Pre-existing Reactivity to an IgG4 Fc-Epitope: Characterization and Mitigation of Interference in a Bridging Anti-drug Antibody Assay.

Twenty percent of baseline patient samples exhibited a pre-existing response in a bridging anti-drug antibody (ADA) assay for a human IgG4 monoclonal antibody (mAb) therapeutic. In some cases, assay signals were more than 100-fold higher than background, potentially confounding detection of true treatment-emergent ADA responses. The pre-existing reactivity was mapped by competitive inhibition experiments using recombinant proteins or chimeric human mAbs with IgG4 heavy chain regions swapped for IgG1 sequences. These experiments demonstrated that the majority of the samples had reactivity to an epitope containing leucine 445 in the CH3 domain of human IgG4. The pre-existing reactivity in baseline patient samples was mitigated by replacing the ADA assay capture reagent with a version of the drug containing a wild type IgG1 proline substitution at residue 445 without impacting detection of drug-specific, treatment-emergent ADA. Finally, purification on Protein G or anti-human IgG (H + L) columns indicated the pre-existing response was likely due to immunoglobulins in patient samples.

RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics.

Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.

Recombinant immunotoxin targeting GPC3 is cytotoxic to H446 small cell lung cancer cells.

Glypican-3 (GPC3) is a cell membrane glycoprotein that regulates cell growth and proliferation. Aberrant expression or distribution of GPC3 underlies developmental abnormalities and the development of solid tumours. The strongest evidence for the participation of GPC3 in carcinogenesis stems from studies on hepatocellular carcinoma and lung squamous cell carcinoma. To the best of our knowledge, the role of the GPC3 protein and its potential therapeutic application have never been studied in small cell lung carcinoma (SCLC), despite the known involvement of associated pathways and the high mortality caused by this disease. Therefore, the aim of the present study was to examine GPC3 targeting for SCLC immunotherapy. An immunotoxin carrying an anti-GPC3 antibody (hGC33) and Pseudomonas aeruginosa exotoxin A 38 (PE38) was generated. This hGC33-PE38 protein was overexpressed in E. coli and purified. ADP-ribosylation activity was tested in vitro against eukaryotic translation elongation factor 2. Cell internalisation ability was confirmed by confocal microscopy. Cytotoxicity was analysed by treating liver cancer (HepG2, SNU-398 and SNU-449) and lung cancer (NCI-H510A, NCI-H446, A549 and SK-MES1) cell lines with hGC33-PE38 and estimating viable cells number. A BrdU assay was employed to verify anti-proliferative activity of hGC33-PE38 on treated cells. Fluorescence-activated cell sorting was used for the detection of cell membrane-bound GPC3. The hGC33-PE38 immunotoxin displayed enzymatic activity comparable to native PE38. The protein was efficiently internalised by GPC3-positive cells. Moreover, hGC33-PE38 was cytotoxic to HepG2 cells but had no effect on known GPC3-negative cell lines. The H446 cells were sensitive to hGC33-PE38 (IC50, 70.6±4.6 ng/ml), whereas H510A cells were resistant. Cell surface-bound GPC3 was abundant on the membranes of H446 cells, but absent on H510A. Altogether, the present findings suggested that GPC3 could be considered as a potential therapeutic target for SCLC immunotherapy.

Probability assessment of intracerebral hemorrhage in prehospital emergency patients.

