands) for endovascularly treated large vessel occlusions were included. Thrombus volume (0.1 mL) and length (0.1 mm) based on manual segmentations and measurements were related to reperfusion grade (expanded Treatment in Cerebral Infarction score) after endovascular treatment, the number of retrieval attempts, symptomatic intracranial hemorrhage, and a shift for functional outcome at 90 days measured with the reverted ordinal modified Rankin Scale (odds ratio >1 implies a favorable outcome). Univariable and multivariable linear and logistic regression were used to report common odds ratios (cORs)/adjusted cOR and regression coefficients (B/aB) with 95% CIs. Furthermore, a multiplicative interaction term was used to analyze the relationship between first-line device choice, stent retrievers versus aspiration device, thrombus volume, and outcomes. Results: Thrombus volume was associated with functional outcome (adjusted cOR, 0.83 [95% CI, 0.71–0.97]) and number of retrieval attempts (aB, 0.16 [95% CI, 0.16–0.28]) but not with the other outcome measures. Thrombus length was only associated with functional independence (adjusted cOR, 0.45 [95% CI, 0.24–0.85]). Patients with more voluminous thrombi had worse functional outcomes if endovascular treatment was based on first-line stent retrievers (interaction cOR, 0.67 [95% CI, 0.50–0.89]; P =0.005; adjusted cOR, 0.74 [95% CI, 0.55–1.0]; P =0.04). Conclusions: In this study, patients with a more voluminous thrombus required more endovascular thrombus retrieval attempts and had a worse functional outcome. Patients with a lengthier thrombus were less likely to achieve functional independence at 90 days. For more voluminous thrombi, first-line stent retrieval compared with first-line aspiration might be associated with worse functional outcome.

Background and Purpose: The benefit of endovascular treatment (EVT) for posterior circulation stroke (PCS) remains uncertain, and little is known on treatment outcomes in clinical practice. This study evaluates outcomes of a large PCS cohort treated with EVT in clinical practice. Simultaneous to this observational study, several intervention centers participated in the BASICS trial (Basilar Artery International Cooperation Study), which tested the efficacy of EVT for basilar artery occlusion in a randomized setting. We additionally compared characteristics and outcomes of patients treated outside BASICS in trial centers to those from nontrial centers. Methods: We included patients with PCS from the Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands Registry: a prospective, multicenter, observational study of patients who underwent EVT in the Netherlands between 2014 and 2018. Primary outcome was a score of 0 to 3 on the modified Rankin Scale at 90 days. Secondary outcomes included reperfusion status and symptomatic intracranial hemorrhage. For outcome comparison between patients treated in trial versus nontrial centers, we used ordinal logistic regression analysis. Results: We included 264 patients of whom 135 (51%) had received intravenous thrombolysis. The basilar artery was most often involved (77%). Favorable outcome (modified Rankin Scale score 0–3) was observed in 115/252 (46%) patients, and 109/252 (43%) patients died. Successful reperfusion was achieved in 178/238 (75%), and symptomatic intracranial hemorrhage occurred in 9/264 (3%). The 154 nontrial patients receiving EVT in BASICS trial centers had similar characteristics and outcomes as the 110 patients treated in nontrial centers (modified Rankin Scale adjusted cOR: 0.77 [95% CI, 0.5–1.2]). Conclusions: Our study shows that high rates of favorable clinical outcome and successful reperfusion can be achieved with EVT for PCS, despite high mortality. Characteristics and outcomes of patients treated in trial versus nontrial centers were similar indicating that our cohort is representative of clinical practice in the Netherlands. Randomized studies using modern treatment approaches are needed for further insight in the benefit of EVT for PCS.

