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Background: The “1-3-6-12-day rule” for starting direct oral anticoagulants (DOACs) in patients with nonvalvular atrial fibrillation after acute ischemic stroke or transient ischemic attack recommends timings that may be later than used in clinical practice. We investigated more practical optimal timing of DOAC initiation according to stroke severity. Methods: The combined data of prospective registries in Japan, Stroke Acute Management with Urgent Risk-factor Assessment and Improvement-nonvalvular atrial fibrillation (September 2011 to March 2014) and RELAXED (February 2014 to April 2016) were used. Patients were divided into transient ischemic attack and 3 stroke subgroups by the National Institutes of Health Stroke Scale score: mild (0–7), moderate (8–15), and severe (≥16). The early treatment group was defined as patients starting DOACs earlier than the median initiation day in each subgroup. Outcomes included a composite of recurrent stroke or systemic embolism, ischemic stroke, and severe bleeding within 90 days. Six European prospective registries were used for validation. Results: In the 1797 derivation cohort patients, DOACs were started at median 2 days after transient ischemic attack and 3, 4, and 5 days after mild, moderate, and severe strokes, respectively. Stroke or systemic embolism was less common in Early Group (n=785)—initiating DOACS within 1, 2, 3, and 4 days, respectively—than Late Group (n=1012) (1.9% versus 3.9%; adjusted hazard ratio, 0.50 [95% CI, 0.27–0.89]), as was ischemic stroke (1.7% versus 3.2%, 0.54 [0.27–0.999]). Major bleeding was similarly common in the 2 groups (0.8% versus 1.0%). On validation, both ischemic stroke (2.4% versus 2.2%) and intracranial hemorrhage (0.2% versus 0.6%) were similarly common in Early (n=547) and Late (n=1483) Groups defined using derivation data. Conclusions: In Japanese and European populations, early DOAC initiation within 1, 2, 3, or 4 days according to stroke severity seemed to be feasible to decrease the risk of recurrent stroke or systemic embolism and no increase in major bleeding. These findings support ongoing randomized trials to better establish the optimal timing of DOAC initiation.

Dimethylsulfoniopropionate (DMSP) is an abundant marine organosulfur compound[1] with roles in stress protection[2, 3], chemotaxis[4], nutrient and sulfur cycling[5] and, potentially, climate regulation[6, 7]. Marine algae and bacteria are considered significant DMSP producers, but many diverse representatives lack known DMSP synthesis genes/enzymes[8, 9]. Here, new DMSP biosynthesis enzymes were identified that considerably increase the number and diversity of potential DMSP-producing organisms, inferring new and significant global DMSP producers. A novel bifunctional DMSP biosynthesis enzyme, DsyGD, identified in the rhizobacterium Gynuella sunshinyii, produces DMSP at levels higher than any other bacterium from methylthiohydroxybutyrate (MTHB) via an N-terminal MTHB S-methyltransferase domain (termed DsyG) and a C-terminal dimethylsulfoniohydroxybutyrate (DMSHB) decarboxylase domain (termed DsyD, which is the first reported enzyme with this activity). DsyGD is also found in some filamentous cyanobacteria, not previously known to produce DMSP. Regulation of DMSP production and dsyGD transcription was consistent with their role in osmoprotection. Indeed, cloned dsyGD conferred osmotolerance to bacteria deficient in osmolyte production, something not previously demonstrated for any known DMSP synthesis gene, and which could be exploited for biotechnology e.g., engineering salt tolerance. DsyGD characterisation led to identification of phylogenetically distinct DsyG-like proteins, termed DSYE, with MTHB S-methyltransferase activity, in diverse and environmentally abundant Chlorophyta, Chlorachniophyta, Ochraphyta, Haptophyta and Bacillariophyta algae. These algae comprise a mix of low, high and previously unknown DMSP producers[10]. Algae containing DSYE, particularly bloom-forming Pelagophyceae species, which we showed to accumulate medium-high intracellular DMSP levels, were globally more abundant DMSP producers than Haptophyta, Dinophyta and Bacillariophyta with DSYB and/or TpMMT. This highlights the potential importance of Pelagophyceae and other DSYE containing algae in global DMSP production and sulfur cycling.

