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Background: Endovascular treatment has become standard of care for the treatment of acute ischemic stroke with large vessel occlusion. However, patients treated in clinical practice differ from the selected populations randomized in clinical trials., Aims: The German Stroke Registry Endovascular Treatment (GSR-ET) aims at a systematic evaluation of outcome, safety, and process parameters of endovascular stroke treatment in standard of care in Germany., Methods: The GSR-ET is an academic, independent, prospective, multicenter, observational registry study. Participating stroke centers from all over of Germany consecutively enroll patients transferred to the angiography suite with an intention to be treated with endovascular stroke treatment. Patients receive regular care. Data are collected as part of clinical routine. Baseline clinical and procedural information and clinical follow-up information after 90 days are recorded. Here, we present an analysis of baseline data of the first 1662 patients included in the GSR-ET., Results: The registry was established in June 2015. By 31 December 2017, 1662 patients were enrolled in 23 active sites. Mean age was 72 ± 13 years, 50% were female, and median National Institutes of Health Stroke Scale on admission was 15 (IQR 10-19), 88% had anterior circulation occlusion. Median ASPECT score was 8 (IQR 7-10) prior to intervention. Fifty-nine percent of patients received intravenous thrombolysis prior to thrombectomy. Mean "onset-to-groin" time was 224 ± 176 min., Conclusions: Baseline characteristics of stroke patients undergoing thrombectomy in clinical practice differ from those in the randomized trials. The GSR-ET will provide valuable insights into practices of endovascular treatment in routine care of acute ischemic stroke. (GSR-ET ClinicalTrials.gov Identifier: NCT03356392.).

Background and Purpose- Using a novel study design with virtual comparators based on predictive modeling, we investigated whether next-generation mechanical thrombectomy devices improve outcomes in patients with ischemic stroke. We hypothesized that this new study design shows that a next-generation mechanical thrombectomy system is superior to intravenous tPA (tissue-type plasminogen activator) therapy (IVT) alone. Methods- ERASER (Eric Acute Stroke Recanalization) was an investigator-initiated, prospective, multicenter, single-arm (virtual 2-arm) study that evaluated the effectiveness of a new recanalization device together with a specific intermediate catheter (Embolus Retriever with Interlinked Cages/SOFIA, Microvention) in stroke patients with internal carotid artery or middle cerebral artery occlusions. The primary end point was the volume of saved tissue. Volume of saved tissue was defined as the difference of actual infarct volume and brain volume predicted to develop infarction using a machine learning model based on data from intravenous tPA therapy patients. Results- Eighty-one patients were enrolled. The median patient age was 71 years (interquartile range, 61-77). National Institutes of Health Stroke Scale score was 14 (interquartile range, 12-18). The actual infarct volume was smaller than predicted by the intravenous tPA therapy model, with a median volume of saved tissue of 50 mL (interquartile range, 19-103; P<0.0001). Good clinical outcome (modified Rankin Scale, 0-2 at 90 days) was observed in 48 out of 69 (70%). The recanalization rate (Thrombolysis in Cerebral Infarction 2b/3) was 95%. Conclusions- ERASER is the first mechanical thrombectomy study with a primary end point based on predictive analytics enabling intraindividual virtual comparisons. The next-generation mechanical thrombectomy method resulted in smaller infarcts than predicted after intravenous tPA therapy alone and showed a high rate of good clinical outcome. The novel study design with virtual comparisons is promising for further application and testing in the neurovascular arena. Clinical Trial Registration- URL: https://www.clinicaltrials.gov . Unique identifier: NCT02534701.

Cerebral amyloid angiopathy (CAA) associated with inflammation is a rare form of a potentially reversible encephalopathy in a subgroup of patients with CAA. The cerebral amyloid deposition can in isolated cases induce an inflammation predominantly of the cerebral blood vessels and a multifocal edema of the cerebral white matter. The courses can occur as monophasic, relapsing remitting and primarily progressive forms. We present seven cases with different courses of the disease and give an overview of the pathophysiology, clinical aspects and treatment of the disease with reference to the current literature. The cases presented show a very different and often difficult differential diagnostic clinical picture and all showed a significant improvement under steroid medication without signs of recurrence of the disease during the course. The recognition and early consistent treatment of inflammatory forms of CAA with and without direct inflammatory involvement of vessels can be decisive for successful treatment.

Stroke in young adults is a diagnostic and therapeutic challenge for all persons involved. Approximately 15% of ischemic strokes occur in young adults. Lack of awareness of the symptoms in emergency departments often results in delayed diagnosis and access to specific therapeutic options, such as revascularization. The causes are often heterogeneous and necessitate specific investigations. The etiology of juvenile stroke includes drug abuse, vasculitis and arteriopathies, such as reversible vasoconstriction syndrome and posterior reversible encephalopathy syndrome, although the prevalence of classical vascular risk factors is high. The most frequent causes of ischemic stroke in young adults are cardioembolism and microangiopathy; furthermore, dissection of vessels of the neck are more frequent compared to older patients. According to the results of currently available studies reperfusion strategies, such as intravenous fibrinolysis and mechanical thrombectomy are efficacious and safe in young patients.

Objective: Many patients with stroke cannot receive intravenous thrombolysis because the time of symptom onset is unknown. We tested whether computed tomography (CT)-based quantification of water uptake in the ischemic tissue can identify patients with stroke onset within 4.5 hours, the time window of thrombolysis., Methods: Perfusion CT was used to identify ischemic brain tissue, and its density was measured in native CT and related to the density of the corresponding area of the contralateral hemisphere to quantify lesion water uptake. The optimal cutoff value of water uptake distinguishing stroke onset within and beyond 4.5 hours was calculated in patients with proximal middle cerebral artery occlusion (derivation cohort) with known time of symptom onset. The so-derived cutoff value was validated in a prospective cohort from other stroke centers., Results: Of 178 patients of the derivation cohort, 147 (82.6%) had CT within 4.5 hours. Percentage water uptake was significantly lower in patients with stroke onset within compared to beyond 4.5 hours. The area under the receiver operating characteristic curve for distinguishing these patient groups according to percentage water uptake was 0.999 (95% confidence interval = 0.996-1.000, p < 0.001) with an optimal cutoff value of 11.5%. Applying this cutoff to the validation cohort of 240 patients, sensitivity was 98.6%, specificity 90.5%, positive predictive value 99.1%, and negative predictive value 86.4%., Interpretation: Quantification of brain water uptake identifies stroke patients with symptom onset within 4.5 hours with high accuracy and may guide the decision to use thrombolysis in patients with unknown time of stroke onset. Ann Neurol 2016;80:924-934., (© 2016 American Neurological Association.)