Supplemental Digital Content is available in the text., Background and Purpose: Benefit of early endovascular treatment (EVT) for ischemic stroke varies considerably among patients. The MR PREDICTS decision tool, derived from MR CLEAN (Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands), predicts outcome and treatment benefit based on baseline characteristics. Our aim was to externally validate and update MR PREDICTS with data from international trials and daily clinical practice. Methods: We used individual patient data from 6 randomized controlled trials within the HERMES (Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials) collaboration to validate the original model. Then, we updated the model and performed a second validation with data from the observational MR CLEAN Registry. Primary outcome was functional independence (defined as modified Rankin Scale score 0–2) 3 months after stroke. Treatment benefit was defined as the difference between the probability of functional independence with and without EVT. Discriminative performance was evaluated using a concordance (C) statistic. Results: We included 1242 patients from HERMES (633 assigned to EVT, 609 assigned to control) and 3156 patients from the MR CLEAN Registry (all of whom underwent EVT within 6.5 hours). The C-statistic for functional independence was 0.74 (95% CI, 0.72–0.77) in HERMES and, after model updating, 0.80 (0.78–0.82) in the Registry. Median predicted treatment benefit of routinely treated patients (Registry) was 10.3% (interquartile range, 5.8%–14.4%). Patients with low (

Background: Symptomatic intracranial hemorrhage (sICH) is a serious complication after endovascular treatment for ischemic stroke. We aimed to identify determinants of its occurrence and location. Methods: We retrospectively analyzed data from the Dutch MR CLEAN trial (Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands) and MR CLEAN registry. We included adult patients with a large vessel occlusion in the anterior circulation who underwent endovascular treatment within 6.5 hours of stroke onset. We used univariable and multivariable logistic regression analyses to identify determinants of overall sICH occurrence, sICH within infarcted brain tissue, and sICH outside infarcted brain tissue. Results: SICH occurred in 203 (6%) of 3313 included patients and was located within infarcted brain tissue in 50 (25%), outside infarcted brain tissue in 23 (11%), and both within and outside infarcted brain tissue in 116 (57%) patients. In 14 patients (7%), data on location were missing. Prior antiplatelet use, baseline systolic blood pressure, baseline plasma glucose levels, post-endovascular treatment modified treatment in cerebral ischemia score, and duration of procedure were associated with all outcome parameters. In addition, determinants of sICH within infarcted brain tissue included history of myocardial infarction (adjusted odds ratio, 1.65 [95% CI, 1.06–2.56]) and poor collateral score (adjusted odds ratio, 1.42 [95% CI, 1.02–1.95]), whereas determinants of sICH outside infarcted brain tissue included level of occlusion on computed tomography angiography (internal carotid artery or internal carotid artery terminus compared with M1: adjusted odds ratio, 1.79 [95% CI, 1.16–2.78]). Conclusions: Several factors, some potentially modifiable, are associated with sICH occurrence. Further studies should investigate whether modification of baseline systolic blood pressure or plasma glucose level could reduce the risk of sICH. In addition, determinants differ per location of sICH, supporting the hypothesis of varying underlying mechanisms. Registration: URL: https://www.isrctn.com/ ; Unique identifier: ISRCTN10888758.

Background: In patients with large vessel occlusion stroke of the anterior circulation, underlying cause is a determinant of outcome. Whether this is the case for posterior circulation large vessel occlusion stroke has yet to be determined. We aimed to report on cause in patients with posterior circulation stroke treated with endovascular thrombectomy and to analyze the association with functional outcome. Methods: We used data of patients with posterior circulation stroke included in the MR CLEAN (Multicenter Randomized Controlled Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands) registry, a prospective multicenter observational study, between 2014 and 2018. Stroke cause was categorized into large artery atherosclerosis (LAA), cardioembolism, arterial dissection, embolic stroke of undetermined source (ESUS), other determined cause, or undetermined cause. For primary analysis on the association between cause and outcome, we used multivariable ordinal logistic regression analysis to estimate the adjusted common odds ratio for a shift towards a better functional outcome on the modified Rankin Scale at 90 days with LAA as a reference group. Secondary outcomes included favorable functional outcome (modified Rankin Scale score 0–3), National Institutes of Health Stroke Scale score at 24 to 48 hours, reperfusion on digital subtraction angiography, and stroke progression. Results: Of 264 patients with posterior circulation stroke, 84 (32%) had LAA, 48 (18%) cardioembolism, 31 (12%) dissection, and 14 (5%) ESUS. Patients with a dissection were younger (48 [interquartile range, 43–60] years) and had a lower National Institutes of Health Stroke Scale at baseline (12 [interquartile range, 6–31]) than patients with other cause. Functional outcome was better for patients with cardioembolism and ESUS compared to LAA (modified Rankin Scale adjusted common odds ratio, 2.4 [95% CI, 1.1–5.2], respectively adjusted common odds ratio, 3.1 [95% CI, 1.0–9.3]). Patients with a dissection had a lower chance of successful reperfusion compared with LAA (adjusted odds ratio, 0.20 [95% CI, 0.06–0.70]). Conclusions: Unlike the anterior circulation, most frequent cause in our posterior large vessel occlusion stroke cohort is LAA followed by cardioembolism, dissection, and ESUS. Patients with cardioembolism and ESUS have a better prognosis for functional outcome after endovascular thrombectomy than patients with LAA.