BACKGROUND: Routing of patients with intracerebral hemorrhage (ICH) and acute ischemic stroke (AIS) to the most appropriate hospital is challenging for emergency medical services particularly when specific treatment options are only provided by specialized hospitals and determination of the exact diagnosis is difficult. We aimed to develop a prehospital score - called prehospital-intracerebral hemorrhage score (ph-ICH score) - to assist in discriminating between both conditions. METHODS: The ph-ICH score was developed with data from patients treated aboard a mobile stroke unit in Berlin, Germany, between 2011 and 2013 (derivation cohort) and in 2018 (validation cohort). Diagnosis of ICH or AIS was established using clinical data and neuroradiological cerebral imaging. Diagnostic accuracy was measured with significance testing, Cohen's d and receiver-operating-characteristics. RESULTS: We analyzed 416 patients (32 ICH, 224 AIS, 41 transient ischemic attack, 119 stroke mimic) in the derivation cohort and 285 patients (33 ICH and 252 AIS) in the validation cohort. Systolic blood pressure, level of consciousness and severity of neurological deficits (i. e. certain items of the National Institutes of Health Stroke Scale) were used to calculate the ph-ICH score that showed higher values in the ICH compared to the AIS group (derivation cohort: 1.8 ± 1.2 vs. 1.0 ± 0.9 points; validation cohort: 1.8 ± 0.9 vs. 0.8 ± 0.7 points; d = 0.9 and 1.4, both p < 0.01). Receiver-operating-characteristics showed fair and good accuracy with an area under the curve of 0.71 for the derivation and 0.81 for the validation cohort. CONCLUSIONS: The ph-ICH score can assist medical personnel in the field to assess the likelihood of ICH and AIS in emergency patients.

Discriminative value of glial fibrillar acidic protein (GFAP) as a diagnostic tool in acute stroke. Individual patient data meta-analysis.

Glial fibrillar acidic protein (GFAP) in serum has been evaluated as a promising biomarker to differentiate between intracerebral hemorrhage (ICH) and acute ischemic stroke (AIS). We assessed its value as diagnostic and prognostic tool for ICH through a literature systematic review and individual patient data (IPD) meta-analysis.We performed a systematic search in PubMed database until November 2018 for publications that evaluated GFAP to differentiate AIS and ICH within 4.5 hours after symptoms onset. Thereafter, we invited authors of selected studies to participate in this work by providing IPD from their cohorts. We used standardized individual subject's data to evaluate the association of GFAP concentrations with stroke subtype, demographics, stroke characteristics and factors related with GFAP measurement.From 4 selected studies, we collected data of 340 patients (236 AIS and 104 ICH). Standardized GFAP blood levels were significantly elevated in ICH compared with those with AIS (median and IQR: 0.84 (0.781-1.24), 0.79 (0.74-0.81); p<0.0001). In both stroke types, GFAP concentrations correlated with baseline stroke severity (r=0.27, p<0.0001; r=0.36, p<0.001; for AIS and ICH, respectively) but no correlation was found regarding time to sampling. Limited data precluded the evaluation of GFAP levels and functional outcome.These findings demonstrate substantially different levels of GFAP in the blood of patients with ICH compared with patients with AIS soon after the event, while no association was found with outcome. In summary, GFAP could be a valuable diagnostic tool to assist in medical decision-making and to optimize management of stroke in the acute setting.

Insulin Reduces the Efficacy of Vemurafenib and Trametinib in Melanoma Cells.