Purpose: The treatment mode in acute vertebrobasilar occlusion (VBO) remains uncertain. We analyzed efficacy and safety of intravenous glycoprotein IIb/IIIa inhibitor (IV GPI) plus subsequent intra-arterial thrombolysis with or without additional endovascular mechanical therapy (percutaneous transluminal angioplasty/stenting or thrombus aspiration) and sought treatment factors that predict good clinical outcome., Methods: We retrospectively analyzed 120 cases of patients with angiographically proven acute VBO. Multivariate logistic regression was used to identify independent predictors for clinical outcome and included level of consciousness, age, sex, time to angiography, GPI agent, admission mode, occlusion type, recanalization success, and endovascular treatment mode. Clinical follow-up was dichotomized in no to moderate disability (modified Rankin scale (mRS) 0-3) vs. severe disability or death (mRS 4-6)., Results: Median National Institutes of Health stroke scale (NIHSS) score on admission was 32, and mean NIHSS score was 24. A total of 49 patients (41 %) developed no to moderate disability (mRS 0-3), and 39 patients (33 %) died. Thrombolysis in myocardial infarction 2/3 recanalization success was achieved in 97 patients (80.8 %). Symptomatic intracerebral hemorrhages occurred in 11 patients (9 %). Mild impairment of consciousness (p < 0.001) and embolic occlusion type (p = 0.01) were significant predictors of favorable outcome. Clinical outcome in recanalized patients was better, but not statistically significant (p = 0.055)., Conclusions: Our results indicate that combined therapy with IV GPI and subsequent endovascular therapy may be a valid treatment strategy in acute VBO. With this treatment approach, a preserved vigilance before treatment and an embolic occlusion type are associated with no to moderate disability.

Introduction: Basilar artery (BA) perforator aneurysms may lead to severe subarachnoid hemorrhage (SAH). The acute management is uncertain. The anatomic approach is challenging both for coiling and clipping, and flow diverter stenting may be dangerous due to the required antiplatelet therapy. We report on our experiences in eight patients., Methods: We retrospectively analyzed eight patients with ruptured BA perforator aneurysm, including clinical characteristics, imaging data, treatment regimen, clinical course, and long-term outcome., Results: Patients presented with major SAH and World Federation of Neurosurgical Societies (WFNS) scores of I in three, II in two, and V in three cases. In four patients, the aneurysm was detected in the initial angiography, in four only in follow-up angiography. Five patients were treated conservatively and three patients had endovascular therapy. In the conservative group, the aneurysm spontaneously thrombosed in three cases. One patient suffered from a re-SAH and stayed permanently dependent due to an associated perforator stroke (modified Rankin Scale (mRS) 5). The remaining four patients recovered well (mRS 0 and 1 in two cases, each) including three patients also exhibiting perforator strokes. Regarding the endovascular group, one parent vessel was an angioma feeder and embolized with Onyx. The second aneurysm spontaneously thrombosed periinterventionally. The third patient underwent coiling. Two parent vessels were occluded postinterventionally, resulting in perforator strokes. Final mRS scores were 0, 2, and 2, respectively., Conclusion: Conservative management of ruptured BA aneurysms might be a first-line treatment option with common spontaneous aneurysm occlusion, low rate of re-SAH, and promising clinical outcome.

There is an ongoing discussion about reimbursement of stent-angioplasty for the treatment of intracranial stenoses in Germany. The discussion was initiated by the statutory health insurance companies after publication of the SAMMPRIS study results, which were in favor for medical management compared to stent-angioplasty with the Wingspan® stent system. A report (Rapid report N14-01) mainly based on SAMMPRIS was written by the German Institute for Quality and Efficiency in Health Care (IQWiG) and serves as a basis for the decision-making process. This report was previously commented by the medical societies involved. Limitations of the SAMMPRIS trial and vital indications for intracranial stenting were outlined in this comment (acute vessel occlusion, hemodynamic impairment, recurrent symptoms under medical treatment). Currently also emergency stent procedures are a matter of debate. In this context a second IQWiG report was commissioned (GA 15 - 02) addressing the results of the VISSIT trial, the transferability of the results of the first report to emergency treatments and the practice of emergency intracranial stent treatment in Germany. Regarding transferability of results the main conclusion was that there was no evidence that the results of the studies analyzed for the first report (mainly SAMMPRIS) could not be transferred to emergency treatments. From a medical professional and scientific standpoint it is inacceptable to compare outcomes of a secondary prophylactic treatment with emergency procedures. The analysis of emergency treatments in Germany based on retrospective case series with a cumulative number of 31 patients. Since most emergency procedures are performed in a clinical context and are not necessarily subject to scientific evaluation, this does not reflect current practice in Germany. The first part of this statement briefly outlines the design of SAMMPRIS and VISSIT and the interpretation of the trial results from a professional perspective. The current state of discussion regarding reimbursement of intracranial stenting is summarized. The second section contains a detailed comment on the current IQWiG report GA15-02 "Stents for the treatment of intracranial artery stenosis: VISSIT study and acute treatment in Germany".

There is an ongoing discussion about reimbursement of stent-angioplasty for the treatment of intracranial stenoses in Germany. The discussion was initiated by the statutory health insurance companies after publication of the SAMMPRIS study results, which were in favor for medical management compared to stent-angioplasty with the Wingspan® stent system. A report (Rapid report N14-01) mainly based on SAMMPRIS was written by the German Institute for Quality and Efficiency in Health Care (IQWiG) and serves as a basis for the decision-making process. This report was previously commented by the medical societies involved. Limitations of the SAMMPRIS trial and vital indications for intracranial stenting were outlined in this comment (acute vessel occlusion, hemodynamic impairment, recurrent symptoms under medical treatment).Currently also emergency stent procedures are a matter of debate. In this context a second IQWiG report was commissioned (GA 15 - 02) addressing the results of the VISSIT trial, the transferability of the results of the first report to emergency treatments and the practice of emergency intracranial stent treatment in Germany6. Regarding transferability of results the main conclusion was that there was no evidence that the results of the studies analyzed for the first report (mainly SAMMPRIS) could not be transferred to emergency treatments. From a medical professional and scientific standpoint it is inacceptable to compare outcomes of a secondary prophylactic treatment with emergency procedures. The analysis of emergency treatments in Germany based on retrospective case series with a cumulative number of 31 patients. Since most emergency procedures are performed in a clinical context and are not necessarily subject to scientific evaluation, this does not reflect current practice in Germany.The first part of this statement briefly outlines the design of SAMMPRIS and VISSIT and the interpretation of the trial results from a professional perspective. The current state of discussion regarding reimbursement of intracranial stenting is summarized. The second section contains a detailed comment on the current IQWiG report GA15-02 "Stents for the treatment of intracranial artery stenosis: VISSIT study and acute treatment in Germany".

Background and Purpose: Clopidogrel resistance is blamed for thromboembolic complications in neurovascular stent placement. Platelet-function assays are weakly standardized. The aim of this study was to correlate the results of 3 different platelet-inhibition measurements (from light transmission aggregometry, the VerifyNow P2Y12 test, and the Multiplate analyzer) and their relation to periprocedural thromboembolic complications in elective neurovascular stent placement., Materials and Methods: Clopidogrel resistance was determined on the day of the intervention according to predefined platelet reactivity cutoff values. All 3 tests were performed in 103 consecutive neurovascular stent-placement procedures in 97 patients (extracranial, n = 77; intracranial, n = 26)., Results: The clopidogrel resistance rates were 47.6% (light transmission aggregometry), 50.5% (VerifyNow), and 35.9% (Multiplate). In 67% of the patients, clopidogrel resistance was present according to at least one method. The correlations of qualitative results that classified a patient as responsive or resistant to clopidogrel were 67.9% for light transmission aggregometry with VerifyNow, 77.7% for light transmission aggregometry with the Multiplate, and 66% for VerifyNow with the Multiplate. Periprocedural thromboembolic complications (n = 9) occurred more frequently in patients who were determined by all 3 methods to be clopidogrel resistant. The difference was most pronounced with light transmission aggregometry (complication rates, 14.4% [clopidogrel-resistant patients] vs 3.7% [clopidogrel-responsive patients]). Sensitivity and specificity rates of clopidogrel resistance in relation to embolic complications were, respectively, 78% and 55% for light transmission aggregometry, 67% and 51% for VerifyNow, and 44% and 67% for the Multiplate., Conclusions: Clopidogrel resistance is a frequent finding in patients who undergo neurovascular stent placement. The correlations among the different testing methods are only modest and differ considerably. Light transmission aggregometry results seem to correlate with thromboembolic complications more accurately than with VerifyNow and Multiplate point-of-care methods., (© 2015 by American Journal of Neuroradiology.)