Background: We evaluated data from all patients in the Netherlands who underwent endovascular treatment for acute ischemic stroke in the past 3.5 years, to identify nationwide trends in time to treatment and procedural success, and assess their effect on clinical outcomes. Methods: We included patients with proximal occlusions of the anterior circulation from the second and first cohorts of the MR CLEAN (Multicenter Randomized Clinical trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands) Registry (March 2014 to June 2016; June 2016 to November 2017, respectively). We compared workflow times and rates of successful reperfusion (defined as an extended Thrombolysis in Cerebral Infarction score of 2B-3) between cohorts and chronological quartiles (all included patients stratified in chronological quartiles of intervention dates to create equally sized groups over the study period). Multivariable ordinal logistic regression was used to assess differences in the primary outcome (ordinal modified Rankin Scale at 90 days). Results: Baseline characteristics were similar between cohorts (second cohort n=1692, first cohort n=1488) except for higher age, poorer collaterals, and less signs of early ischemia on computed tomography in the second cohort. Time from stroke onset to groin puncture and reperfusion were shorter in the second cohort (median 185 versus 210 minutes; P P P Conclusions: Clinical outcomes after endovascular treatment for acute ischemic stroke in routine clinical practice have improved over the past years, likely resulting from improved workflow times and higher successful reperfusion rates.

Background Time to reperfusion in patients with ischemic stroke is strongly associated with functional outcome and may differ between hospitals and between patients within hospitals. Improvement in time to reperfusion can be guided by between‐hospital and within‐hospital comparisons and requires insight in specific targets for improvement. We aimed to quantify the variation in door‐to‐reperfusion time between and within Dutch intervention hospitals and to assess the contribution of different time intervals to this variation. Methods and Results We used data from the MR CLEAN (Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands) Registry. The door‐to‐reperfusion time was subdivided into time intervals, separately for direct patients (door‐to‐computed tomography, computed tomography‐to‐computed tomography angiography [CTA], CTA‐to‐groin, and groin‐to‐reperfusion times) and for transferred patients (door‐to‐groin and groin‐to‐reperfusion times). We used linear mixed models to distinguish the variation in door‐to‐reperfusion time between hospitals and between patients. The proportional change in variance was used to estimate the amount of variance explained by each time interval. We included 2855 patients of 17 hospitals providing endovascular treatment. Of these patients, 44% arrived directly at an endovascular treatment hospital. The between‐hospital variation in door‐to‐reperfusion time was 9%, and the within‐hospital variation was 91%. The contribution of case‐mix variables on the variation in door‐to‐reperfusion time was marginal (2%–7%). Of the between‐hospital variation, CTA‐to‐groin time explained 83%, whereas groin‐to‐reperfusion time explained 15%. Within‐hospital variation was mostly explained by CTA‐to‐groin time (33%) and groin‐to‐reperfusion time (42%). Similar results were found for transferred patients. Conclusions Door‐to‐reperfusion time varies between, but even more within, hospitals providing endovascular treatment for ischemic stroke. Quality of stroke care improvements should not only be guided by between‐hospital comparisons, but also aim to reduce variation between patients within a hospital, and should specifically focus on CTA‐to‐groin time and groin‐to‐reperfusion time.