BACKGROUND: Despite the progress made in the clinical management of metastatic melanoma, a patient's response to treatment cannot be fully predicted, and intrinsic or acquired resistance that is developed in most melanoma patients warrants further research efforts. In addition to genetic factors, microenvironmental input should be considered to explain the diversity of response to treatment among melanoma patients. In this study, we evaluated the impact of insulin on patient-derived BRAFV600E melanoma cells, either untreated or treated with vemurafenib or trametinib, inhibitors of BRAFV600 and MEK1/2, respectively. METHODS: Cells were cultured in serum-free conditions, either with or without insulin. The activity of the MAPK/ERK and PI3K/AKT pathways was assessed by Western blotting, cell viability, and percentages of Ki-67- and NGFR-positive cells by flow cytometry. Transcript levels were analyzed using qRT-PCR, and γ-H2AX levels by immunoblotting and confocal microscopy. A luminescence-based assay was used to measure glutathione content. RESULTS: While insulin did not influence the MAPK/ERK pathway activity, it had a strong influence on melanoma cells, in which this pathway was suppressed by either vemurafenib or trametinib. In the presence of insulin, both drugs were much less efficient in 1) inhibiting proliferation and reducing the percentage of Ki-67-positive cells, and 2) inducing apoptosis and phosphorylation of histone H2AX in melanoma cells. Changes induced by vemurafenib and trametinib in glutathione homeostasis and DNA repair gene expression were also attenuated by insulin. Moreover, insulin impaired the combined effects of targeted drugs and doxorubicin in melanoma cells. In addition to insulin-induced PI3K/AKT activity, which was either transient or sustainable depending on the cell line, an insulin-triggered increase in the percentage of cells expressing NGFR, a marker of neural crest stem-like cells, may contribute to the reduced drug efficacy. CONCLUSION: Our results demonstrate the role of insulin in reducing the efficacy of vemurafenib and trametinib. This needs clinical assessment.

Direct Measurement of Kinetic Parameters of ABCG2-Dependent Transport of Natural Flavonoids Using a Fluorogenic Substrate.

Flavonoids are an important part of the human diet since plant-derived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ATP-binding cassette transporter ABCG2, expressed in gut epithelium and other barrier tissues. While flavonoids were previously identified as substrates and/or inhibitors of this protein, precise enzyme kinetic calculations of affinity and activity parameters are rare due to the lack of suitable experimental models. We present a novel method that allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate, which reacts with intracellular flavonoids forming a fluorescent, nonmembrane-permeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in Madin-Darby canine kidney II-derived cell lines expressing human ABCG2 and involve substrate efflux from whole, unmodified cells, precluding the need for plasma membrane vesicle preparation. We present methods for calculation of enzyme kinetic constants by measuring substrate concentration at efflux-influx equilibrium or during efflux from preloaded cells, and we obtained K m values of 137 µM for quercetin, 36 µM for kaempferol, and 348 µM for luteolin. Our method also allows direct verification of the transport inhibition mechanism and potentially the structure-activity relationship in substrates. SIGNIFICANCE STATEMENT: The study presents the first direct calculation of kinetic constants for enzyme-mediated active transport of natural flavonoids in a whole-cell assay, using a fluorogenic compound to measure intracellular substrate concentrations at specific time points. It has implications for nutriceutical use of polyphenols, mechanisms of food-drug interactions, and studies on absorption/distribution-determining membrane transporters, allowing a quantitative approach to pharmacokinetics of flavonoid transport across barrier tissues.

Telemedicine in Prehospital Acute Stroke Care.

Background Mobile stroke units ( MSU s), equipped with an integrated computed tomography scanner, can shorten time to thrombolytic treatment and may improve outcome in patients with acute ischemic stroke. Original (German) MSU s are staffed by neurologists trained as emergency physicians, but patient assessment and treatment decisions by a remote neurologist may offer an alternative to neurologists aboard MSU . Methods and Results Remote neurologists examined and assessed emergency patients treated aboard the MSU in Berlin, Germany. Audiovisual quality was rated by the remote neurologist from 1 (excellent) to 6 (insufficient), and duration of video examinations was assessed. We analyzed interrater reliability of diagnoses, scores on the National Institutes of Health Stroke Scale and treatment decisions (intravenous thrombolysis) between the MSU neurologist and the remote neurologist. We included 90 of 103 emergency assessments (13 patients were excluded because of either failed connection, technical problems, clinical worsening during teleconsultation, or missing data in documentation) in this study. The remote neurologist rated audiovisual quality with a median grade for audio quality of 3 (satisfactory) and for video quality of 2 (good). Mean time for completion of teleconsultations was about 19±5 minutes. The interrater reliabilities between the onboard and remote neurologist were high for diagnoses (Cohen's κ=0.86), National Institutes of Health Stroke Scale sum scores (intraclass correlation coefficient, 0.87) and treatment decisions (16 treatment decisions agreed versus 2 disagreed; Cohen's κ=0.93). Conclusions Remote assessment and treatment decisions of emergency patients are technically feasible with satisfactory audiovisual quality. Agreement on diagnoses, neurological examinations, and treatment decisions between onboard and remote neurologists was high.