Dimethylsulfoxonium propionate (DMSOP) is a recently identified and abundant marine organosulfur compound with purported roles in oxidative stress protection, global carbon and sulfur cycling1. Diverse algae and bacteria synthesise DMSOP from dimethylsulfoniopropionate (DMSP), which potentially limits the production of climate-active gases e.g., dimethylsulfide (DMS) generated from microbial DMSP cleavage1. Here, DMSOP was found at mM levels in saltmarsh sediment, >10-fold higher than DMSP, and orders of magnitude higher than DMSOP levels previously reported in seawater1. Moreover, we showed bacteria could utilise DMSOP as an osmoprotectant. Some bacteria also cleave DMSOP liberating dimethyl sulfoxide (DMSO), an ubiquitous marine metabolite and acrylate1, but the enzymes responsible and their environmental importance were unknown. Here, we elucidated the DMSOP cleavage mechanism/s in diverse heterotrophic bacteria, e.g., SAR11 clade and Roseobacters, and fungi and phototrophic algae, e.g., Emiliania huxleyi, not previously known to have this activity. All these diverse organisms utilised their DMSP lyase ‘Ddd’ or ‘Alma1’ enzymes, that span five protein families, to cleave DMSOP with similar specific activities to DMSP. Bacteria with DMSP lyases that used DMSP as a carbon source likewise used DMSOP. Furthermore, ddd gene transcription in these bacteria was induced by DMSOP, including dddK in SAR11 strain HTCC1062. We determined the structure of DddK bound to DMSOP and concluded that the catalytic mechanisms of DMSOP cleavage by Ddd enzymes were like those for DMSP. Given the predicted teragram DMSOP production budget1, its newly found abundance in marine sediments, and the abundance of microbial DMSP lyase genes and transcripts in marine environments, DMSOP cleavage is likely a globally significant process influencing global carbon and sulfur fluxes and marine ecological interactions.

Despite extensive use of micelles in materials and colloidal science, their supramolecular organization as well as host-guest interactions within these dynamic assemblies are poorly understood. Small guest molecules in the presence of micelles undergo constant exchange between a micellar aggregate and the surrounding solution, posing a considerable challenge for their molecular level characterisation. In this work we reveal the interaction maps between small guest molecules and surfactants forming micelles via novel applications of NMR techniques supported with state-of-the-art analytical methods used in colloidal science. Model micelles composed of structurally distinct surfactants (block non-ionic polymer Pluronic® F-127, non-ionic surfactant Tween 20 or Tween 80 and ionic surfactant SLS, sodium lauryl sulphate) were selected and loaded with model small molecules of biochemical relevance (i.e. the drugs fluconazole, FLU or indomethacin, IMC) known to have different partition coefficients. Molecular level organization of FLU or IMC within hydrophilic and hydrophobic domains of micellar aggregates was established using combination of NMR methods (1D 1H NMR, 1D 19F NMR, 2D 1H-1H NOESY and 2D 1H-19F HOESY, and the multifrequency-STD NMR) and corroborated with molecular dynamics (MD) simulations. This is the first application of multifrequency-STD NMR to colloidal systems, enabling us to elucidate intricately detailed patterns of drug/micelle interactions in a single NMR experiment within minutes. Importantly, our results indicate that flexible surfactants, such as block copolymers and polysorbates, form micellar aggregates with a surface composed of both hydrophilic and hydrophobic domains and do not follow the classical core-shell model of the micelle. We propose that the magnitude of the changes in 1H chemical shifts corroborated with interaction maps obtained from DEEP-STD NMR and 2D NMR experiments can be used as an indicator of the strength of the guest-surfactant interactions. This NMR toolbox can be adopted for the analysis of broad range colloidal host-guest systems from soft materials to biological systems.

Background and Purpose: Clinical trials on stroke prevention in patients with atrial fibrillation have consistently shown clinical benefit from either warfarin or non–vitamin K antagonist oral anticoagulants (NOACs). NOAC-treated patients have consistently reported to be at lower risk for intracerebral hemorrhage (ICH) than warfarin-treated patients. The aims of this prospective, multicenter, multinational, unmatched, case-control study were (1) to investigate for risk factors that could predict ICH occurring in patients with atrial fibrillation during NOAC treatment and (2) to evaluate the role of CHA 2 DS 2 -VASc and HAS-BLED scores in the same setting. Methods: Cases were consecutive patients with atrial fibrillation who had ICH during NOAC treatment. Controls were consecutive patients with atrial fibrillation who did not have ICH during NOAC treatment. As within the CHA 2 DS 2 -VASc and HAS-BLED scores there are some risk factors in common, several multivariable logistic regression models were performed to identify independent prespecified predictors for ICH events. Results: Four hundred nineteen cases (mean age, 78.8±8.1 years) and 1526 controls (mean age, 76.0±10.3 years) were included in the study. From the different models performed, independent predictors of ICH were increasing age, concomitant use of antiplatelet agents, active malignancy, high risk of fall, hyperlipidemia, low clearance of creatinine, peripheral artery disease, and white matter changes. Low doses of NOACs (given according to label or not) and congestive heart failure were inversely associated with the risk of ICH. HAS-BLED and CHA 2 DS 2 -VASc scores performed poorly in predicting ICH with areas under the curves of 0.496 (95% CI, 0.468–0.525) and 0.530 (95% CI, 0.500–0.560), respectively. Conclusions: Several risk factors were associated to ICH in patients treated with NOACs for stroke prevention but not HAS-BLED and CHA 2 DS 2 -VASc scores.

