Early versus Later Anticoagulation for Stroke with Atrial Fibrillation.

BACKGROUND: The effect of early as compared with later initiation of direct oral anticoagulants (DOACs) in persons with atrial fibrillation who have had an acute ischemic stroke is unclear. METHODS: We performed an investigator-initiated, open-label trial at 103 sites in 15 countries. Participants were randomly assigned in a 1:1 ratio to early anticoagulation (within 48 hours after a minor or moderate stroke or on day 6 or 7 after a major stroke) or later anticoagulation (day 3 or 4 after a minor stroke, day 6 or 7 after a moderate stroke, or day 12, 13, or 14 after a major stroke). Assessors were unaware of the trial-group assignments. The primary outcome was a composite of recurrent ischemic stroke, systemic embolism, major extracranial bleeding, symptomatic intracranial hemorrhage, or vascular death within 30 days after randomization. Secondary outcomes included the components of the composite primary outcome at 30 and 90 days. RESULTS: Of 2013 participants (37% with minor stroke, 40% with moderate stroke, and 23% with major stroke), 1006 were assigned to early anticoagulation and 1007 to later anticoagulation. A primary-outcome event occurred in 29 participants (2.9%) in the early-treatment group and 41 participants (4.1%) in the later-treatment group (risk difference, -1.18 percentage points; 95% confidence interval [CI], -2.84 to 0.47) by 30 days. Recurrent ischemic stroke occurred in 14 participants (1.4%) in the early-treatment group and 25 participants (2.5%) in the later-treatment group (odds ratio, 0.57; 95% CI, 0.29 to 1.07) by 30 days and in 18 participants (1.9%) and 30 participants (3.1%), respectively, by 90 days (odds ratio, 0.60; 95% CI, 0.33 to 1.06). Symptomatic intracranial hemorrhage occurred in 2 participants (0.2%) in both groups by 30 days. CONCLUSIONS: In this trial, the incidence of recurrent ischemic stroke, systemic embolism, major extracranial bleeding, symptomatic intracranial hemorrhage, or vascular death at 30 days was estimated to range from 2.8 percentage points lower to 0.5 percentage points higher (based on the 95% confidence interval) with early than with later use of DOACs. (Funded by the Swiss National Science Foundation and others; ELAN ClinicalTrials.gov number, NCT03148457.).

Neptune: a bioinformatics tool for rapid discovery of genomic variation in bacterial populations.

The ready availability of vast amounts of genomic sequence data has created the need to rethink comparative genomics algorithms using 'big data' approaches. Neptune is an efficient system for rapidly locating differentially abundant genomic content in bacterial populations using an exact k-mer matching strategy, while accommodating k-mer mismatches. Neptune's loci discovery process identifies sequences that are sufficiently common to a group of target sequences and sufficiently absent from non-targets using probabilistic models. Neptune uses parallel computing to efficiently identify and extract these loci from draft genome assemblies without requiring multiple sequence alignments or other computationally expensive comparative sequence analyses. Tests on simulated and real datasets showed that Neptune rapidly identifies regions that are both sensitive and specific. We demonstrate that this system can identify trait-specific loci from different bacterial lineages. Neptune is broadly applicable for comparative bacterial analyses, yet will particularly benefit pathogenomic applications, owing to efficient and sensitive discovery of differentially abundant genomic loci. The software is available for download at: http://github.com/phac-nml/neptune.

If you build it, will they eat it? Consumer preferences for plant-based and cultured meat burgers.

In a hypothetical choice experiment consumers were given the option of purchasing burgers that were made from beef, plant-based protein, or cultured meat. Willingness to purchase plant-based and cultured meat burgers is linked to age, sex, views of other food technologies, and attitudes towards the environment and agriculture. Although consumers were told that all burgers tasted the same, there was a marked preference for beef burgers. A mixed-logit model predicts that, if prices were equal, 65% of consumers would purchase the beef burger, 21% would purchase the plant-based burger, 11% would purchase the cultured meat burger, and 4% would make no purchase. Preferences for plant-based and cultured meat burgers are found to be highly, but not perfectly, correlated.

Mannose metabolism in recombinant CHO cells and its effect on IgG glycosylation.