Evaluation of a score for the prehospital distinction between cerebrovascular disease and stroke mimic patients.

BACKGROUND: Patients with a sudden onset of focal neurological deficits consistent with stroke, who turn out to have alternative conditions, have been labeled stroke mimics. AIMS: We assessed a recently validated telemedicine-based stroke mimic score (TeleStroke mimic score; TM-score) and individual patient characteristics with regard to its discriminative value between cerebrovascular disease and stroke mimic patients in the in-person, pre-hospital setting. METHODS: We evaluated patients cared for in a mobile stroke unit in Berlin, Germany. We investigated whether the TM-score (comprising six parameters), Face Arm Speech Time test, and individual patient characteristics were able to differentiate cerebrovascular disease from stroke mimic patients. RESULTS: We included 423 patients (299 (70.7%) cerebrovascular disease and 124 (29.3%) stroke mimic) in the final analysis. A TM-score > 30 indicated a high probability of a cerebrovascular disease and a score ≤15 of a stroke mimic. The TM-score performed well to identify stroke mimics (area under the curve of 0.74 under receiver-operating characteristic curve analysis). The cerebrovascular disease patients were older (74.8 vs. 69.8 years, p = 0.001), had more often severe strokes (NIHSS > 14 25.8% vs. 11.3%, p = 0.001), presented more often with weakness of the face (70.9% vs. 42.7%, p = 0.001) or arm (60.9% vs. 33.9%, p = 0.001), dysarthria (59.5% vs. 40.3%, p < 0.001), history of atrial fibrillation (38.1% vs. 21.0%, p = 0.001), arterial hypertension (78.9% vs. 53.2%, p < 0.001), and less often with seizure (0.7% vs. 21.0%, p < 0.001). CONCLUSIONS: The TM-score and certain patient characteristics can help paramedics and emergency physicians in the field to identify stroke mimic patients and select the most appropriate hospital destination.

Ultraearly Intravenous Thrombolysis for Acute Ischemic Stroke in Mobile Stroke Unit and Hospital Settings.

Background and Purpose- Mobile stroke units (MSUs) are known to increase the proportion of acute ischemic stroke (AIS) patients treated with intravenous thrombolysis (IVT) in the first golden hour (GH) after onset compared with hospital settings (HS). However, because of the low number of AIS patients treated with intravenous thrombolysis within this ultraearly time window in conventional care, characteristics, and outcome of this subgroup of AIS patients have not been compared between MSU and HS. Methods- MSU-GH patients were selected from the Berlin-based MSU (STEMO [Stroke Emergency Mobile]), whereas HS-GH patients were selected from the SITS-EAST (Safe Implementation of Treatments in Stroke-East) registry. The outcome events of interest included the rates of favorable functional outcome (modified Rankin Scale scores of 0 or 1), distribution of the modified Rankin Scale scores, and mortality after 3 months between MSU-GH and HS-GH groups. Results- We identified 117 MSU-GH (38.4% of 305 MSU-treated patients) and 136 HS-GH (0.9% of 15 591 HS-treated patients) eligible patients without prestroke disability. No significant differences were documented in the rates of favorable functional outcome (51.3% versus 46.2%, P=0.487) and mortality (7.7% versus 9.9%, P=0.576) at 3 months, or in the distribution of 3-month modified Rankin Scale scores between the 2 groups ( P=0.196). In multivariable logistic regression analyses, adjusting for potential confounders, MSU treatment was not associated with a significantly different likelihood of favorable functional outcome (odds ratio, 1.84 for MSU patients; 95% CI, 0.86-3.96) or mortality (odds ratio, 0.95; 95% CI, 0.28-3.20) at 3 months. Conclusions- There is no evidence that safety and efficacy of ultraearly intravenous thrombolysis for AIS differs when used in MSUs or in HS.

