The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Dietary Crude Lecithin Increases Systemic Availability of Dietary Docosahexaenoic Acid with Combined Intake in Rats

Crude lecithin, a mixture of mainly phospholipids, potentially helps to increase the systemic availability of dietary omega-3 polyunsaturated fatty acids (n-3 PUFA), such as docosahexaenoic acid (DHA). Nevertheless, no clear data exist on the effects of prolonged combined dietary supplementation of DHA and lecithin on RBC and plasma PUFA levels. In the current experiments, levels of DHA and choline, two dietary ingredients that enhance neuronal membrane formation and function, were determined in plasma and red blood cells (RBC) from rats after dietary supplementation of DHA-containing oils with and without concomitant dietary supplementation of crude lecithin for 2–3 weeks. The aim was to provide experimental evidence for the hypothesized additive effects of dietary lecithin (not containing any DHA) on top of dietary DHA on PUFA levels in plasma and RBC. Dietary supplementation of DHA-containing oils, either as vegetable algae oil or as fish oil, increased DHA, eicosapentaenoic acid (EPA), and total n-3 PUFA, and decreased total omega-6 PUFA levels in plasma and RBC, while dietary lecithin supplementation alone did not affect these levels. However, combined dietary supplementation of DHA and lecithin increased the changes induced by DHA supplementation alone. Animals receiving a lecithin-containing diet also had a higher plasma free choline concentration as compared to controls. In conclusion, dietary DHA-containing oils and crude lecithin have synergistic effects on increasing plasma and RBC n-3 PUFA levels, including DHA and EPA. By increasing the systemic availability of dietary DHA, dietary lecithin may increase the efficacy of DHA supplementation when their intake is combined.Nutricia Researc

A Metabolically Integrated Lossen Rearrangement in Escherichia coli

Biocompatible chemistry enables abiotic reactions to be interfaced with metabolic pathways in living microorganisms. This includes both native and de novo biosynthetic processes to access abiotic feedstocks, intermediates and products in vivo. Herein we report a biocompatible Lossen rearrangement that is catalysed by phosphate in the bacterium Escherichia coli for the chemical transformation of activated acyl hydroxamates to primary amines in living cells. Using a para-carboxylated substrate, the biocompatible reaction can be used to generate the metabolite para-aminobenzoic acid (PABA) to rescue ∆pabA/B and ∆aroC auxotrophy and enable cell growth. The Lossen substrate can also be synthesised from polyethylene terephthalate (PET) and applied to whole-cell biocatalytic reactions and fermentations generating industrial small molecule products – including the analgesic and antipyretic drug paracetamol – paving the way for a general strategy to addict E. coli and other industrial chassis strains to PET plastic waste as a bioremediation strategy and for the upcycling of plastic waste using engineered biology. Together, this work showcases how non-enzymatic biocompatible reactions can be interfaced and cooperate with microbial metabolism to expand the available toolbox of metabolic chemistry for small molecule synthesis in native and engineered cellular systems

Isolation of adipose tissue derived regenerative cells from human subcutaneous tissue with or without the use of an enzymatic reagent.

Freshly isolated, uncultured, autologous adipose derived regenerative cells (ADRCs) have emerged as a promising tool for regenerative cell therapy. The Transpose RT system (InGeneron, Inc., Houston, TX, USA) is a system for isolating ADRCs from adipose tissue, commercially available in Europe as a CE-marked medical device and under clinical evaluation in the United States. This system makes use of the proprietary, enzymatic Matrase Reagent for isolating cells. The present study addressed the question whether the use of Matrase Reagent influences cell yield, cell viability, live cell yield, biological characteristics, physiological functions or structural properties of the ADRCs in final cell suspension. Identical samples of subcutaneous adipose tissue from 12 subjects undergoing elective lipoplasty were processed either with or without the use of Matrase Reagent. Then, characteristics of the ADRCs in the respective final cell suspensions were evaluated. Compared to non-enzymatic isolation, enzymatic isolation resulted in approximately twelve times higher mean cell yield (i.e., numbers of viable cells/ml lipoaspirate) and approximately 16 times more colony forming units. Despite these differences, cells isolated from lipoaspirate both with and without the use of Matrase Reagent were independently able to differentiate into cells of all three germ layers. This indicates that biological characteristics, physiological functions or structural properties relevant for the intended use were not altered or induced using Matrase Reagent. A comprehensive literature review demonstrated that isolation of ADRCs from lipoaspirate using the Transpose RT system and the Matrase Reagent results in the highest viable cell yield among published data regarding isolation of ADRCs from lipoaspirate

Two Functional Motifs Define the Interaction, Internalization and Toxicity of the Cell-Penetrating Antifungal Peptide PAF26 on Fungal Cells

The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW) is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW) and PAF96 (RKKAAA), were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i) its interaction with the cell envelope; (ii) its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii) its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the cytoplasm, which coincides with cell death. Peptide containment within vacuoles preserves fungal cells from peptide toxicity

Activity and localization of PAF peptides in <i>A. fumigatus</i>.

<p>(A) Influence of PAF peptides on conidial germination. Percentage of germinated conidia was quantified by microscopy ∼5 h after treatment with the peptides at different concentrations. (B) Confocal microscopy of the localization of 5 µM TMR-labeled peptides (in red) in germlings of <i>A. fumigatus</i> expressing H1-GFP (in green) 1 h after treatment with the three peptides. Note that the left hand panels show brightfield images of germlings and the right panels show corresponding GFP-labeled nuclei in the same cells. The germlings were all treated for 1 h with the different PAF peptides before imaging. Note that the whole germlings treated with TMR-PAF26 are labeled by the fluorescent peptide, lack nuclei and are dead. This contrasts with both the TMR-PAF95 and TMR-PAF96 treated germlings that possess nuclei and are alive. The TMR-PAF95 treated germlings lack any labeling with the fluorescent peptide whilst TMR-PAF96 is bound only to the cell envelope of the germlings. Bar: 10 µm.</p

Saccharomyces cerevisiae gene deletion mutants used in this study.

<p>Saccharomyces cerevisiae gene deletion mutants used in this study.</p

Differential localization of TMR-PAF26 in different <i>S. cerevisiae</i> strains.

<p>(A) Graph showing the percentage of cells that show TMR-PAF26 fluorescence limited to the cell envelope (class 1, white bars), in intracellular organelles (class 2, light grey bars), or filling whole cells (class 3, black bars), after treatment for 1 h with the peptide for each strain (as labeled on the x-axis). Cell location was determined by confocal microscopy. Results represent the means ± standard deviations of three experiments, each performed in triplicate. Dots above bars indicate statistical significance as compared to each class in the BY4741 parental strain control. Black dots indicate percentage lower than BY4741 and white dots higher. Two dots indicate p<0.01, and one dot p<0.05. (B) Representative confocal microscopy images are shown from the data set quantified in (A). Two examples of classes 1, 2 and 3 are labelled in strain BY4741. Bar: 10 µm.</p