Benefit of endovascular recanalization beyond established treatment time windows likely exists in select stroke patients. However, there is currently no imaging model that predicts infarction adjusting for elapsed time between the pathologic snapshot of admission imaging until endovascular recanalization. We trained and cross validated a multivariate generalized linear model (GLM) that uses computer tomography perfusion and clinical data to quantify patient-specific dynamic change of tissue infarction depending on degree and time of recanalization. Multicenter data of 161 patients with proximal anterior circulation occlusion undergoing endovascular therapy were included. Multivariate voxelwise infarct probability was calculated within the GLM. The effect of increasing time to treatment and degree of recanalization on voxelwise infarction was calculated in each patient. Tissue benefit of successful relative to unsuccessful recanalization was shown up to 15 hours after onset in individual patients and decreased nonlinearly with time. On average, the relative reduction of infarct volume at the treatment interval of 5 hours was 53% and this salvage effect decreased by 5% units per hour to <5% after 10 additional hours to treatment. Treatment time-adjusted multivariate prediction of infarction by perfusion and clinical status may identify patients who benefit from extended time to recanalization therapy.

Background and Purpose: The purpose of this article is to report on the long-term success rates of Silk flow-diverter (FD) treatment in a multicenter prospective study for the treatment of complex aneurysms., Methods: Between May 2008 and January 2011, all consecutive patients featuring complex intracranial aneurysms eligible for FD treatment with the Silk in three neurovascular centers were included. Clinical and imaging data were assessed during hospitalization and follow-up., Results: Five patients were initially asymptomatic, 20 patients showed various neurological symptoms. Twenty-eight FDs were implanted in 25 patients treating 28 aneurysms. The immediate procedure-related morbidity was 8% (two of 25), mortality 0%. One procedure-related death was observed during follow-up (in-stent thrombosis). Compared to the immediate result nearly two of three aneurysms improved during follow-up; all angiographically confirmed inflow changes took place within six months after treatment. Final anatomic outcome in 24 aneurysms of 22 patients comprised 14 (59%) with complete occlusion, seven (29%) with a neck remnant, two (8%) with residual filling <50%, none with residual filling >50% and one (4%) unchanged in comparison to its pretreatment status. Postinterventional recanalizations were seen in three of 13 (23%) aneurysms treated with FD alone; none were observed in 15 aneurysms treated with adjunctive coiling., Conclusion: Anatomic presentation and location are key for successful FD treatment. The rate of successful occlusion increases during follow-up. Postinterventional monitoring for at least six months is paramount, as anatomic outcome is not reliably predictable and recanalizations may occur in initially completely occluded aneurysms., (© The Author(s) 2015.)

Quaternary chalcogenides, particular compounds with the stannite structure-type, are of interest for thermoelectrics applications however tellurium-containing compositions have not been extensively investigated. We report on the synthesis and high temperature thermoelectric properties of p-type stannites Cu2.2Zn0.8SnSe4-xTex (x = 0.1, 0.2, 0.3, and 0.4). The compositions for each specimen were confirmed with a combination of Rietveld refinement and elemental analysis. Hall measurements indicate that holes are the dominant charge carriers in these materials. The electrical resistivity shows little temperature dependence up to 500 K and then increases with increasing temperature. The thermal conductivity decreases with increasing temperature with no indication of increase at higher temperatures suggesting a minimal bipolar diffusion effect in the thermal conductivity although these materials possess relatively small band-gaps as compared to that of other stannite compositions. A maximum ZT value of 0.56 was obtained at 700 K for Cu2.2Zn0.8SnSe3.7Te0.3 due to a relatively high Seebeck coefficient and low thermal conductivity.

Purpose: Infections with Enterohaemorrhagic Escherichia coli typically occur in children causing haemolytic uraemic syndrome (HUS) and neurological symptoms in 20-50 %. Little information is available on the morphology of brain manifestations in adults. The purpose of this study was to identify a characteristic magnetic resonance imaging (MRI) pattern during the outbreak of a novel mutation of Escherichia coli O104:H4., Methods: Patients were recruited from two hospitals between May and July 2011. The MRI protocol included standard anatomical, diffusion-weighted, and susceptibility-sensitive sequences., Results: A total of 104 MRIs of 57 (32 female, 25 male) patients (mean 45.5 ± 18.4 years) showed abnormal signal intensity on 51 MRIs (49 %). Bilateral thalamus (39 %), bilateral pons (35 %), centrum semiovale and splenium of corpus callosum (33 %) were most often involved. Acute lesions were reversible in 81 % of cases. There was no statistically significant association between symptom onset and the MRI findings (P = 0.2)., Conclusions: Neuroimaging findings in this adult patient cohort were non-specific and similar to previous findings in children. A characteristic neuroimaging pattern of an infection with Escherichia coli O104:H4 was not identified. However, bilateral symmetric T2 hyperintense lesions of the thalami and dorsal pons characterized by restricted diffusion suggest a metabolic toxic effect of the disease on the brain.

Background: Successful management of patients with hematologic malignancies depends upon accurate and timely diagnosis, which frequently requires integration and interpretation of multiple tests. Our retrospective analysis compared diagnostic uncertainty, resource utilization, and costs for patients with diagnostic bone marrow (BM) tests managed by commercial laboratories., Methods: Patients with BM biopsies and suspected hematologic cancer/condition were identified from claims (2005-2011) within a large US health plan (coverage ≥6 pre- and ≥3-months post-biopsy). Cohorts defined by laboratories performing BM morphologic assessment/directing testing sequence: Genoptix (GX, specialty hematology-testing laboratory), large commercial laboratories (LL), other laboratories (OL). One-year post-biopsy changes in diagnosis or treatments, tests performed, and diagnostic/treatment medical costs (measured as per-patient-per-month [PPPM]) were examined., Results: The study population included 1,387 GX, 4,162 LL, and 19,115 OL patients with suspected hematologic malignancy/disease and BM morphology assessment. GX had lower diagnostic uncertainty measured between 2 time periods by diagnostic stability (no conditions the same; 6.16% GX, 8.04% LL, 9.73% OL; p < 0.001) and changes (≥1 condition different; 7.88% GX, 11.19% LL, and 14.08% OL; p < 0.001), fewer repeat BM biopsies, and fewer chemotherapy changes (30-days and 60-days post-initiation). One-year PPPM costs adjusted for patient characteristics differences were $8,202 GX, $7,711 LL, and $10,302 OL (p < 0.05); adjusted PPPM costs (excluding testing period) were $6,019 GX, $6,649 LL, and $7,801 OL (p < 0.05)., Conclusions: Our data suggests that a hematopathology specialty laboratory may result in earlier final diagnosis, fewer subsequent diagnosis changes, reduced need for follow-on testing requiring repeat biopsy procedures, and may result in lower downstream healthcare costs. Further evaluations using medical chart abstractions or registries will be valuable.