Supplemental Digital Content is available in the text., Background: Cerebral white matter lesions (WMLs) have been associated with a greater risk of poor functional outcome after ischemic stroke. We assessed the relations between WML burden and radiological and clinical outcomes in patients treated with endovascular treatment in routine practice. Methods: We analyzed data from the MR CLEAN Registry (Multicenter Randomized Controlled Trial of Endovascular Treatment for Acute Ischaemic Stroke in the Netherlands)—a prospective, multicenter, observational cohort study of patients treated with endovascular treatment in the Netherlands. WMLs were graded on baseline noncontrast computed tomography using a visual grading scale. The primary outcome was the score on the modified Rankin Scale at 90 days. Secondary outcomes included early neurological recovery, successful reperfusion (extended Thrombolysis in Cerebral Infarction ≥2b), futile recanalization (modified Rankin Scale score ≥3 despite successful reperfusion), and occurrence of symptomatic intracranial hemorrhage. We used multivariable logistic regression models to assess associations between WML severity and outcomes, taking the absence of WML on noncontrast computed tomography as the reference category. Results: Of 3180 patients included in the MR CLEAN Registry between March 2014 and November 2017, WMLs were graded for 3046 patients and categorized as none (n=1855; 61%), mild (n=608; 20%), or moderate to severe (n=588; 19%). Favorable outcome (modified Rankin Scale score, 0–2) was achieved in 838 patients (49%) without WML, 192 patients (34%) with mild WML, and 130 patients (24%) with moderate-to-severe WML. Increasing WML grades were associated with a shift toward poorer functional outcome in a dose-dependent manner (adjusted common odds ratio, 1.34 [95% CI, 1.13–1.60] for mild WML and 1.67 [95% CI, 1.39–2.01] for moderate-to-severe WML; Ptrend

Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations (βT84Y, αL91Y and βF85Y) also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (< 100 μM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (βT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of βT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, βT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. βT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product., Graphical abstract fx1, Highlights • Cell free Hemoglobin Based Oxygen Carriers can deliver oxygen to tissue. • Oxidation of ferrous hemoglobin to ferric and ferryl leads to oxidative toxicity. • Insertion of tyrosine residues enhances reduction of ferryl to ferric hemoglobin. • Some mutants (e.g. βT84Y) also enhance ferric to ferrous reduction by ascorbate. • βT84Y decreases cellular oxidative stress and enhances vascular retention in vivo.

BackgroundRandomised controlled trials with perfusion selection have shown benefit of endovascular treatment (EVT) for ischaemic stroke between 6 and 24 hours after symptom onset or time last seen well. However, outcomes after EVT in these late window patients without perfusion imaging are largely unknown. We assessed their characteristics and outcomes in routine clinical practice.MethodsThe Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands Registry, a prospective, multicentre study in the Netherlands, included patients with an anterior circulation occlusion who underwent EVT between 2014 and 2017. CT perfusion was no standard imaging modality. We used adjusted ordinal logistic regression analysis to compare patients treated within versus beyond 6.5 hours after propensity score matching on age, prestroke modified Rankin Scale (mRS), National Institutes of Health Stroke Scale, Alberta Stroke Programme Early CT Score (ASPECTS), collateral status, location of occlusion and treatment with intravenous thrombolysis. Outcomes included 3-month mRS score, functional independence (defined as mRS 0–2), and death.ResultsOf 3264 patients who underwent EVT, 106 (3.2%) were treated beyond 6.5 hours (median 8.5, IQR 6.9–10.6), of whom 93 (87.7%) had unknown time of stroke onset. CT perfusion was not performed in 87/106 (80.2%) late window patients. Late window patients were younger (mean 67 vs 70 years, pConclusionsWithout the use of CT perfusion selection criteria, EVT in the 6.5–24-hour time window was not associated with poorer outcome in selected patients with favourable clinical and CT/CT angiography characteristics. randomised controlled trials with lenient inclusion criteria are needed to identify more patients who can benefit from EVT in the late window.