Understanding the causes of high-mannose (HM) glycosylation of recombinant IgG in CHO cells would facilitate the production of therapeutics. CHO cells grown with mannose as the major carbon source demonstrated a dramatic increase in total HM glycosylation in recombinant IgG, with no effect on cell growth, viability, or titer. Quantitative metabolomics and (13) C flux analysis were used to explore the mechanism for increased HM glycosylation and understand the metabolism of mannose in CHO cells. It was demonstrated that mannose was a good carbon source for CHO cell growth and IgG production, readily entering both glycolysis and the TCA Cycle. Previous mechanisms for increased HM glycosylation during antibody production have been attributed to changes in pH, osmolality, increased specific productivity, and nutrient limitation. The results from this study propose a novel mechanism where an increased carbon flux in the GDP-mannose synthetic pathway increased the intracellular concentration of mannose-containing metabolites. The abnormally high concentration of mannose and mannose-metabolites were shown to inhibit α-mannosidase activity and it was proposed that this inhibition in the ER and Golgi caused the production of IgG with increased high-mannose glycosylation. Biotechnol. Bioeng. 2016;113: 1468-1480. © 2016 Wiley Periodicals, Inc.

Quantitation of AAV in a dual-vector system using SV-AUC.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become the "gold standard" for characterization of the empty, partial, and full capsids of gene therapy products (e.g., AAV and Adenovirus vectors). Other techniques, such SEC-MALS, TEM, and mass photometry, are commonly used for capsid quantitation, however, the resolving power of these techniques is lacking. In this body of work, SV-AUC was implemented in the characterization of a dual-vector AAV system where the difference in packaged genomes was ∼400 nucleotides. SV-AUC instrument parameters and analysis were optimized to accurately quantitate both AAV vectors with less than 8% error and highly correlated linearity (R2 > 0.99) as compared to ddPCR. The results of this work highlight the resolution and accuracy of dual-vector capsid quantitation by SV-AUC and demonstrate the use of the powerful Bayesian analysis implemented in the SEDFIT analysis software.

Recovery kinetics of dual AAV-mediated human otoferlin expression.

Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3' portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression.

A filtered database search algorithm for endogenous serum protein carbonyl modifications in a mouse model of inflammation.

During inflammation, the resulting oxidative stress can damage surrounding host tissue, forming protein-carbonyls. The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to reactive oxygen and nitrogen species from inflammation. The goals of this study were to identify the major serum proteins modified with a carbonyl functionality and to identify the types of carbonyl adducts. To select for carbonyl-modified proteins, serum proteins were reacted with an aldehyde reactive probe that biotinylated the carbonyl modification. Modified proteins were enriched by avidin affinity and identified by two-dimensional liquid chromatography tandem MS. To identify the carbonyl modification, tryptic peptides from serum proteins were subjected to avidin affinity and the enriched modified peptides were analyzed by liquid chromatography tandem MS. It was noted that the aldehyde reactive probe tag created tag-specific fragment ions and neutral losses, and these extra features in the mass spectra inhibited identification of the modified peptides by database searching. To enhance the identification of carbonyl-modified peptides, a program was written that used the tag-specific fragment ions as a fingerprint (in silico filter program) and filtered the mass spectrometry data to highlight only modified peptides. A de novo-like database search algorithm was written (biotin peptide identification program) to identify the carbonyl-modified peptides. Although written specifically for our experiments, this software can be adapted to other modification and enrichment systems. Using these routines, a number of lipid peroxidation-derived protein carbonyls and direct side-chain oxidation proteins carbonyls were identified in SJL mouse serum.

Specialist Perspectives on the Imaging Selection of Large Vessel Occlusion in the Late Window.