Infection of Escherichia coli by the filamentous phage fd starts with the binding of the N2 domain of the phage gene-3-protein to an F pilus. This interaction triggers partial unfolding of the gene-3-protein, cis → trans isomerization at Pro-213, and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA. The trans-proline sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. We elucidated the changes in structure and local stability that lead to partial unfolding and thus to the activation of the gene-3-protein for phage infection. Protein folding and TolA binding experiments were combined with real-time NMR spectroscopy, amide hydrogen exchange measurements, and phage infectivity assays. In combination, the results provide a molecular picture of how a local unfolding reaction couples with prolyl isomerization not only to generate the activated state of a protein but also to maintain it for an extended time.

Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the seven proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labelling. Fitting with high-resolution structures reveals the organization of TERT, TER and p65 in the ribonucleoprotein (RNP) catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.

This article reports an analysis that aims to quantify the effect of fingolimod, an oral treatment for relapsing remitting multiple sclerosis (MS), on disability progression. The standard approach utilizes survival analysis methods, which may be problematic for MS studies that assess disability at only a few time points and include as a cardinal feature both relapses and remissions. Instead, a Markov transition model, originally developed in the framework of longitudinal data, is fit, and its special probabilistic properties are used to estimate survival curves for time to disability progression. The transition approach models the whole disability process and uses all available transition data for inference, while survival methods concentrate on a single event of interest and use only time to event data. This article compares the transition model approach to survival analysis methods, and discusses the differences in the interpretations of the estimated parameters. It applies both models to data obtained from two phase 3 clinical trials and finds that both yield positive effects for the new treatment compared to placebo, and provide similar estimates for the probability of disability progression over time. The transition model enables calculation of covariate-specific transition matrices that describe the short-term effect of treatment and other covariates on the disability process., (Copyright © 2013, The International Biometric Society.)

Background: Multiple sclerosis (MS) is a common cause of neurological disability in young adults. The TRIBUNE study provides a detailed exploration of costs in relation to relapses and disease severity, and assesses the quality of life impact on MS patients in terms of utilities, fatigue and activities of daily living (ADL)., Methods: Patients in five European countries (France, Germany, Italy, Spain and the United Kingdom) completed a self-administered web-based questionnaire capturing information on demographics, disease characteristics and severity (EDSS), co-morbidities, relapses, resource consumption, utilities, fatigue, and activities of daily living., Results: In total, 1261 MS patients completed the questionnaire. More than half of the patients (68%) had the relapsing-remitting form of the disease; 87% of the sample reported receiving MS treatments. Costs were higher with advancing disease severity; for mild patients (EDSS score ≤ 3) the costs ranged between €13,534 and €22,461 across countries; for moderate (EDSS score 4 - 6.5) between €28,524 and €43,948; for severe (EDSS ≥ 7) between €39,592 and €65,395. Relapses were also associated with increasing costs; the difference in the cost per patient per year for relapsing-remitting patients with EDSS score ≤ 5 that did experience at least one relapse during the past 12 months and those who did not ranged between €3321 and €9430. The quality of life of patients decreased with disease progression and existence of relapses., Conclusion: The TRIBUNE study provides an important update on the economic burden of MS in an era of more widespread use of disease-modifying therapies. It explores the cost of MS linked to relapses and disease severity, and examines the impact of MS on additional health outcomes beyond utilities such as ADL and fatigue.

Background and Objective: Streptococcosis/lactococcosis is the cause of high morbidity and mortality in aquaculture sector and to date a number of species of Streptococcus and Lactococcus genera including S. iniae, S. agalactiae, S. dysagalactiae, S. parauberis, S. feacalis, L. garvieae and L. lactis have been discriminated as the cause of disease in aquatic animals. Despite the use of diagnostic molecular methods for each of these bacterial species, no data is available on a suitable, rapid and simple simultaneous detection tool for these pathogens. This paper describes a simultaneous detection method which is PCR based on a reverse line blot (RLB) for rapid detection and differentiation of four species of genera of Streptococcus and Lactococcus genera consisting of S. iniae, S. agalactiae, S. parauberis and L. garvieae the most important agents of the disease in fish., Materials and Methods: A reverse line blot (RLB) assay was developed for the simultaneously identification of four species of Streptococcus/lactococcusconsisting of S. iniae, S. parauberis, S. agalactiaeand Lactococcusgarvieae. The assay employs one set of primer pair for specific amplification of the 16S rRNA gene. These were designed based on the nucleotide sequences of 16S rRNA gene sharing a homology region with Streptococcus spp. and Lactococcus spp. DNA was extracted from the pure bacterial colonies and amplified. A membrane was prepared with specific oligonucleotide for each bacterial species. PCR products were then hybridized to a membrane., Results and Conclusion: The amplification resulted in PCR product of 241 bp in length. No cross-reactions were observed between any of the tested bacterial species, and mixed DNAs from these four bacterial species were correctly identified. This RLB method is a suitable technique for a simultaneous detection of these species of bacterial fish pathogens that are some of the main causes of streptococcal/lactococcal infections in both freshwater and marine aquatic animals, and so we recommend its use for integrated epidemiological monitoring of streptococcosis/lactococcis in aquaculture industry.

Objectives: Previous systematic reviews and meta-analyses of treatments in relapsing-remitting multiple sclerosis (RRMS) derived their findings from either placebo-controlled studies only or separately from head-to-head and comparative studies. The purpose of this study is to compare annualized relapse rates (ARR) of fingolimod versus all of the commonly used first-line treatments in RRMS using evidence from both placebo-controlled and head-to-head studies. In absence of the head-to-head data between fingolimod and the other treatments, these comparisons were formed using meta-analysis techniques for indirect treatment comparisons., Methods: A systematic literature review was conducted by searching MEDLINE, EMBASE, and the Cochrane Library with no limitations applied on publication language or dates. Included studies were randomized controlled trials evaluating one or more of fingolimod, interferon beta-1a, interferon beta-1b, or glatiramer acetate in RRMS populations. Primary outcome was ARR. Data extraction included author, year, treatment, dosage, mean age, percentage females, duration of disease, Expanded Disability Status Scale (EDSS) score at baseline, relapses in 2 years prior to baseline, trial duration, relapse-related outcome, and definition of relapse. The indirect treatment comparisons were performed using a mixed-treatment comparison framework. ARR was analyzed as a Poisson outcome., Results: The relative ARRs, for each treatment versus fingolimod, estimated from our meta-analyses were 1.43 (glatiramer acetate 20 mg), 1.51 (interferon beta-1b 250 mcg), 1.55 (interferon beta-1a 44 mcg), 1.67 (interferon beta-1a 22 mcg), 1.93 (interferon beta-1a 30 mcg), and 2.32 (placebo). None of the 95% confidence intervals for these estimates overlapped unity, implying statistical significance of these findings., Limitations: The key limitations of this study are the persisting heterogeneity even after adjusting for covariates and the variability in outcome definition across the included trials., Conclusions: Our study demonstrated that fingolimod significantly reduces relapse frequency in patients with RRMS compared with current first-line disease-modifying therapies.