Effects of Prehospital Thrombolysis in Stroke Patients With Prestroke Dependency.

BACKGROUND AND PURPOSE: Data on effects of intravenous thrombolysis on outcome of patients with ischemic stroke who are dependent on assistance in activities of daily living prestroke are scarce. Recent registry based analyses in activities of daily -independent patients suggest that earlier start of intravenous thrombolysis in the prehospital setting leads to better outcomes when compared with the treatment start in hospital. We evaluated whether these observations can be corroborated in patients with prestroke dependency. METHODS: This observational, retrospective analysis included all patients with acute ischemic stroke depending on assistance before stroke who received intravenous thrombolysis either on the Stroke Emergency Mobile (STEMO) or through conventional in-hospital care (CC) in a tertiary stroke center (Charité, Campus Benjamin Franklin, Berlin) during routine care. Prespecified outcomes were modified Rankin Scale scores of 0 to 3 and survival at 3 months, as well as symptomatic intracranial hemorrhage. Outcomes were adjusted in multivariable logistic regression. RESULTS: Between February 2011 and March 2015, 122 of 427 patients (28%) treated on STEMO and 142 of 505 patients (28%) treated via CC needed assistance before stroke. Median onset-to-treatment times were 97 (interquartile range, 69-159; STEMO) and 135 (interquartile range, 98-184; CC; P<0.001) minutes. After 3 months, modified Rankin Scale scores of 0 to 3 was observed in 48 STEMO patients (39%) versus 35 CC patients (25%; P=0.01) and 86 (70%, STEMO) versus 85 (60%, CC) patients were alive (P=0.07). After adjustment, STEMO care was favorable with respect to modified Rankin Scale scores of 0 to 3 (odds ratio, 1.99; 95% confidence interval, 1.02-3.87; P=0.042) with a nonsignificant result for survival (odds ratio, 1.73; 95% confidence interval, 0.95-3.16; P=0.07). Symptomatic intracranial hemorrhage occurred in 5 STEMO versus 12 CC patients (4.2% versus 8.5%; P=0.167). CONCLUSIONS: The results of this study suggest that earlier, prehospital (as compared with in-hospital) start of intravenous thrombolysis in acute ischemic stroke may translate into better clinical outcome in patients with prestroke dependency. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02358772.

Exogenous growth factors bFGF, EGF and HGF do not influence viability and phenotype of V600EBRAF melanoma cells and their response to vemurafenib and trametinib in vitro.

It has been shown that the response of V600EBRAF melanoma cells to targeted therapeutics is affected by growth factors. We have investigated the influence of three different growth factors, bFGF, EGF and HGF used either alone or in combination, on the response of V600EBRAF melanoma cell populations established from surgical specimens to vemurafenib and trametinib, targeting V600EBRAF and MEK1/2, respectively. We report that proliferation and phenotype of V600EBRAF melanoma cell populations were not detectably influenced by exogenous growth factors. Neither cell distribution in cell cycle and CCND1 expression nor activity of signaling pathways crucial for melanoma development and maintenance, including the RAF/MEK/ERK pathway, WNT/ß-catenin pathway and NF-κB signaling, were affected by the presence of different growth factors. We furthermore show that vemurafenib and trametinib abrogated the activity of ERK1/2, arrested cells in G0/G1 cell cycle phase, triggered apoptosis, induced changes in the expression of CXCL8, CCND1 and CTGF and the frequency of Ki-67high and CD271high cells. These effects were, however, similar in the presence of different growth factors. Interestingly, comparable results were also obtained for melanoma cells grown without exogenous growth factors bFGF, EGF and HGF for a period as long as 4 months prior the drug treatment. We conclude that the composition or lack of exogenous growth factors bFGF, EGF and HGF do not markedly influence viability and phenotype of V600EBRAF melanoma cells and their response to vemurafenib and trametinib in vitro. Our results question the necessity of these growth factors in the medium that is used for culturing V600EBRAF melanoma cells.