Weak protein–ligand interactions have been demonstrated to play key roles in biological processes, particularly in those involving quick cellular responses after certain stimuli (e.g. signal transduction). Although powerful biophysical techniques are available to gain high-resolution structural information of protein–ligand complexes of high affinity, NMR spectroscopy has been demonstrated to stand out among them for protein–ligand studies within the limit of weak affinity. In particular, ligand-based NMR techniques allow the detection and quantification of weak biomolecular binding processes, where saturation transfer difference (STD) NMR techniques have demonstrated through the years their strong ability to not only detect binding processes but also to provide structural information about the ligand mode of binding in the receptor-binding pocket, a highly valuable piece of information for the further development of enhanced binders along the process of drug discovery. This is particularly useful in fragment-based drug-discovery approaches, where the binding of the identified initial small fragments tends to fall within the low affinity range of the spectrum. In this chapter we briefly introduce the different classical STD NMR approaches, and later focus in detail on novel developments based on multi-frequency STD NMR experiments, which expand further the analytical capabilities of STD NMR, exemplified by the DEEP-STD NMR protocol, by providing also some key information on the nature of the protein residues in contact with the ligands in the bond state.

Introduction The aim of this study in patients with acute posterior ischaemic stroke (PS) and atrial fibrillation (AF) was to evaluate (1) the risks of recurrent ischaemic event and severe bleeding and (2) these risks in relation with oral anticoagulant therapy (OAT) and its timing. Materials and Methods Patients with PS were prospectively included; the outcome events of these patients were compared with those of patients with anterior stroke (AS) which were taken from previous registries. The primary outcome was the composite of stroke recurrence, transient ischaemic attack, symptomatic systemic embolism, symptomatic cerebral bleeding and major extracranial bleeding occurring within 90 days from acute stroke. Results A total of 2470 patients were available for the analysis: 473 (19.1%) with PS and 1997 (80.9%) with AS. Over 90 days, 213 (8.6%) primary outcome events were recorded: 175 (8.7%) in patients with AS and 38 (8.0%) in those with PS. In patients who initiated OAT within 2 days, the primary outcome occurred in 5 out of 95 patients (5.3%) with PS compared to 21 out of 373 patients (4.3%) with AS (OR 1.07; 95% CI 0.39–2.94). In patients who initiated OAT between days 3 and 7, the primary outcome occurred in 3 out of 103 patients (2.9%) with PS compared to 26 out of 490 patients (5.3%) with AS (OR 0.54; 95% CI 0.16–1.80). Discussion our findings suggest that, when deciding the time to initiate oral anticoagulation, the location of stroke, either anterior or posterior, does not predict the risk of outcome events. Conclusions Patients with PS or AS and AF appear to have similar risks of ischaemic or haemorrhagic events at 90 days with no difference concerning the timing of initiation of OAT.

Background: In patients with atrial fibrillation who suffered an ischemic stroke while on treatment with nonvitamin K antagonist oral anticoagulants, rates and determinants of recurrent ischemic events and major bleedings remain uncertain. Methods: This prospective multicenter observational study aimed to estimate the rates of ischemic and bleeding events and their determinants in the follow-up of consecutive patients with atrial fibrillation who suffered an acute cerebrovascular ischemic event while on nonvitamin K antagonist oral anticoagulant treatment. Afterwards, we compared the estimated risks of ischemic and bleeding events between the patients in whom anticoagulant therapy was changed to those who continued the original treatment. Results: After a mean follow-up time of 15.0±10.9 months, 192 out of 1240 patients (15.5%) had 207 ischemic or bleeding events corresponding to an annual rate of 13.4%. Among the events, 111 were ischemic strokes, 15 systemic embolisms, 24 intracranial bleedings, and 57 major extracranial bleedings. Predictive factors of recurrent ischemic events (strokes and systemic embolisms) included CHA 2 DS 2 -VASc score after the index event (odds ratio [OR], 1.2 [95% CI, 1.0–1.3] for each point increase; P =0.05) and hypertension (OR, 2.3 [95% CI, 1.0–5.1]; P =0.04). Predictive factors of bleeding events (intracranial and major extracranial bleedings) included age (OR, 1.1 [95% CI, 1.0–1.2] for each year increase; P =0.002), history of major bleeding (OR, 6.9 [95% CI, 3.4–14.2]; P =0.0001) and the concomitant administration of an antiplatelet agent (OR, 2.8 [95% CI, 1.4–5.5]; P =0.003). Rates of ischemic and bleeding events were no different in patients who changed or not changed the original nonvitamin K antagonist oral anticoagulants treatment (OR, 1.2 [95% CI, 0.8–1.7]). Conclusions: Patients suffering a stroke despite being on nonvitamin K antagonist oral anticoagulant therapy are at high risk of recurrent ischemic stroke and bleeding. In these patients, further research is needed to improve secondary prevention by investigating the mechanisms of recurrent ischemic stroke and bleeding.