Telomerase extends chromosome ends by the addition of single-stranded telomeric repeats. To support processive repeat synthesis, it has been proposed that coordination occurs between DNA interactions with the telomerase RNA template, the active site in the telomerase reverse transcriptase (TERT) core, a TERT N-terminal (TEN) domain, and additional subunits of the telomerase holoenzyme required for telomere elongation in vivo. The roles of TEN domain surface residues in primer binding and product elongation have been studied largely using assays of minimal recombinant telomerase enzymes, which lack holoenzyme subunits that properly fold and conformationally stabilize the ribonucleoprotein and/or control its association with telomere substrates in vivo. Here, we use Tetrahymena telomerase holoenzyme reconstitution in vitro to assess TEN domain sequence requirements in the physiological enzyme context. We find that TEN domain sequence substitutions in the Tetrahymena telomerase holoenzyme influence synthesis initiation and elongation rate but not processivity. Functional and direct physical interaction assays pinpoint a conserved TEN domain surface required for holoenzyme subunit association and for high repeat addition processivity. Our results add to the understanding of telomerase holoenzyme architecture and TERT domain functions with direct implications for the unique mechanism of single-stranded repeat synthesis.

Babesiosis is a haemoparasitic disease with high economical losses in livestock industry worldwide. The early diagnosis and successful therapy of babesiosis belong to the key steps of control and health management of livestock. Ethanol-fixed blood samples of 400 sheep were analyzed for Babesia infection. Reverse line blot (RLB) was established specifically for Theileria lestoquardi, Theileria (China 1), Theileria (China 2), Theileria ovis, Theileria separata, Babesia ovis, Babesia motasi, Babesia crassa, and Babesia (Lintan). The DNA was extracted from the ethanol-fixed blood samples and amplified with a common primer pair derived from 18S rRNA gene, amplifying both Theileria spp. as well as Babesia spp. Regarding the differences in the length of nucleotide sequences of the polymerase chain reaction (PCR) products obtained from Theileria spp. and Babesia spp., the PCR products derived from Babesia spp. were out screened and analyzed by RLB. The RLB analysis showed that 28 samples within the 400 blood samples were B. ovis positive. No B. motasi, B. crassa, or Babesia (Lintan) could be detected. The sequence analysis of one PCR product as a representative for other B. ovis-positive PCR products confirmed the results of RLB. Our results and the results of other investigators showed that B. ovis could be considered as a main causative agent of sheep babesiosis in Iran. Furthermore, our results also showed that RLB can be used as a reliable method for a simultaneous differentiation of Theileria and Babesia species from each other.

In order to identify and differentiate Theileria orientalis in cattle which may be infected with Theileria annulata simultaneously, a semi-nested PCR was performed. Thus, 160 blood samples were collected from apparently healthy native cattle in Golestan province of northern Iran, during 2009 to 2011. The Tbs-S/Tbs-A primer set derived from the 18S rRNA encoding gene was used for first PCR amplification, and the amplified sequence weight by this primer set for Theileria sp. was 426-430 bp. Then, DNA solution from purified PCR product was used for the semi-nested PCR analysis. The first PCR product amplified using T. orientalis primer set (To-S/Tbs-A) derived from the 18SrRNA encoding gene, and this specific primer weight was 235 bp. Also, the first PCR product amplified using T. annulata primer set (Ta-S/Tbs-A) derived from the 18SrRNA encoding gene and this specific primer weight was 193 bp. Having extracted DNA of each sample, using Tbs-S/Tbs-A primer set for PCR and analyzing the PCR products on the 2% agarose gel electrophoresis, 13 out of 160 blood samples (8.12%) were positive for Theileria sp. Meanwhile, performing semi-nested PCR with T. orientalis-specific primers, 9 out of 13 blood samples (5.62%) were positive and performing semi-nested PCR with T. annulata-specific primers, 12 out of 13 blood samples (7.5%) were also positive. This molecular assay approves the presence of T. orientalis in the native cattle of northern parts of Iran for the first time. In addition, this procedure will detect the concurrent infection of T. orientalis and T. annulata in the cattle too.

Background: Because of the strong immunologic responses of surface protein TaSp in Theileria annulata infected host, we tried to characterize this protein in a T. annulata isolate from Iran., Methods: The RNA prepared from T. annulata infected cells was used to produce SMART-DS-cDNA. The Double strand cDNA was then amplified with primers derived from TaSp mRNA sequences. The PCR product was cloned in pTZ57R/T vector, sequenced and registered under accession no. JQ003240 in GenBank., Results: The sequence analysis showed 90%-94% nucleotide sequence identity and 68%-94% amino acid homology to the corresponding sequences of TaSp gene by T. annulata, T. sp. china I, T. sp. china and T. lestoquardi and three T. annulata reported from Iran respectively. Interestingly, the sequence analysis also showed small nucleotide sequence region near the 5' end in which the presented TaSp protein differed very strongly from the other known TaSp sequences. For the preparation of the recombinant protein, the cDNA was cloned in pQE-32 vector, the recombinant protein was prepared and assayed by Theileria infected bovine serum., Conclusion: The polymorphism in TaSp gene could be detected in intra- as well as inter species. The different characterized TaSp proteins had a common identic region, which may be helpful for development of broad band vaccine based on the recombinant proteins. The polymorphism in this gene, make this protein also interesting for the diagnostic purposes.

Introduction: Using balloon-expandable stents (BES) for treatment of intracranial stenoses, high inflation pressures and rigidity of the device are regarded as major drawbacks limiting feasibility and safety of the procedure. Self-expanding stents (SES) were developed to facilitate lesion access and to allow for less aggressive dilatation. We analyzed data of the INTRASTENT multicentric registry to assess whether self-expanding stents significantly reduced peri-interventional complication rates., Methods: Records of intracranial stent procedures were entered consecutively into the registry. Datasets were divided into two groups according to the type of stent used. For outcome measurement, we chose three categories: TIA/minor stroke [modified Rankin score (mRS) <2], disabling stroke, and patient death. Clinical outcome was compared between BES and SES. We analyzed types of adverse events occurring in each group in addition., Results: Of 409 atherosclerotic lesions, 254 were treated with BES and 155 with SES. Technical success rates were 97.6% and 98.7%, respectively. Adverse event rates were 4.9%, 3.7%, and 0.8% for TIA/nondisabling stroke, disabling stroke, and death in the BES group compared with 5.3%, 6.0%, and 4.0% in the SES group. The differences were not statistically significant. We observed more perforator strokes after use of BES, but thromboembolic events occurred more often in the SES treatment group., Conclusion: Data of the INTRASTENT registry do not support the hypothesis that introduction of SES lowered the overall complication rate of intracranial stent procedures. There might be an advantage using self-expanding stents in vessel segments with important perforating arteries.