Background and Purpose: Thrombectomy with stent retriever and direct aspiration are equally effective in the endovascular treatment of anterior circulation acute ischemic stroke. We report efficacy and safety of initial treatment technique per occlusion segment. Methods: For this study, we analyzed data from the MR CLEAN Registry, a prospective, observational study in all centers that perform endovascular therapy in the Netherlands. We used ordinal logistic regression analysis to compare clinical and technical results of first line direct aspiration treatment with that of stent retriever thrombectomy stratified for occlusion segment. Primary outcome measure was functional outcome at 3 months. Secondary outcome measures included reperfusion grade expressed as the extended Thrombolysis in Cerebral Infarction score, periprocedural complication risk, time to reperfusion, and mortality. Results: Of the 2282 included patients, 1658 (73%) were initially treated with stent retriever and 624 (27%) with aspiration. Four hundred sixty-two patients had an occlusion of the intracranial part of the carotid artery, 1349 of the proximal middle cerebral artery, and 471 of the distal parts of the middle cerebral artery. There was no difference in functional outcome between aspiration and stent retriever thrombectomy (odds ratio, 1.0 [95% CI, 0.9–1.2]) in any of the occlusion segments ( P value for interaction=0.2). Reperfusion rate was higher in the aspiration group (odds ratio, 1.4 [95% CI, 1.1–1.6]) and did not differ between occlusion segments ( P value for interaction=0.6). Procedure times were shorter in the aspiration group (minutes 50 versus 65 minutes; P Conclusions: In unselected patients with anterior circulation infarcts, we observed equal functional outcome of aspiration and stent retriever thrombectomy in all occlusion segments. When aspiration was the first line treatment modality, reperfusion rates were higher and procedure times shorter in all occlusion segments.

Contains fulltext : 220823.pdf (Publisher’s version ) (Closed access) Before 2015, endovascular treatment (EVT) for acute ischemic stroke was considered a promising treatment option. Based on limited evidence, it was performed in several dedicated stroke centers worldwide on selected patients. Since 2015, EVT for patients with intracranial large vessel occlusion has quickly been implemented as standard treatment in many countries worldwide, supported by the revised international guidelines based on solid evidence from multiple clinical trials. We describe the development in use of EVT in the Netherlands before, during, and after the pivotal EVT trials. We used data from all patients who were treated with EVT in the Netherlands from January 2002 until December 2018. We undertook a time-series analysis to examine trends in the use of EVT using Poisson regression analysis. Incidence rate ratios per year with 95% CIs were obtained to demonstrate the impact and implementation after the publication of the EVT trial results. We made regional observation plots, adjusted for stroke incidence, to assess the availability and use of the treatment in the country. In the buildup to the MR CLEAN (Multicenter Clinical Trial of Endovascular Treatment of Acute Ischemic Stroke in the Netherlands), a slow increase of EVT patients was observed, with 0.2% of all ischemic stroke patients receiving EVT. Before the trial results were formally announced, a statistically significant increase in EVT-treated patients per year was observed (incidence rate ratio, 1.72 [95% CI, 1.46-2.04]), and after the trial publication, an immediate steep increase was seen, followed by a more gradual increase (incidence rate ratio, 2.14 [95% CI, 1.77-2.59]). In 2018, the percentage of ischemic stroke patients receiving EVT increased to 5.8%. A well-developed infrastructure, a pragmatic approach toward the use of EVT in clinical practice, in combination with a strict adherence by the regulatory authorities to national evidence-based guidelines has led to successful implementation of EVT in the Netherlands. Ongoing efforts are directed at further increasing the proportion of stroke patients with EVT in all regions of the country.

Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the β-subunit (F41). We therefore replaced this residue with a tyrosine (βF41Y, Hb Mequon). The βF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, βF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the β-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in βF41Y. NO bioavailability was enhanced in βF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the β-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.