Background and Purpose— Despite treatment with oral anticoagulants, patients with nonvalvular atrial fibrillation (AF) may experience ischemic cerebrovascular events. The aims of this case-control study in patients with AF were to identify the pathogenesis of and the risk factors for cerebrovascular ischemic events occurring during non–vitamin K antagonist oral anticoagulants (NOACs) therapy for stroke prevention. Methods— Cases were consecutive patients with AF who had acute cerebrovascular ischemic events during NOAC treatment. Controls were consecutive patients with AF who did not have cerebrovascular events during NOACs treatment. Results— Overall, 713 cases (641 ischemic strokes and 72 transient ischemic attacks; median age, 80.0 years; interquartile range, 12; median National Institutes of Health Stroke Scale on admission, 6.0; interquartile range, 10) and 700 controls (median age, 72.0 years; interquartile range, 8) were included in the study. Recurrent stroke was classified as cardioembolic in 455 cases (63.9%) according to the A-S-C-O-D (A, atherosclerosis; S, small vessel disease; C, cardiac pathology; O, other causes; D, dissection) classification. On multivariable analysis, off-label low dose of NOACs (odds ratio [OR], 3.18; 95% CI, 1.95–5.85), atrial enlargement (OR, 6.64; 95% CI, 4.63–9.52), hyperlipidemia (OR, 2.40; 95% CI, 1.83–3.16), and CHA 2 DS 2 -VASc score (OR, 1.72 for each point increase; 95% CI, 1.58–1.88) were associated with ischemic events. Among the CHA 2 DS 2 -VASc components, age was older and presence of diabetes mellitus, congestive heart failure, and history of stroke or transient ischemic attack more common in patients who had acute cerebrovascular ischemic events. Paroxysmal AF was inversely associated with ischemic events (OR, 0.45; 95% CI, 0.33–0.61). Conclusions— In patients with AF treated with NOACs who had a cerebrovascular event, mostly but not exclusively of cardioembolic pathogenesis, off-label low dose, atrial enlargement, hyperlipidemia, and high CHA 2 DS 2 -VASc score were associated with increased risk of cerebrovascular events.

Background and Purpose— Bridging therapy with low-molecular-weight heparin reportedly leads to a worse outcome for acute cardioembolic stroke patients because of a higher incidence of intracerebral bleeding. However, this practice is common in clinical settings. This observational study aimed to compare (1) the clinical profiles of patients receiving and not receiving bridging therapy, (2) overall group outcomes, and (3) outcomes according to the type of anticoagulant prescribed. Methods— We analyzed data of patients from the prospective RAF and RAF-NOACs studies. The primary outcome was defined as the composite of ischemic stroke, transient ischemic attack, systemic embolism, symptomatic cerebral bleeding, and major extracerebral bleeding observed at 90 days after the acute stroke. Results— Of 1810 patients who initiated oral anticoagulant therapy, 371 (20%) underwent bridging therapy with full-dose low-molecular-weight heparin. Older age and the presence of leukoaraiosis were inversely correlated with the use of bridging therapy. Forty-two bridged patients (11.3%) reached the combined outcome versus 72 (5.0%) of the nonbridged patients ( P =0.0001). At multivariable analysis, bridging therapy was associated with the composite end point (odds ratio, 2.3; 95% CI, 1.4–3.7; P P =0.005) and hemorrhagic (odds ratio, 2.4; 95% CI, 1.2–4.9; P =0.01) end points separately. Conclusions— Our findings suggest that patients receiving low-molecular-weight heparin have a higher risk of early ischemic recurrence and hemorrhagic transformation compared with nonbridged patients.