Background: Health-related quality of life (HRQoL) worsens with multiple sclerosis (MS) relapses and disease progression. Common symptoms including depression and fatigue may contribute to poor HRQoL., Objectives: To report exploratory analyses assessing the impact of fingolimod (FTY720) on HRQoL and depression in a phase II study of relapsing MS., Methods: The Hamburg Quality of Life Questionnaire in MS (HAQUAMS) and Beck Depression Inventory second edition (BDI-II) scores were assessed during a 6-month, placebo-controlled study and optional extension., Results: HAQUAMS total score improved with fingolimod and worsened with placebo. Mean score change from baseline to month 6 was -0.02 with fingolimod 1.25 mg (p < 0.05 versus placebo), -0.01 with fingolimod 5.0 mg and + 0.12 with placebo. Categorical data supported a clinically important effect of fingolimod on HRQoL. Fingolimod 1.25 mg was also beneficial over placebo in the fatigue/thinking HAQUAMS sub-domain (p < 0.05 versus placebo). Change in mean BDI-II scores from baseline to month 6 and the proportion of patients with BDI-II scores indicative of clinical depression favored fingolimod 1.25 mg over placebo (p < 0.05 for both). At month 4, mean BDI-II and HAQUAMS total scores appeared to be maintained in fingolimod-treated patients., Conclusion: Fingolimod 1.25 mg may improve HRQoL and depression at 6 months compared with placebo in patients with relapsing MS.

The innate immune system detects infection by using germline-encoded receptors that are specific for conserved microbial molecules. The recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFNs), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of inducing IFN production, and this process requires signalling through TANK binding kinase 1 (TBK1) and its downstream transcription factor, IFN-regulatory factor 3 (IRF3). In addition, a transmembrane protein called STING (stimulator of IFN genes; also known as MITA, ERIS, MPYS and TMEM173) functions as an essential signalling adaptor, linking the cytosolic detection of DNA to the TBK1-IRF3 signalling axis. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signalling molecules in bacteria, have also been shown to induce a STING-dependent type I IFN response. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP), and we show that unlabelled cyclic dinucleotides, but not other nucleotides or nucleic acids, compete with c-di-GMP for binding to STING. Furthermore, we identify mutations in STING that selectively affect the response to cyclic dinucleotides without affecting the response to DNA. Thus, STING seems to function as a direct sensor of cyclic dinucleotides, in addition to its established role as a signalling adaptor in the IFN response to cytosolic DNA. Cyclic dinucleotides have shown promise as novel vaccine adjuvants and immunotherapeutics, and our results provide insight into the mechanism by which cyclic dinucleotides are sensed by the innate immune system.

Background and Objective: Advances in statistical modelling have allowed the creation of smoothly changing spirometry reference ranges that apply across a wide age range and better define the lower limit of normal. The objective of this study was to assess the agreement of the Stanojevic 2009 all-age reference ranges to contemporary lung function data to verify the appropriateness of this reference for clinical use in Australia and New Zealand., Methods: Spirometry data from healthy Caucasians measured between 2000-2009 in Australia and New Zealand were collected. Z-scores were calculated for the standard spirometry outcomes based on the all-age reference ranges., Results: Spirometry from 2066 subjects aged 4-80 years (55% male) from 14 centres were eligible. Statistically, the collated contemporary dataset differed from the all-age reference ranges, but these differences were relatively small and clinically irrelevant representing differences of approximately 3% predicted. Significant differences were also observed between some centres and equipment, potentially indicating varying influence of equipment or subject selection., Conclusions: Spirometry from contemporary Australasian healthy subjects fits the all-age reference ranges well. While the current study supports the use of the all-age reference ranges, the between-centre differences highlight the need for spirometry to be used in conjunction with other clinical findings., (© 2011 The Authors. Respirology © 2011 Asian Pacific Society of Respirology.)

Introduction: Acute stroke multimodal CT imaging (MMCT: non-enhanced CT, CT angiography, and CT perfusion (CTP)) may show normal results despite persistent clinical stroke. We prospectively evaluated the sensitivity/specificity of MMCT infarct detection and the clinical outcome in patients with normal MMCT findings., Methods: From April 2007 to April 2008, all patients with acute hemispheric stroke within 6 h of symptom onset who underwent complete MMCT and MRI follow-up imaging were included. MMCT analysis included occlusion type, early infarct hypodensities (EIH), mean transit time (MTT), and cerebral blood volume (CBV) maps according to Alberta Stroke Program Early CT Score (ASPECTS). Clinical assessment included symptom onset to CT scan (≤3 h/>3 h), the National Institute of Health Stroke Scale score (admission/discharge), and the modified Rankin scale (mRS) 90 days after stroke onset., Results: One hundred seven were included (mean age, 68.4 years; ≤3 h, n = 84; >3 h, n = 23; intravenous thrombolysis (IVT), n = 51; ≤3 h, n = 40; >3 h, n = 11). In patients with normal MMCT on admission (n = 54), follow-up MRT detected brain infarctions in 23 patients (lacunar strokes, n = 16; infratentorial strokes, n = 4; territorial infarction, n = 3). Sensitivity/specificity/positive predictive value/negative predictive value of any infarct detection was 69.5%/99.8%/99.9%/57.2% and of a any territorial infarct detection was 93.9%/99.9%/99.9%/93.6%, respectively. In univariate regression analysis (time to CT scan, ≤3 h/>3 h; IVT: yes/no; ASPECTS EIH/CBV/MTT, 10/<10), only the evidence of normal CTP (ASPECTS MTT = 10) had a statistically significant impact (p = 0.02) on a good outcome (mRS 0.1)., Conclusion: MMCT sensitivity in acute lacunar or infratentorial stroke was poor. But, we found a high specifity and a fairly good sensitivity in territorial infarct detection. In acute stroke patients with normal MMCT findings on admission, a good clinical prognosis can be expected.

Background: The PRIMUS is a Multiple Sclerosis (MS)-specific suite of outcome measures including assessments of QoL (PRIMUS QoL, scored 0-22) and activity limitations (PRIMUS Activities, scored 0-30). The U-FIS is a measure of fatigue impact (scored 0-66). These measures have been fully validated previously using an MS sample with mixed diagnoses. The aim of the present study was to validate the measures further in a specifically Relapse Remitting MS (RRMS) sample and to provide preliminary evidence of the responder definitions (RD; also known as minimal important difference) for these instruments., Methods: Data were derived from a multi-country efficacy trial of MS patients with assessments at baseline and 12 months. Baseline data were used to assess the internal reliability and validity of the measures. Both anchor-based and distribution-based approaches were employed for estimating RD. Anchor-based estimates were based on published RD values for the EQ-5D and were assessed for those improving and deteriorating separately. Distribution-based estimates were based on standard error of measurement (SEM), change score equivalent to 0.30, and change score equivalent to 0.50, effect sizes (ES)., Results: The sample included 911 RRMS patients (67.3% female, age mean (SD) 36.2 (8.4) years, duration of MS mean (SD) 4.8 (5.2) years). Results showed that the PRIMUS and U-FIS had good internal consistency. Appropriate correlations were observed with comparator instruments and both measures were able to distinguish between participants based on Expanded Disability Status Scale scores and time since diagnosis. The anchor-based and distribution-based RD estimates were: PRIMUS Activities range = 1.2-2.3, PRIMUS QoL range = 1.0-2.2, and U-FIS range = 2.4-7.0., Conclusions: The results show that the PRIMUS and U-FIS are valid instruments for use with RRMS patients. The analyses provide preliminary information on how to interpret scores on the scales. These data will be useful for assessing treatment efficacy and for powering clinical studies. TRIAL REFERENCE NUMBER: ClinicalTrials.gov Identifier NCT00340834.