Vemurafenib and trametinib reduce expression of CTGF and IL-8 in V600EBRAF melanoma cells.

Clinical evidence has revealed that while RAS/RAF/MEK/ERK pathway is a crucial component of melanomagenesis, other signaling pathways can also contribute to the malignant growth and development of resistance to targeted therapies. We explored the response of V600EBRAF melanoma cells derived from surgical specimens and grown in stem cell medium to vemurafenib and trametinib, drugs targeting the activity of V600EBRAF and MEK1/2, respectively. Cell growth and apoptosis were monitored by real-time imaging system, immunophenotype and cell cycle by flow cytometry, gene expression by quantitative real-time PCR, immunoblotting and enzyme-linked immunosorbent assay. The V600EBRAF melanoma cell populations were diverse. Differences in morphology, pigmentation, cell cycle profiles, and immunophenotype were observed. At the molecular level, melanoma cells differed in the phosphorylation of ERK1/2, NF-κB, and ß-catenin, and expression of several relevant genes, including MITF-M, DKK1, CCND1, BRAF, CXCL8, and CTGF. Despite having different characteristics, melanoma cells responded similarly to vemurafenib and trametinib. Both drugs reduced ERK1/2 phosphorylation and percentages of cells expressing Ki-67 at high level, inhibited expression of CCND1 and induced cell cycle arrest in the Go/G1 phase. These expected cytostatic effects were accompanied by increased CD271 expression, a marker of stem-like cells. NF-κB activity was reduced by both drugs, however, not completely abolished, whereas the level of active ß-catenin was increased by drugs in three out of six cell populations. Interestingly, expression of IL-8 and CTGF was significantly reduced by treatment with vemurafenib and trametinib. Simultaneous inhibition of NF-κB activity and induction of ERK1/2 phosphorylation revealed that CTGF expression depends on ERK1/2 activity but not on NF-κB activity. Both, the positive effects of treatment with vemurafenib and trametinib such as the newly identified CTGF suppression and undesired effects such as increased CD271 expression suggesting selection of melanoma stem-like cells should be considered in the development of combination treatment for melanoma patients.

Glial Fibrillary Acidic Protein for Prehospital Diagnosis of Intracerebral Hemorrhage.

BACKGROUND: Both, acute ischemic stroke (AIS) and hemorrhage stroke (intracerebral hemorrhage, ICH) require early attention but different treatment strategies. Plasma glial fibrillary acidic protein (GFAP) levels were found to be elevated in ICH patients after they arrived in the hospital. Because treatment options differed, we sought to determine whether GFAP can be used to accurately differentiate between of AIS and ICH in the prehospital setting. METHODS: We assessed acute stroke patients in the Stroke Emergency Mobile (STEMO). STEMO is a stroke ambulance staffed by a specialized team including a neurologist and equipped with a computed tomography scanner plus a point-of-care laboratory. The STEMO ambulance is integrated in the emergency medical system of Berlin, Germany. Following prehospital stroke diagnosis, blood was drawn and subsequently analysed using research assays from Roche diagnostics. The clinical accuracy of plasma GFAP was tested using a cut-off value of 0.29 ng/ml. RESULTS: Blood samples of 74 patients were analysed. Twenty-five patients had ICH (mean age 69 ± 11 years, median National Institutes of Health Stroke Scale (NIHSS) 15) and 49 IS (mean age 75 ± 10 years, median NIHSS 6). Nine ICH (0 IS patients) had GFAP-levels above 0.29 ng/ml. The sensitivity and specificity of GFAP for differentiating between ICH and AIS were 36.0 and 100%. The sensitivity for ICH volume >15 ml was 61.5%. ICH patients without GFAP elevation had significantly smaller hemorrhage volumes (median 4.5 vs. 37.6 ml, p = 0.004) and were less likely to deteriorate (19 vs. 56%, p = 0.087). CONCLUSIONS: GFAP levels >0.29 ng/ml were seen only in ICH, thus confirming the diagnosis of ICH during prehospital care. However, sensitivity is low particularly in smaller hemorrhages.