Introduction To describe clinical, neuroimaging, and laboratory features of a large cohort of Italian patients with reversible cerebral vasoconstriction syndrome. Methods In the setting of the multicenter Italian Project on Stroke at Young Age (IPSYS), we retrospectively enrolled patients with a diagnosis of definite reversible cerebral vasoconstriction syndrome according to the International Classification of Headache Disorders (ICHD)-3 beta criteria (6.7.3 Headache attributed to reversible cerebral vasoconstriction syndrome, imaging-proven). Clinical manifestations, neuroimaging, treatment, and clinical outcomes were evaluated in all patients. Characteristics of reversible cerebral vasoconstriction syndrome without typical causes (“idiopathic reversible cerebral vasoconstriction syndrome”) were compared with those of reversible cerebral vasoconstriction syndrome related to putative causative factors (“secondary reversible cerebral vasoconstriction syndrome”). Results A total of 102 patients (mean age, 47.2 ± 13.9 years; females, 85 [83.3%]) qualified for the analysis. Thunderclap headache at presentation was reported in 69 (67.6%) patients, and it typically recurred in 42 (60.9%). Compared to reversible cerebral vasoconstriction syndrome cases related to putative etiologic conditions (n = 21 [20.6%]), patients with idiopathic reversible cerebral vasoconstriction syndrome (n = 81 [79.4%]) were significantly older (49.2 ± 13.9 vs. 39.5 ± 11.4 years), had more frequently typical thunderclap headache (77.8% vs. 28.6%) and less frequently neurological complications (epileptic seizures, 11.1% vs. 38.1%; cerebral infarction, 6.1% vs. 33.3%), as well as concomitant reversible brain edema (25.9% vs. 47.6%). Conclusions Clinical manifestations and putative etiologies of reversible cerebral vasoconstriction syndrome in our series are slightly different from those observed in previous cohorts. This variability might be partly related to the coexistence of precipitating conditions with a putative etiologic role on disease occurrence.

Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms.

The ability to custom-modify cell surface glycans holds great promise for treatment of a variety of diseases. We propose a glycomimetic ofl-fucose that markedly inhibits the creation of sLeXby FTVI and FTVII, but has no effect on creation of LeXby FTIX. Our findings thus indicate that selective suppression of sLex display can be achieved, and STD-NMR studies surprisingly reveal that the mimetic does not compete with GDP-fucose at the enzymatic binding site.

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-l-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-l-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1–3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3′-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.

We have screened small molecule libraries specifically for inhibitors that target WWP2, an E3 ubiquitin ligase associated with tumour outgrowth and spread. Selected hits demonstrated dose‐dependent WWP2 inhibition, low micromolar IC50 values, and inhibition of PTEN substrate‐specific ubiquitination. Binding to WWP2 was confirmed by ligand‐based NMR spectroscopy. Furthermore, we used a combination of STD NMR, the recently developed DEEP‐STD NMR approach, and docking calculations, to propose for the first time an NMR‐validated 3D molecular model of a WWP2‐inhibitor complex. These first generation WWP2 inhibitors provide a molecular framework for informing organic synthetic approaches to improve activity and selectivity.

Objective:\ud It is not known whether patients with atrial fibrillation (AF) with ischemic stroke despite oral anticoagulant therapy are at increased risk for further recurrent strokes or how ongoing secondary prevention should be managed.\ud \ud Methods:\ud We conducted an individual patient data pooled analysis of 7 prospective cohort studies that recruited patients with AF and recent cerebral ischemia. We compared patients taking oral anticoagulants (vitamin K antagonists [VKA] or direct oral anticoagulants [DOAC]) prior to index event (OACprior ) with those without prior oral anticoagulation (OACnaive ). We further compared those who changed the type (ie, from VKA or DOAC, vice versa, or DOAC to DOAC) of anticoagulation (OACchanged ) with those who continued the same anticoagulation as secondary prevention (OACunchanged ). Time to recurrent acute ischemic stroke (AIS) was analyzed using multivariate competing risk Fine-Gray models to calculate hazard ratios (HRs) and 95% confidence intervals (CIs).\ud \ud Results:\ud We included 5,413 patients (median age = 78 years [interquartile range (IQR) = 71-84 years]; 5,136 [96.7%] had ischemic stroke as the index event, median National Institutes of Health Stroke Scale on admission = 6 [IQR = 2-12]). The median CHA2 DS2 -Vasc score (congestive heart failure, hypertension, age≥ 75 years, diabetes mellitus, stroke/transient ischemic attack, vascular disease, age 65-74 years, sex category) was 5 (IQR = 4-6) and was similar for OACprior (n = 1,195) and OACnaive (n = 4,119, p = 0.103). During 6,128 patient-years of follow-up, 289 patients had AIS (4.7% per year, 95% CI = 4.2-5.3%). OACprior was associated with an increased risk of AIS (HR = 1.6, 95% CI = 1.2-2.3, p = 0.005). OACchanged (n = 307) was not associated with decreased risk of AIS (HR = 1.2, 95% CI = 0.7-2.1, p = 0.415) compared with OACunchanged (n = 585).\ud \ud Interpretation:\ud Patients with AF who have an ischemic stroke despite previous oral anticoagulation are at a higher risk for recurrent ischemic stroke despite a CHA2 DS2 -Vasc score similar to those without prior oral anticoagulation. Better prevention strategies are needed for this high-risk patient group.