Shrinking lung syndrome (SLS) is a rare manifestation of systemic lupus erythematosus (SLE). We report the case of a 27-year-old woman with SLE presenting with a 2-year history of chest pain and progressive dyspnea. Respiratory function tests demonstrated severe restrictive ventilatory impairment. Chest x-ray demonstrated elevated hemi diaphragms and chest computed tomography showed no evidence of interstitial fibrosis, significant pleural disease or pulmonary emboli. Based on a diagnosis of SLS the patient received 4 months of high dose corticosteroids, mycophenolate and pain management with opiates. Her condition deteriorated and she was given a trial of rituximab. This resulted in marked improvement of the clinical condition and respiratory function tests that was maintained for 18 months. Subsequently, the patient represented with a similar clinical picture and another course of rituximab again produced remission. This is the first case report of reproducible remission of SLS in SLE treated with rituximab.

Unlabelled: Spinal cord-injured subjects were assessed during the acute admission for sleep-disordered breathing. Polysomnography demonstrated a high incidence of sleep apnoea that persisted during the acute phase. There was no correlation with respiratory function tests or measures of ventilatory control. Screening of this population is worthwhile although the clinical significance is uncertain., Background and Objective: Previous studies have demonstrated an increased incidence of sleep apnoea in spinal cord-injured patients. Many of these studies were performed in long-term, stable spinal cord injury (SCI). The aims of this study were: (i) to determine the prevalence of sleep-disordered breathing (SDB) in acute SCI; (ii) to document the change in SDB over time during the rehabilitation period; and (iii) to correlate the degree of SDB with ventilatory parameters., Methods: Sixteen subjects with an acute SCI level T12 and above with complete motor impairment (American Spinal Injury Association impairment scale A or B) were recruited. Assessment, including polysomnography, respiratory function testing, and hypoxic and hypercapnic ventilatory responses, were performed 6-8 weeks post SCI, and repeated 6 months post SCI., Results: Eleven of 16 subjects (73%) had evidence of sleep apnoea, five of whom were moderate to severe. This high incidence persisted during the acute admission, with 9 of 12 subjects (75%) having sleep apnoea on polysomnography 20 weeks following injury. There was no correlation between the severity of SDB and other measures, such as level or completeness of injury, respiratory function tests or measures of ventilatory responses., Conclusions: We have demonstrated a high incidence of sleep apnoea in the acute phase of SCI that persisted during the acute admission. Despite the high incidence of sleep apnoea, patients were relatively asymptomatic. Screening of this population would appear worthwhile given the high prevalence, although the significance of the sleep apnoea and clinical impact is not known.

Purpose: Evaluation of hospital resource allocation in intracranial aneurysm treatment in a medium-volume neurovascular center., Materials and Methods: Retrospective data analysis included 653 procedures performed on 598 patients with 667 aneurysms (A) from 1990 to 2004. 515 treatments were carried out in ruptured A (clip: n = 370; coil: n = 145) and 138 procedures in non-ruptured A (clip: n = 51, coil: n = 87). Patient management data included procedure time (min), length of stay in the intensive care unit (days), total length of hospital stay (days), and discharge to home ratio., Results: Clinical admission grade (rupt. A: Hunt and Hess grade 1 - 3: clip: 73 % coil: 72 %) and clinical outcome at discharge (good neurological outcome/mortality rate: rupt. A: clip: 51.1/13.8 % coil: 45.5 / 10.3 % non-rupt. A: 88.2/0 % coil: 88.5/1.3 %) were similar for both treatment modes. The coil procedure time was found to be significantly shorter (rupt. A: coil: 145 min; clip: 203 min; p < 0.01; non-rupt. A: coil: 164 min, clip: 200 min; p < 0.01). Coiling reduced the length of stay in the ICU (rupt. A: coil: 5.3 d; clip: 6 d, p < 0.01; non-rupt. A: coil: 1.5 d; clip: 2 d; p = 0.21) and coiling significantly reduced the length of hospital stay (rupt. A: coil: 21.4 d; clip: 26.8 d, p < 0.01; non-rupt. A: coil: 9.2 d; clip: 17.5 d; p = 0.01).The discharge to home ratio did not differ (rupt. A: clip: 31.6 % coil: 29.7 % non-rupt. A: clip: 74.5 % coil: 80.5 %)., Conclusion: In a medium-volume neurovascular center, coiling significantly reduced the procedure time, the stay in the ICU, and the length of hospital stay suggesting favorable resource allocation in endovascular therapy., (Georg Thieme Verlag KG Stuttgart-New York.)

Background: The multidimensional assessment of fatigue is complicated by the interrelation of its multiple causes and effects., Objective: The purpose of the research was to develop a unidimensional assessment of fatigue (U-FIS)., Methods: Data collected with the Fatigue Impact Scale (FIS) were subjected to Rasch analysis to identify potential problems with the scale. Additional items for the U-FIS were generated from interviews with UK MS patients. The U-FIS was tested for face and content validity in patient interviews and included in a validation survey to determine dimensionality (Rasch model), reliability and validity., Results: The original FIS was not unidimensional when subscale items were combined. The modification of the FIS and addition of a number of items allowed the development of a 22-item unidimensional scale (U-FIS) that was reliable (Cronbach Alpha = 0.96; test-retest = 0.86,) and valid given correlations with the Nottingham Health Profile and ability to distinguish between MS severity groups. There was no significant difference in U-FIS scores according to MS type., Conclusion: It is valid to conceptualize the functional impact of fatigue as unidimensional. The U-FIS is a reliable and valid questionnaire that will allow the measurement of this construct in clinical studies.

Prolyl cis/trans isomerizations determine the rates of protein folding reactions and can serve as molecular switches and timers. In the gene-3-protein of filamentous phage, Pro-213 trans --> cis isomerization in a hinge region controls the assembly of the 2 domains N1 and N2 and, in reverse, the activation of the phage for infection. We elucidated the structural and energetic basis of this proline-limited domain assembly at the level of individual residues by real-time 2D NMR. A local cluster of inter-domain hydrogen bonds, remote from Pro-213, is stabilized up to 3,000-fold by trans --> cis isomerization. This network of hydrogen bonds mediates domain assembly and is connected with Pro-213 by rigid backbone segments. Thus, proline cis/trans switching is propagated in a specific and directional fashion to change the protein structure and stability at a distant position.