BACKGROUND: The proper imaging modality for use in the selection of patients for endovascular thrombectomy (EVT) presenting in the late window remains controversial, despite current guidelines advocating the use of advanced imaging in this population. We sought to understand if clinicians with different specialty training differ in their approach to patient selection for EVT in the late time window. METHODS: We conducted an international survey of stroke and neurointerventional clinicians between January and May 2022 with questions focusing on imaging and treatment decisions of large vessel occlusion (LVO) patients presenting in the late window. Interventional neurologists, interventional neuroradiologists, and endovascular neurosurgeons were defined as interventionists whereas all other specialties were defined as non-interventionists. The non-interventionist group was defined by all other specialties of the respondents: stroke neurologist, neuroradiologist, emergency medicine physician, trainee (fellows and residents) and others. RESULTS: Of 3000 invited to participate, 1506 (1027 non-interventionists, 478 interventionists, 1 declined to specify) physicians completed the study. Interventionist respondents were more likely to proceed directly to EVT (39.5% vs. 19.5%; p < 0.0001) compared to non-interventionist respondents in patients with favorable ASPECTS (Alberta Stroke Program Early CT Score). Despite no difference in access to advanced imaging, interventionists were more likely to prefer CT/CTA alone (34.8% vs. 21.0%) and less likely to prefer CT/CTA/CTP (39.1% vs. 52.4%) for patient selection (p < 0.0001). When faced with uncertainty, non-interventionists were more likely to follow clinical guidelines (45.1% vs. 30.2%) while interventionists were more likely to follow their assessment of evidence (38.7% vs. 27.0%) (p < 0.0001). CONCLUSION: Interventionists were less likely to use advanced imaging techniques in selecting LVO patients presenting in the late window and more likely to base their decisions on their assessment of evidence rather than published guidelines. These results reflect gaps between interventionists and non-interventionists reliance on clinical guidelines, the limits of available evidence, and clinician belief in the utility of advanced imaging.

Identifying the CHO secretome using mucin-type O-linked glycosylation and click-chemistry.

Chinese hamster ovary cells (CHO) are the most common cell line used in the production of therapeutic proteins. Understanding the complex pattern of secreted host cell proteins (HCP) that are released by CHO cells will facilitate the development of new recombinant protein production processes. In this study, we have adapted the N-azido-galactosamine (GalNAz) metabolic labeling method to enable the mass spectrometry identification and quantification of secreted proteins in cell culture media. CHO DG44 and CHO-S cells were cultured in media containing GalNAz, which was metabolically incorporated into mucin-type O-linked glycans of secreted proteins. These proteins were effectively enriched using click-chemistry from the cell culture media, allowing for the analysis of secreted proteins across multiple days of cell growth. When compared to the standard method for secretome analysis, the GalNAz method not only increased the total number of proteins identified but dramatically improved the quality of data by decreasing the number of background proteins (cytosolic or nuclear) to essentially zero.

Research protocol - Assessing Post-Stroke Psychology Longitudinal Evaluation (APPLE) study: A prospective cohort study in stroke.

Background: Cognitive and mood problems have been highlighted as priorities in stroke research and guidelines recommend early screening. However, there is limited detail on the preferred approach.We aimed to (1) determine the optimal methods for evaluating psychological problems that pre-date stroke; (2) assess the test accuracy, feasibility and acceptability of brief cognitive and mood tests used at various time-points following stroke; (3) describe temporal changes in cognition and mood following stroke and explore predictors of change. Methods: We established a multi-centre, prospective, observational cohort with acute stroke as the inception point - Assessing Post-stroke Psychology Longitudinal Evaluation (APPLE). We approached patients admitted with stroke or transient ischaemic attack (TIA) from 11 different hospital sites across the United Kingdom. Baseline demographics, clinical, functional, cognitive, and mood data were collected. Consenting stroke survivors were followed up with more extensive evaluations of cognition and mood at 1, 6, 12 and 18 months. Results: Continuous recruitment was from February 2017 to February 2019. With 357 consented to full follow-up. Eighteen-month assessments were completed in September 2020 with permissions in-place for longer term in-person or electronic follow-up. A qualitative study has been completed, and a participant sample biobank and individual participant database are both available. Discussion: The APPLE study will provide guidance on optimal tool selection for cognitive and mood assessment both before and after stroke, as well as information on prognosis and natural history of neuropsychological problems in stroke. The study data, neuroimaging and tissue biobank are all available as a resource for future research.

The effects of apixaban on clot characteristics in atrial fibrillation: A novel pharmacodynamic biomarker.