STD NMR is a powerful ligand-based tool for screening small molecules and low molecular weight fragments for their interaction with a given macromolecule. Such information is invaluable both in the drug discovery sector and in understanding fundamental biological interactions. Recently, powerful methods have been developed to extract a greater wealth of information from the STD NMR experiment, including ligand binding epitopes, dissociation constant determination, and mapping of binding site properties. Herein we describe these STD NMR experiments, giving practical examples for each approach, and highlight the important parameters and common pitfalls that must be considered for a successful experiment.

STD NMR is a powerful ligand-based tool for screening small molecules and low molecular weight fragments for their interaction with a given macromolecule. Such information is invaluable both in the drug discovery sector and in understanding fundamental biological interactions. Recently, powerful methods have been developed to extract a greater wealth of information from the STD NMR experiment, including ligand binding epitopes, dissociation constant determination, and mapping of binding site properties. Herein we describe these STD NMR experiments, giving practical examples for each approach, and highlight the important parameters and common pitfalls that must be considered for a successful experiment.

Fluid biphasic systems are one of the most interesting dynamic systems in chemistry and biochemistry. In nuclear magnetic resonance (NMR) spectroscopy, the study of the solute dynamics across fluid biphasic systems requires the introduction of dedicated NMR methods, due to their intrinsic heterogeneity. Diffusion and spatially resolved NMR techniques represent a useful approach for dealing with the study of solutes in biphasic systems and have been applied lately with success. Nevertheless, other potential applications of NMR spectroscopy for biphasic systems remain to be explored. In this proof of-concept communication, we specifically aimed to investigate whether solute exchange between two immiscible phases can be followed by NMR experiments involving transfer of magnetization. To that aim, we have used spatially resolved saturation transfer difference NMR (SR-STD NMR) experiments to analyze solute exchange by transfer of saturation from one phase to the other in a biphasic system and have explored which are the underlying mechanisms leading to the transfer of magnetization between phases and the limits of the approach. We hereby demonstrate that SR-STD NMR is feasible and that it might be implemented in pharmacological screening for binders of biological receptors or in the study of chemical and biochemical reactions occurring at interfaces.

15 pags, 5 figs, The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. Here we report that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face Si mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens., This work was supported by Grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (NRF- 2016R1A2B2013305, 2016R1A5A1010764, 2014R1A4A1008625, 2017M3A9F6029755 and 2017R1A2B3006704), the Strategic Initiative for Microbiomes in Agriculture and Food funded by Ministry of Agriculture, Food and Rural Affairs (918012-4) and Brain Korea 21 PLUS Project for Medical Science. This work was also supported by Grants AI093913 and AI127973 from the National Institutes of Health (to P.R.H.). We also thank ARAID and MEC (CTQ2013-44367-C2-2-P, BFU2016-75633-P to R.H-G.), and the DGA (group number E34_R17) for financial support. S.M. acknowledges a postgraduate studentship from the School of Pharmacy of the University of East Anglia. J.A. and S W. acknowledge funding from BBSRC through a research grant (BB/P010660/1) and a DTP PhD studentship, respectively.

Background and Purpose: The aim of this study was to investigate for a possible association between both prestroke CHA(2)DS(2)-VASc score and the severity of stroke at presentation, as well as disability and mortality at 90 days, in patients with acute stroke and atrial fibrillation (AF). Methods: This prospective study enrolled consecutive patients with acute ischemic stroke, AF, and assessment of prestroke CHA2DS2-VASc score. Severity of stroke was assessed on admission using the National Institutes of Health Stroke Scale (NIHSS) score (severe stroke: NIHSS >= 10). Disability and mortality at 90 days were assessed by the modified Rankin Scale (mRS = 3). Multiple logistic regression was used to correlate prestroke CHA(2)DS(2)-VASc and severity of stroke, as well as disability and mortality at 90 days. Results: Of the 1020 patients included in the analysis, 606 patients had an admission NIHSS score lower and 414 patients higher than 10. At 90 days, 510 patients had mRS >= 3. A linear correlation was found between the prestroke CHA(2)DS(2)-VASc score and severity of stroke (P = .001). On multivariate analysis, CHA(2)DS(2)-VASc score correlated with severity of stroke (P = .041) and adverse functional outcome (mRS = 3) (P = .001). A logistic regression with the receiver operating characteristic graph procedure (C-statistics) evidenced an area under the curve of .60 (P = .0001) for severe stroke. Furthermore, a correlation was found between prestroke CHA(2)DS(2)-VASc score and lesion size. Conclusions: In patients with AF, in addition to the risk of stroke, a high CHA(2)DS(2)-VASc score was independently associated with both stroke severity at onset and disability and mortality at 90 days.