Heme mediated oxidative toxicity has been linked to adverse side effects in Hemoglobin Based Oxygen Carriers (HBOC), initiated by reactive ferryl (FeIV) iron and globin based free radical species. We recently showed that the addition of a redox active tyrosine residue in the beta subunit (βF41Y) of recombinant hemoglobin had the capability to decrease lipid peroxidation by facilitating the reduction of FeIV iron by plasma antioxidants such as ascorbate. In order to explore this functionality further we created a suite of tyrosine mutants designed to be accessible for both reductant access at the protein surface, yet close enough to the heme cofactor to enable efficient electron transfer to the FeIV. The residues chosen were: βF41Y; βK66Y; βF71Y; βT84Y; βF85Y; and βL96Y. As with βF41Y, all mutants significantly enhanced the rate of ferryl (FeIV) to ferric (FeIII) reduction by ascorbate. However, surprisingly a subset of these mutations (βT84Y, and βF85Y) also enhanced the further reduction of ferric (FeIII) to ferrous (FeII) heme, regenerating functional oxyhemoglobin. The largest increase was seen in βT84Y with the percentage of oxyhemoglobin formed from ferric hemoglobin in the presence of 100 μM ascorbate over a time period of 60 min increasing from 10% in βF41Y to over 50% in βT84Y. This increase was accompanied by an increased rate of ascorbate consumption. We conclude that the insertion of novel redox active tyrosine residues may be a useful component of any recombinant HBOC designed for longer functional activity without oxidative side effects.

Nucleotide excision repair (NER) is the primary mechanism for removal of ultraviolet light (UV)-induced DNA photoproducts and is mechanistically conserved across all kingdoms of life. Bacterial NER involves damage recognition by UvrA2 and UvrB, followed by UvrC-mediated incision either side of the lesion. Here, using a combination of in vitro and in vivo single-molecule studies we show that a UvrBC complex is capable of lesion identification in the absence of UvrA. Single-molecule analysis of eGFP-labelled UvrB and UvrC in living cells showed that UV damage caused these proteins to switch from cytoplasmic diffusion to stable complexes on DNA. Surprisingly, ectopic expression of UvrC in a uvrA deleted strain increased UV survival. These data provide evidence for a previously unrealized mechanism of survival that can occur through direct lesion recognition by a UvrBC complex.

In this study we describe a new methodology to physically probe individual complexes formed between proteins and DNA. By combining nanoscale, high speed physical force measurement with sensitive fluorescence imaging we investigate the complex formed between the prokaryotic DNA repair protein UvrA2 and DNA. This approach uses a triangular, optically-trapped “nanoprobe” with a nanometer scale tip protruding from one vertex. By scanning this tip along a single DNA strand suspended between surface-bound micron-scale beads, quantum-dot tagged UvrA2 molecules bound to these ‘”DNA tightropes” can be mechanically interrogated. Encounters with UvrA2 led to deflections of the whole nanoprobe structure, which were converted to resistive force. A force histogram from all 144 detected interactions generated a bimodal distribution centered on 2.6 and 8.1 pN, possibly reflecting the asymmetry of UvrA2’s binding to DNA. These observations successfully demonstrate the use of a highly controllable purpose-designed and built synthetic nanoprobe combined with fluorescence imaging to study protein-DNA interactions at the single molecule level.

The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5'-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can 'turnover' in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase-nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed.

We investigated how Escherichia coli ClpXP targets the helicase-nuclease (HsdR) subunit of the bacterial Type I restriction–modification enzyme EcoKI during restriction alleviation (RA). RA is a temporary reduction in endonuclease activity that occurs when Type I enzymes bind unmodified recognition sites on the host genome. These conditions arise upon acquisition of a new system by a naïve host, upon generation of new sites by genome rearrangement/mutation or during homologous recombination between hemimethylated DNA. Using recombinant DNA and proteins in vitro, we demonstrate that ClpXP targets EcoKI HsdR during dsDNA translocation on circular DNA but not on linear DNA. Protein roadblocks did not activate HsdR proteolysis. We suggest that DNA translocation lifetime, which is elevated on circular DNA relative to linear DNA, is important to RA. To identify the ClpX degradation tag (degron) in HsdR, we used bioinformatics and biochemical assays to design N- and C-terminal mutations that were analysed in vitro and in vivo. None of the mutants produced a phenotype consistent with loss of the degron, suggesting an as-yet-unidentified recognition pathway. We note that an EcoKI nuclease mutant still produces cell death in a clpx− strain, consistent with DNA damage induced by unregulated motor activity.