Atrial fibrillation (AF) is a major risk factor for stroke. We aim to characterize AF patients and the effects of apixaban therapy in terms of clot microstructure using gel point analysis, a novel biomarker. Seventy-eight patients were included in the study, 50 Stroke with AF (AF-S), and 28 AF without stroke (AF). Pre- and post-anticoagulation samples were collected: gel point (GP) analysis was performed to obtain (i) TGP (the time taken to reach the GP or the clot formation time) and (ii) df, the fractal dimension of the clot, a quantification of clot fibrin microstructure at the GP. At baseline, the AF-S group had a df  = 1.70 (±0.05) and TGP = 306 (±73 s). The AF group had a df = 1.70 ± 0.05 and TGP = 346 ± 78 s, showing a significantly shortened TGP in the stroke group (p = .008). For both groups, apixaban significantly prolonged TGP, p = .005, but resulted in no change in df. Apixaban prolonged clotting time while having no significant impact on the blood's ability to form stable clots (no change in df ). This indicates that apixaban provides protection from the formation of thrombi by reducing clotting kinetics.

Proteins modified by the lipid peroxidation aldehyde 9,12-dioxo-10(E)-dodecenoic acid in MCF7 breast cancer cells.

The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic acid (DODE), which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by DODE. To reduce false positives, three methods were used to identify DODE-modified proteins. First, cells were treated with a synthetically biotinylated 13-HPODE (13-HPODE-biotin). Modified proteins were enriched by neutravidin affinity and identified by two-dimensional liquid chromatography--tandem mass spectrometry (2D LC-MS/MS). Second, cells were treated with native 13-HPODE. Protein carbonyls were biotinylated with an aldehyde reactive probe, and modified proteins were enriched by neutravidin affinity and identified by 2D LC-MS/MS. Third, using a newly developed DODE antibody, DODE-modified proteins were located by 2D sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized by all three methods revealed a significant overlap and identified 32 primary proteins modified by DODE in MCF7 cells. These results demonstrated the feasibility for the cellular formation of DODE protein-carbonyl adducts that may be future indicators of oxidative stress.

Factors affecting the retrieval of famous names.

Tests of famous faces are used to study language and memory. Yet, the effect of stimulus properties on performance has not been fully investigated. To identify factors influencing proper name retrieval and to probe stimulus-specific parameters within proper name lexicon, we analysed the results obtained by 300 healthy participants on a test of famous faces that includes 74 personalities. A factor analysis yielded five main factors that were characterized by language (national or foreign names), epoch of peak popularity (current, recent or past) and occupation (politicians, entertainment and sports) of the personalities. Multiple regression analysis showed that participants' education, age and gender accounted for 10-32% of the variance in factor scores. These results indicate that there are variables of the stimulus and participants' that must be taken into account in proper name testing and in designing tests aimed to differentiate age-associated difficulties from cognitive decline.

Neurocognitive correlates of positive and negative perfectionism.

The present study examined the neurocognitive correlates of positive and negative perfectionism. A clinical sample of 160 patients undergoing standard neuropsychological testing was administered the Positive and Negative Perfectionism Scale (PANPS), a 40-item questionnaire measure of positive and negative perfectionism. The main question addressed in the study was how individual differences in positive and negative perfectionism relate to differences in neurocognitive performance. The general hypotheses to be tested were that positive perfectionism would be associated primarily with tests that relied on mental and physical "effort," while negative perfectionism would be associated with tests involving both "speed and accuracy." The results of the study provided general support for these hypotheses. Implications for the perfectionism literature and sports psychology are briefly discussed.

The serial use of child neurocognitive tests: development versus practice effects.

When serial neurocognitive assessments are performed, 2 main factors are of importance: test-retest reliability and practice effects. With children, however, there is a third, developmental factor, which occurs as a result of maturation. Child tests recognize this factor through the provision of age-corrected scaled scores. Thus, a ready-made method for estimating the relative contribution of developmental versus practice effects is the comparison of raw (developmental and practice) and scaled (practice only) scores. Data from a pool of 507 Portuguese children enrolled in a study of dental amalgams (T. A. DeRouen, B. G. Leroux, et al., 2002; T. A. DeRouen, M. D. Martin, et al., 2006) showed that practice effects over a 5-year period varied on 8 neurocognitive tests. Simple regression equations are provided for calculating individual retest scores from initial test scores.

Challenges in reducing TIA clinic waiting times from 9 to 3 days in an acute Welsh hospital.