Embolic complications of intravenous thrombolysis for acute ischemic stroke are increasingly recognized, and to date several cases of myocardial infarction soon after the administration of alteplase have been reported. We describe a case of early anterior myocardial infarction during intravenous thrombolysis for stroke and review the current literature.

Background and Purposes— This study was designed to derive and validate a score to predict early ischemic events and major bleedings after an acute ischemic stroke in patients with atrial fibrillation. Methods— The derivation cohort consisted of 854 patients with acute ischemic stroke and atrial fibrillation included in prospective series between January 2012 and March 2014. Older age (hazard ratio 1.06 for each additional year; 95% confidence interval, 1.00–1.11) and severe atrial enlargement (hazard ratio, 2.05; 95% confidence interval, 1.08–2.87) were predictors for ischemic outcome events (stroke, transient ischemic attack, and systemic embolism) at 90 days from acute stroke. Small lesions (≤1.5 cm) were inversely correlated with both major bleeding (hazard ratio, 0.39; P =0.03) and ischemic outcome events (hazard ratio, 0.55; 95% confidence interval, 0.30–1.00). We assigned to age ≥80 years 2 points and between 70 and 79 years 1 point; ischemic index lesion >1.5 cm, 1 point; severe atrial enlargement, 1 point (ALESSA score). A logistic regression with the receiver-operating characteristic graph procedure (C statistic) showed an area under the curve of 0.697 (0.632–0.763; P =0.0001) for ischemic outcome events and 0.585 (0.493–0.678; P =0.10) for major bleedings. Results— The validation cohort consisted of 994 patients included in prospective series between April 2014 and June 2016. Logistic regression with the receiver-operating characteristic graph procedure showed an area under the curve of 0.646 (0.529–0.763; P =0.009) for ischemic outcome events and 0.407 (0.275–0.540; P =0.14) for hemorrhagic outcome events. Conclusions— In acute stroke patients with atrial fibrillation, high ALESSA scores were associated with a high risk of ischemic events but not of major bleedings.

Purpose: Acute basilar artery occlusion (BAO) is a devastating neurological condition associated with a poor clinical outcome and a high mortality rate. Mechanical thrombectomy using a retrievable stent applied shortly after symptom onset could increase a good functional outcome, improving survival rate in patients with acute BAO. First clinical trials using stent retrievers have shown promising high recanalization rates. This study aimed to evaluate the feasibility, safety and efficacy of mechanical thrombectomy. Material and Methods: Seven consecutive patients (2 female, 5 male) with a mean age of 59,4 (range 40-82) with acute BAO undergone to endovascular therapy: all patients were treated with mechanical thrombectomy using a retrievable stent (in 5 patients with Solitaire-Covidien system, in 2 patients Revive-Codman system); two of these received in addition a intra-arterial thrombolysis (IAT), one was additionally treated with intra-venous thrombolysis (IVT). Successful recanalization was defined as Thrombolysis in Cerebral Infarction (TICI) grade 2b or 3. Good outcome was defined as modified Rankin Scale (mRS) score of 0–2 at 3 months. Results: Median NIHSS score at onset was 15,8 (range 12-22).Median procedure time to maximal recanalization was 72 minutes (range 35-135).Recanalization was achieved in 100% (7/7) of patients. One symptomatic parenchymal hemorrhage occurred in a patient treated additionally with intra-arterial thrombolysis. Median NIHSS score at 7 days from treatment was 7,2 (range 0-20). At 3 months a good outcome (mRS 0–2) was observed in 71,4% (5/7); overall mortality at 3 months was 28,6% (2/7). Conclusion: Mechanical thrombectomy in BAO presents high recanalization rate, with a very low complication rate, improving good outcome and survival rates in patients with BAO.