S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization.

Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.

Functional outcomes of pre-hospital thrombolysis in a mobile stroke treatment unit compared with conventional care: an observational registry study.

BACKGROUND: Specialised CT-equipped mobile stroke treatment units shorten time to intravenous thrombolysis in acute ischaemic stroke by starting treatment before hospital admission; however, direct effects of pre-hospital thrombolysis on clinical outcomes have not been shown. We aimed to compare 3-month functional outcomes after intravenous thrombolysis in patients with acute ischaemic who had received emergency mobile care or and conventional care. METHODS: In this observational registry study, patients with ischaemic stroke received intravenous thrombolysis (alteplase) either within a stroke emergency mobile (STEMO) vehicle (pre-hospital care covering 1·3 million inhabitants of Berlin) or within conventional care (normal ambulances and in-hospital care at the Charité Campus Benjamin Franklin in Berlin). Patient data on treatment, outcome, and demographics were documented in STEMO (pre-hospital) or conventional care (in-hospital) registries. The primary outcome was the proportion of patients who had lived at home without assistance before stroke and had a 3-month modified Rankin Scale (mRS) score of 1 or lower. Our multivariable logistic regression was adjusted for demographics, comorbidities, and stroke severity. This study is registered with ClinicalTrials.gov, number NCT02358772. FINDINGS: Between Feb 5, 2011, and March 5, 2015, 427 patients were treated within the STEMO vehicle and their data were entered into a pre-hospital registry. 505 patients received conventional care and their data were entered into an in-hospital thrombolysis registry. Of these, 305 patients in the STEMO group and 353 in the conventional care group met inclusion criteria and were included in the analysis. 161 (53%) patients in the STEMO group versus 166 (47%) in the conventional care group had an mRS score of 1 or lower (p=0·14). Compared with conventional care, adjusted odds ratios (ORs) for STEMO care for the primary outcome (OR 1·40, 95% CI 1·00-1·97; p=0·052) were not significant. Intracranial haemorrhage (p=0·27) and 7-day mortality (p=0·23) did not differ significantly between treatment groups. INTERPRETATION: We found no significant difference between the proportion of patients with a mRS score of 1 or lower receiving STEMO care compared with conventional care. However, our results suggest that pre-hospital start of intravenous thrombolysis might lead to improved functional outcome in patients. This evidence requires substantiation in future large-scale trials. FUNDING: Zukunftsfonds Berlin, the Technology Foundation Berlin with EU co-financing by the European Regional Development Fund via Investitionsbank Berlin, and the German Federal Ministry for Education and Research via the Center for Stroke Research Berlin.

Influence of Distance to Scene on Time to Thrombolysis in a Specialized Stroke Ambulance.