A patient impact project which successfully reduced the transient ischaemic attack (TIA) clinic waiting time from 9 to 3 days in an acute Welsh hospital, revealing the challenges faced and how alternative thinking and team work improved care given to our service users. Evaluating current situation, careful planning with multiple brainstorming meetings, 4 N chart and driver diagram with change ideas laid the foundation for this service improvement. Run charts, statistical process control and Pareto charts helped to identify the issues that are hindering the progress, which when rectified, reduced the clinic waiting times. Avoiding clinic cancellations by cross covering TIA clinics with mutual agreement among consultants and redeployment of ward staff to support clinics resulted in a positive impact to the patients. The average waiting time to see a patient in TIA clinic dropped from 9 days to just 3 days as a result of this, reflecting the hard-working and proactive nature of a team following a collaborative leadership journey. The service improvement initiative for 'avoiding clinic cancellations' was implemented in January 2017 and has reduced our waiting times by three times. Repeat analysis by six monthly Plan Do Study Act cycles revealed that this improvement is sustained.

Spiroiminodihydantoin as an oxo-atom transfer product of 8-oxo-2'-deoxyguanosine oxidation by chromium(V).

Oxidation of the DNA lesion 8-oxo-2'-deoxyguanosine by the two electron oxidants N,N'-ethylenebis(salicylideneanimato)oxochromium(V) (Cr(V)-salen) and bis(2-ethyl-2-hydroxybutyrato)oxochromium(V) (Cr(V)-ehba) at neutral pH forms spiroiminodihydantoin by an oxo-atom transfer mechanism. The chromium complexes are models of a DNA oxidation pathway caused by the carcinogen chromate.

Body mass and wages: New evidence from quantile estimation.

I estimate the effect of body mass index (BMI) on wages across the unconditional distribution of wages. I find that for whites and Hispanics the effect of BMI is generally decreasing across the wage distribution; at the .9 quantile of the wage distribution, a two standard deviation increase in BMI reduces wages by 8% for white males, 13% for white females, 9% for Hispanic males, and 16% for Hispanic females. Conversely, at the .1 quantile, a two standard deviation increase in BMI affects wages by less than 2% for all these groups. For black males, the effect of BMI is positive, and either increasing or non-linear in wages. For black females, the estimates tend to be more uniform across the wage distribution. I discuss possible explanations for these inter-quantile differences including preference discrimination, productivity differences, and statistical discrimination. The results point to a new explanation for the observed correlation between socioeconomic status and body weight: individuals with higher income earning potential have differential incentives to maintain a lower BMI.

Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases NEIL1 and NEIL2.

8-Oxoguanine (8-oxoG) is an unstable mutagenic DNA lesion that is prone to further oxidation. High valent metals such as Cr(V) and Ir(IV) readily oxidize 8-oxoG to form guanidinohydantoin (Gh), its isomer iminoallantoin (Ia), and spiroiminodihydantoin (Sp). When present in DNA, these lesions show enhanced base misincorporation over the parent 8-oxoG lesion leading to G --> T and G --> C transversion mutations and polymerase arrest. These findings suggested that further oxidized lesions of 8-oxoG are more mutagenic and toxic than 8-oxoG itself. Repair of oxidatively damaged bases, including Sp and Gh/Ia, are initiated by the base excision repair (BER) system that involves the DNA glycosylases Fpg, Nei, and Nth in E. coli. Mammalian homologs of two of these BER enzymes, OGG1 and NTH1, have little or no affinity for Gh/Ia and Sp. Herein we report that two recently identified mammalian glycosylases, NEIL1 and NEIL2, showed a high affinity for recognition and cleavage of DNA containing Gh/Ia and Sp lesions. NEIL1 and NEIL2 recognized both of these lesions in single-stranded DNA and catalyzed the removal of the lesions through a beta- and delta-elimination mechanism. NEIL1 and NEIL2 also recognized and excised the Gh/Ia lesion opposite all four natural bases in double-stranded DNA. NEIL1 was able to excise the Sp lesion opposite the four natural bases in double-stranded DNA, however, NEIL2 showed little cleavage activity against the Sp lesion in duplex DNA although DNA trapping studies show recognition and binding of NEIL2 to this lesion. This work suggests that NEIL1 and NEIL2 are essential in the recognition of further oxidized lesions arising from 8-oxoG and implies that these BER glycosylases may play an important role in the repair of DNA damage induced by carcinogenic metals.