BACKGROUND: The aim of our study was to evaluate in patients with acute ischemic stroke the relationship between immuno-inflammatory variables, clinical outcome and infarct site. MATERIALS AND METHODS: We evaluated plasma levels of IL-1beta, TNF-alpha, IL-6 and IL-10, E-selectin, P-selectin, sICAM-1 ,sVCAM-1 vWF, TPA and PAI-1. RESULTS: Patients with cardioembolic subtype showed significantly higher median plasma levels of TNF-alpha, IL-6, IL-1beta whereas the lacunar subtype showed significantly lower median plasma levels of TNF-alpha, IL-6 and IL-1beta. CONCLUSIONS: A significant association was noted between the severity of neurological deficit at admission, the diagnostic subtype and some inflammatory variables.

The aim of this study was to investigate for a possible association between both prestroke CHAThis prospective study enrolled consecutive patients with acute ischemic stroke, AF, and assessment of prestroke CHAOf the 1020 patients included in the analysis, 606 patients had an admission NIHSS score lower and 414 patients higher than 10. At 90 days, 510 patients had mRS ≥3. A linear correlation was found between the prestroke CHAIn patients with AF, in addition to the risk of stroke, a high CHA

Fish is a common cause of food allergy. The reactions usually occur after its ingestion. In most immunoglobulin E-mediated reactions, the allergens are gastroresistant and heat-stable proteins of low molecularweight (parvalbumin). On the other hand, isolated contact urticaria following the handling of raw fish but without symptoms after its ingestion was found among cooks and professional fish handlers. In these cases, the fish allergens are gastrosensitive and thermolabile, as demonstrated by the decrease in the diameter of the wheal in the skin-prick test using cooked fish. To the best of our knowledge isolated fish contact urticaria in children has not been previously reported. We analyze the features of three pediatric cases of contact urticaria from cod (one of them was sensitized to parvalbumin), with tolerance after ingestion of this fish on oral food challenge.

Background and Purposes: In a case-control study in patients with acute ischemic stroke and extracranial internal carotid artery (eICA) occlusion, thrombolytic treatment was associated with increased mortality. The aim of this cohort study was to assess the efficacy and safety of thrombolysis in patients with eICA occlusion compared to those without eICA occlusion. Methods: Consecutive patients treated with intravenous tissue-type plasminogen activator within 4.5 h from symptom onset included in the Safe Implementation of Thrombolysis in Stroke – International Stroke Thrombolysis Registry (SITS-ISTR) in 20 Italian centres were analyzed. Acute carotid occlusion was diagnosed using ultrasound examination, angio-CT scan or angio-MRI. Since the SITS-ISTR database did not plan to report the site of vessel occlusion, each participating center provided the code of the patient with eICA occlusion. Patients were divided into 2 groups, those with and those without eICA occlusion. Main outcome measures were: death, disability (modified Rankin Scale, mRS, 3–6) and any intracranial bleeding at 3 months. Multiple logistic regression analysis was performed to reveal predictors for main outcomes. The following variables of interest were included in the analysis: presence of eICA occlusion, age, gender, diabetes mellitus, hyperlipidemia, atrial fibrillation, congestive heart failure, previous stroke, current smoking, antiplatelet treatment at stroke onset, baseline NIHSS score, baseline blood glucose, cholesterol and blood pressure, history of hypertension and stroke onset to treatment time. Results: A total of 1,761 patients without eICA occlusion and 137 with eICA occlusion were included in the study. At 3 months, 42 patients were lost to follow-up (3 with eICA occlusion). Death occurred in 30 (22.4%) patients with eICA occlusion and in 175 (10.2%) patients without (p < 0.0001). Death or disability at 3 months occurred in 91 of 134 patients with eICA occlusion (67.9%) compared with 654 of 1,722 patients without eICA occlusion (37.9%, p < 0.0001). No or minimal disability at 3 months (mRS 0–1) was reported in 25 (18.7%) patients with eICA occlusion and in 829 (48.2%) patients without (p < 0.0001). Any intracranial bleeding detected by CT or MRI at posttreatment imaging was seen in 16 (11.7%) patients with eICA occlusion and in 314 (17.8%) of those without (p = 0.09). The proportion of symptomatic intracerebral hemorrhage was 5.8% for patients with eICA occlusion and 8.0% for patients without (p = 0.16). At logistic regression analysis, eICA occlusion was associated with mortality (odds ratio, OR 5.7; 95% confidence interval, CI 2.9–11.1) and mortality or disability (OR 5.0; 95% CI 2.9–8.7) at 90 days. Conclusions: This cohort study in patients with acute ischemic stroke treated with thrombolysis showed an association between eICA occlusion and adverse outcome.