BACKGROUND AND PURPOSE: Specialized computed tomography-equipped stroke ambulances shorten time to intravenous thrombolysis in acute ischemic stroke by starting treatment before hospital arrival. Because of longer travel-time-to-scene, time benefits of this concept are expected to diminish with longer distances from base station to scene. METHODS: We used data from the Prehospital Acute Neurological Treatment and Optimization of Medical Cares in Stroke (PHANTOM-S) trial comparing time intervals between patients for whom a specialized stroke ambulance (stroke emergency mobile) was deployed and patients with conventional emergency medical service. Expected times from base station to scene had been calculated beforehand using computer algorithms informed by emergency medical service routine data. Four different deployment zones with-75% probability-expected arrival within 4, 8, 12, and 16 minutes and total population coverage of ≈1.3 million inhabitants were categorized for stroke emergency mobile deployment. We analyzed times from alarm-to-arrival at scene, to start of intravenous thrombolysis and from onset-to-intravenous thrombolysis. RESULTS: Corresponding to the size of the respective catchment zone, the number of patients cared increased with distance (zone 1: n=30, zone 2: n=127, zone 3: n=156, and zone 4: n=217). Although time to stroke emergency mobile arrival increased with distance (mean: 8.0, 12.5, 15.4, and 18.4 minutes in zones 1-4), time from alarm-to-intravenous thrombolysis (mean: 41.8 versus 76.5; 50.2 versus 79.1; 54.5 versus 76.6; and 59.3 versus 78.0 minutes, respectively; all P<0.01) remained shorter in the stroke emergency mobile group across all zones. CONCLUSIONS: In a metropolitan area such as Berlin, time benefits justify a specialized stroke ambulance service up to a mean travel time of 18 minutes from base station. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01382862.

[Not Available]. / Mieszanki ziolowe o dzialaniu przeciwgoraczkowym jako przyklady tradycyjnego leku roslinnego stosowanego w populacji pediatrycznej.

Herbal medicines with antipyretic action have a long tradition of use in Polish phytotherapy. Over the years, they have proven both their efficacy and safety in a feverishness, which very often have accompanied the upper respiratory tract infections in children. Leading active ingredient of these products is the willow bark (Cortex salicis), which has been the active ingredient in both, the herbal mixture Pyrosan placed on the market in the 50's of last century and Pyrosal syrup marketed in the year 1989. Other herbal substances used in such preparations are elder flower, lime flower and meadowsweet flower. The usage of a pharmaceutical form of syrup is prerequisite when the main group of the patients are children. Such products are continuously in use for nearly 30 years in Poland.

Copeptin Levels in Patients With Acute Ischemic Stroke and Stroke Mimics.

BACKGROUND AND PURPOSE: Copeptin levels are increased in patients diagnosed with stroke and other vascular diseases. Copeptin elevation is associated with adverse outcome, predicts re-events in patients with transient ischemic attack and is used in ruling-out acute myocardial infarction. We evaluated whether copeptin can also be used as a diagnostic marker in the prehospital stroke setting. METHODS: We prospectively examined patients with suspected stroke on the Stroke Emergency Mobile-an ambulance that is equipped with computed tomography and point-of-care laboratory. A blood sample was taken from patients immediately after arrival. We analyzed copeptin levels in patients with final hospital-based diagnosis of stroke or stroke mimics as well as in vascular or nonvascular patients. In addition, we examined the associations of symptom onset with copeptin levels and the prognostic value of copeptin in patients with stroke. RESULTS: Blood samples of 561 patients were analyzed. No significant differences were seen neither between cerebrovascular (n=383) and other neurological (stroke mimic; n=90) patients (P=0.15) nor between vascular (n=391) and nonvascular patients (n=170; P=0.57). We could not detect a relationship between copeptin levels and time from onset to blood draw. Three-month survival status was available in 159 patients with ischemic stroke. Copeptin levels in nonsurviving patients (n=8: median [interquartile range], 27.4 [20.2-54.7] pmol/L) were significantly higher than in surviving patients (n=151: median [interquartile range], 11.7 [5.2-30.9] pmol/L; P=0.024). CONCLUSIONS: In the prehospital setting, copeptin is neither appropriate to discriminate between stroke and stroke mimic patients nor between vascular and nonvascular patients. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01382862. The Pre-Hospital Acute Neurological Therapy and Optimization of Medical Care in Stroke Patients study (PHANTOM-S) was registered (NCT01382862). This sub-study was observational and not registered separately, therefore.