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair.

ABSTRACT PBK/TOPK is a recently cloned serine/threonine kinase which is phosphorylated during mitosis. Earlier work indicated that this kinase is upregulated in a Burkitt's lymphoma cell line (GA-10). To determine whether PBK/TOPK is upregulated in other mitotically active neoplastic cell lines and tissues, Northern analysis was performed on a panel of malignant cell lines and on clinical samples from patients with leukemia or lymphoma. While PBK/TOPK mRNA was not detectable in normal peripheral blood cells and was weakly expressed in hyperplastic tonsillar B-cells, significantly higher levels of mRNA were detected in 8 Burkitt's lymphoma cell lines, 10 other neoplastic cell lines, and 2 clinical samples—one derived from a patient with ALL and a second derived from a patient with relapsed myeloma. In addition, Northern analysis of fetal tissues showed upregulated expression of PBK/TOPK in fetal kidney, lung, spleen, brain, and testis. These data suggest that PBK/TOPK expression is increased in highly proliferative malignant cells and during normal fetal development.

We have cloned and determined the structure of the 5' region of the mouse Nktr gene located on the distal end of mouse chromosome 9. This gene encodes an NK-cell-specific 150-kDa protein (NK-TR) homologous to cyclophilin, Nopp140, and SR-containing proteins. NK-TR expression is important for maintaining the lytic activity of natural killer cells. The region of the Nktr gene cloned in this study spans 25 kb and contains the promoter, eight exons, and a single alternative exon. The boundaries of exons 6-8 and the alternate splicing events in this region are identical to those previously described for the human NKTR gene. The Nktr promoter region has features that are typical of a housekeeping gene, including high G + C content, high frequency of CpG dinucleotides, and a lack of canonical TATAA and CCAAT boxes. The activity of Nktr promoter/beta-gal reporter constructs was equivalent in lymphocyte and fibroblast cell lines, suggesting that NK-TR protein expression is regulated by posttranscriptional mechanisms. In support of this hypothesis, two levels of splicing control have been identified within the Nktr gene. A 10-kb intron was found to remain in mRNAs produced in bone marrow, and an alternative exon capable of interrupting the Nktr open reading frame was found in immature NK cells. A conserved intronic sequence has been identified that may be important for the regulation of the Nktr gene by pre-mRNA splicing.

Direct observation of a multi-protein DNA repair pathway is now possible using single molecule methods. Previously we have shown that UvrA and UvrB belonging to the bacterial nucleotide excision repair pathway collaborate to locate damage. Here we report on the next phase of repair, dual incision by UvrC. However as UvrC is in such short supply in the cell, locating a pre-incision complex at a site of damage becomes the key rate limiting step of NER. By labelling UvrB and UvrC with different coloured quantum dots their interactions with DNA tightropes can be studied at the single molecule level using oblique angle fluorescence microscopy. We found UvrC interacts with DNA and performs a 1D diffusional search (7.4x10−2 μm2s−1). Surprisingly, we also found that UvrC could load UvrB onto DNA generating a previously unseen UvrBC-DNA complex. This UvrBC complex is highly mobile relative to UvrC alone (15% vs. 57% mobility) and engages in unbiased 1D diffusion with a coefficient of 6.6x10−3 μm2s−1. Ionic strength profoundly affects the motion of the proteins; at elevated salt more UvrC molecules slide, however UvrBC mobility remains constant. Furthermore, the inclusion of ATP decreases UvrBC's dwell time on DNA from 175s to 83s. Based on these and further results from the study of various mutants we deduce that UvrB switches UvrC's search mechanism from 3D distributive to 1D sliding. We propose a new chaperoning role for UvrB in NER, where it protects the genome from unwanted UvrC nuclease activity but facilitates UvrC's location of pre-incision UvrB complexes.