This article includes a review of major intravenous and endovascular stroke trials, treatment options, and future aspects of acute stroke treatment in hemispheric and vertebrobasilar stroke. Since the invention of local intraarterial thrombolysis by Hermann Zeumer in 1981, acute stroke diagnostics and treatment have undergone dramatic improvement. This article addresses major topics in recent stroke treatment debates: optimization of patient selection, intravenous versus endovascular therapy, time window limitations, combined treatment with intravenous/intraarterial bridging therapies (intravenous/intraarterial recombinant tissue plasminogen activator [rtPA] bridging and intravenous glycoprotein IIb/IIIa inhibitor/intraarterial rtPA bridging) and modern endovascular treatment modes like percutaneous transluminal angioplasty (PTA)/stenting and mechanical thrombectomy devices. Modern acute stroke therapy networks should optimize their noninvasive diagnostic capacity to early identify candidates for endovascular therapy with rapid access to specialized neuroendovascular centers using standard protocols. The most promising approach in acute stroke treatment seems to be a combination of intravenous and endovascular revascularization procedure, combining early treatment initiation with direct clot manipulation and PTA/stenting in underlying stenosis with atherothrombotic occlusions. Further randomized studies comparing intravenous and endovascular treatment, mainly in the anterior circulation, have to be expected and need wide support of the neurologic and neuroradiologic stroke community.

Herbaspirillum frisingense is a diazotrophic betaproteobacterium isolated from C4-energy plants, for example Miscanthus sinensis. To demonstrate endophytic colonization unequivocally, immunological labeling techniques using monospecific polyclonal antibodies against two H. frisingense strains and green fluorescent protein (GFP)-fluorescence tagging were applied. The polyclonal antibodies enabled specific in situ identification and very detailed localization of H. frisingense isolates Mb11 and GSF30(T) within roots of Miscanthusxgiganteus seedlings. Three days after inoculation, cells were found inside root cortex cells and after 7 days they were colonizing the vascular tissue in the central cylinder. GFP-tagged H. frisingense strains could be detected and localized in uncut root material by confocal laser scanning microscopy and were found as endophytes in cortex cells, intercellular spaces and the central cylinder of barley roots. Concerning the production of potential plant effector molecules, H. frisingense strain GSF30(T) tested positive for the production of indole-3-acetic acid, while Mb11 was shown to produce N-acylhomoserine lactones, and both strains were able to utilize 1-aminocyclopropane-1-carboxylate (ACC), providing an indication of the activity of an ACC-deaminase. These results clearly present H. frisingense as a true plant endophyte and, although initial greenhouse experiments did not lead to clear plant growth stimulation, demonstrate the potential of this species for beneficial effects on the growth of crop plants.

Introduction: A debate is emerging over whether the treatment time window in acute stroke can be extended beyond 6 h if penumbral tissue can be identified. Treatment decisions are very difficult in cases of tandem proximal carotid occlusion with arterioarterial intracranial embolism. We enter this debate with the present report on a case of atherosclerotic proximal carotid occlusion and resulting periocclusional carotid T embolism that was successfully treated 9 h after symptom onset., Methods: The case of a 68-year-old man with fluctuating symptoms of right-hemispheric stroke is presented (NIHSS score 12-20 on admission). CT angiography demonstrated proximal carotid occlusion and periocclusional embolism of the entire internal carotid artery (ICA) including the carotid T segment. Penumbral tissue was diagnosed by nonenhanced and perfusion CT imaging 7.5 h after symptom onset. Treatment was initiated 9 h after symptom onset by passing the proximal occlusion with a microcatheter and local administration of recombinant tissue plasminogen activator (rt-PA) into the carotid T segment at the level of posterior communicating artery (PCoA) origin., Results: Recanalization of the ICA and middle cerebral artery was accomplished within 1 h by flow establishment via the PCoA. The atherosclerotic proximal ICA occlusion was not stented due to the risk of embolism from remnant thrombi in the petrous and cavernous ICA segments. Follow-up MRI showed only mild haemorrhagic infarct transformation of the initial infarct core. The patient was discharged from hospital 18 days after treatment with NIHSS score 5., Conclusion: If penumbral tissue can be conclusively identified, endovascular treatment in proximal and intracranial tandem occlusion can be successful, even in treatments initiated 6-9 h after stroke onset. If the intracranial flow after recanalization can be established via the circle of Willis, the underlying proximal ICA occlusion may not require treatment.

The envelope glycoproteins of Rubella virus, E1 and E2, mediate cell tropism, and E1 in particular plays a pivotal role in the fusion of the virus with the endosomal membrane. Both are the prime targets of the humoral immune response. Recombinant variants of the E1 ectodomain as well as E1 antigen preparations from virus lysates are commonly used to detect anti-Rubella immunoglobulins in human sera. Hitherto, recombinant E1 for diagnostic applications has been produced chiefly in eukaryotic expression systems. Here, we report the high-yield overproduction of an engineered E1 ectodomain in the Escherichia coli cytosol and its simple and convenient renaturation into a highly soluble and immunoreactive conformation. C-Terminal fusion to one or two units of the E. coli chaperone SlyD enhances expression, facilitates in vitro refolding, and improves the overall solubility of Rubella E1. As part of this fusion protein, the E1 ectodomain fragment of residues 201-432 adopts an immunoreactive fold, providing a promising tool for the sensitive and specific detection of anti-E1 IgG in Rubella serology. Two disulfide bonds in the membrane-adjacent part of the E1 ectodomain are sufficient to generate conformations with a high and specific antigenicity. The covalently attached chaperone modules do not impair antibody recognition and binding of Rubella E1 when assessed in a heterogeneous immunoassay. SlyD and related folding helpers are apparently generic tools for the expression and refolding of otherwise unavailable proteins of diagnostic or medical importance.

Unfolding usually leads to the loss of the biological function of a protein. Here, we show that an unfolding reaction activates the gene-3-protein of the filamentous phage fd for its function during the infection of Escherichia coli. Before infection, the gene-3-protein is in a fully folded locked form, in which the binding site for the phage receptor TolA is buried at the domain interface. To expose this binding site, the gene-3-protein must be activated, and previously we identified the cis-to-trans isomerization at Pro213 in the hinge region between the two domains as a key step of activation. We now report that Pro213 isomerization destabilizes the protein and leads to a loss of folded structure, presumably in the hinge region. The partially unfolded form of the gene-3-protein is metastable, and trans-Pro213 arrests the protein in this activated form for an extended time, long enough to find the receptor TolA. The partial unfolding and its timing by prolyl isomerization are essential for the biological function.

The catalytic activity of human FKBP12 as a prolyl isomerase is high towards short peptides, but very low in proline-limited protein folding reactions. In contrast, the SlyD proteins, which are members of the FKBP family, are highly active as folding enzymes. They contain an extra "insert-in-flap" or IF domain near the prolyl isomerase active site. The excision of this domain did not affect the prolyl isomerase activity of SlyD from Escherichia coli towards short peptide substrates but abolished its catalytic activity in proline-limited protein folding reactions. The reciprocal insertion of the IF domain of SlyD into human FKBP12 increased its folding activity 200-fold and generated a folding catalyst that is more active than SlyD itself. The IF domain binds to refolding protein chains and thus functions as a chaperone module. A prolyl isomerase catalytic site and a separate chaperone site with an adapted affinity for refolding protein chains are the key elements for a productive coupling between the catalysis of prolyl isomerization and conformational folding in the enzymatic mechanisms of SlyD and other prolyl isomerases, such as trigger factor and FkpA.