miRNA-197 and miRNA-223 Predict Cardiovascular Death in a Cohort of Patients with Symptomatic Coronary Artery Disease

Background

Circulating microRNAs (miRNAs) have been described as potential diagnostic biomarkers in cardiovascular disease and in particular, coronary artery disease (CAD). Few studies were undertaken to perform analyses with regard to risk stratification of future cardiovascular events. miR-126, miR-197 and miR-223 are involved in endovascular inflammation and platelet activation and have been described as biomarkers in the diagnosis of CAD. They were identified in a prospective study in relation to future myocardial infarction.

Objectives

The aim of our study was to further evaluate the prognostic value of these miRNAs in a large prospective cohort of patients with documented CAD.

Methods

Levels of miR-126, miR-197 and miR-223 were evaluated in serum samples of 873 CAD patients with respect to the endpoint cardiovascular death. miRNA quantification was performed using real time polymerase chain reaction (RT-qPCR).

Results

The median follow-up period was 4 years (IQR 2.78–5.04). The median age of all patients was 64 years (IQR 57–69) with 80.2% males. 38.9% of the patients presented with acute coronary syndrome (ACS), 61.1% were diagnosed with stable angina pectoris (SAP). Elevated levels of miRNA-197 and miRNA-223 reliably predicted future cardiovascular death in the overall group (miRNA-197: hazard ratio (HR) 1.77 per one standard deviation (SD) increase (95% confidence interval (CI) 1.20; 2.60), p = 0.004, C-index 0.78; miRNA-223: HR 2.23 per one SD increase (1.20; 4.14), p = 0.011, C-index 0.80). In ACS patients the prognostic power of both miRNAs was even higher (miRNA-197: HR 2.24 per one SD increase (1.25; 4.01), p = 0.006, C-index 0.89); miRA-223: HR 4.94 per one SD increase (1.42; 17.20), p = 0.012, C-index 0.89).

Conclusion

Serum-derived circulating miRNA-197 and miRNA-223 were identified as predictors for cardiovascular death in a large patient cohort with CAD. These results reinforce the assumption that circulating miRNAs are promising biomarkers with prognostic value with respect to future cardiovascular events.     

Digitomotography in Parkinson’s Disease: A Cross-Sectional and Longitudinal Study

Motor symptoms in Parkinson’s disease (PD) are usually assessed with semi-quantitative tests such as the Unified PD Rating Scale (UPDRS) which are limited by subjectivity, categorical design, and low sensitivity. Particularly bradykinesia as assessed e.g. with speeded index finger tapping exhibits low validity measures. This exploratory study set out to (i) assess whether force transducer-based objective and quantitative analysis of motor coordination in index finger tapping is able to distinguish between PD patients and controls, and (ii) assess longitudinal changes. Sixteen early-stage and 17 mid-stage PD patients as well as 18 controls were included in the cross-sectional part of the study; thirteen, 16 and 16 individuals of the respective groups agreed in a reassessment 12 months later. Frequency, force, rhythmicity, regularity and laterality of speeded and metronome paced tapping were recorded by digitomotography using a quantitative motor system ("Q-Motor"). Analysis of cross-sectional data revealed most consistent differences between PD patients and controls in variability of tap performance across modalities assessed. Among PD patients, variability of taps and the ability to keep a given rhythm were associated with UPDRS motor and finger tapping scores. After 12 months, laterality parameters were reduced but no other parameters changed significantly. This data suggests that digitomotography provides quantitative and objective measures capable to differentiate PD from non-PD in a small cohort, however, the value of the assessment to track PD progression has to be further evaluated in larger cohorts of patients.     

On the effects of the <Emphasis Type="Italic">Fusarium</Emphasis> toxin deoxynivalenol (DON) administered per os or intraperitoneal infusion to sows during days 63 to 70 of gestation

Six pregnant sows of 180.6 ± 5.6 kg were fed either a Fusarium-contaminated (4.42 mg DON and 48.3 μg ZON per kg, DON per os, n = 3) or a control diet (0.15 mg DON and 5 μg ZON/kg) in the period of days 63 and 70 of gestation. On day 63 of gestation, sows fed the control diet were implanted with an intraperitoneal osmotic minipump (delivery rate of 10 μL/h, for 7 days) containing 50 mg pure (98%) DON in 2 ml 50% DMSO (DON ip, n = 3). Frequent plasma samples were taken to estimate the kinetics after oral and ip DON exposure. The intended continuous delivery of DON by the intraperitoneal minipump could not be shown, as there was a plasma peak (C_max) of 4.2–6.4 ng DON/mL either immediately (sow IP-2+3) or 2.5 h (sow IP-1) after implantation of the pump followed by a one-exponential decline with a mean half-time (t_1/2) of 1.75–4.0 h and only negligible DON plasma concentrations after 12 h. Therefore, the DON ip exposure has to be regarded as one single dose 1 week before termination of experiment. The DON per os sows showed a mean basis level (after achieving a steady state) of DON plasma concentration of about 6–8 ng/mL, as also indicated by the plasma DON concentration at the termination of the experiment. On day 70, caesarean section was carried out, the fetuses were killed immediately after birth, and samples of plasma, urine, and bile were taken to analyze the concentration of DON and its metabolite de-epoxy-DON. At necropsy there were no macroscopic lesions observed in any organ of either sows or piglets. Histopathological evaluation of sows liver and spleen revealed no alterations. The proliferation rate of peripheral blood mononuclear cells (PBMC) with or without stimulation was not affected by the kind of DON treatment. The exposure of pregnant sows at mid-gestation (days 63–70, period of organogenesis) to a Fusarium toxin-contaminated diet (4.42 mg DON and 0.048 mg ZON per kg) or pure DON via intraperitoneal osmotic minipump did not cause adverse effects on health, fertility, maintenance of pregnancy, and performance of sows and their fetuses. However, DON was detected in fetus plasma, indicating that this toxin can pass the placental barrier and may cause changes in the proportion of white blood cells (lower monocyte and neutrophil and higher lymphocyte proportion in DON per os fetuses).     

Allicin and derivates are cysteine protease inhibitors with antiparasitic activity

Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure–activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei.     

Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers

Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of “impurities” into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.     

Quantitative NMR spectroscopy of biologically active substances and excipients

Biologically active ingredients and excipients are the essentials of a drug formulation, such as a tablet, dragee, solution, etc. Quality control of such substances thus plays a pivotal role in the production process of pharmaceutical drugs. Since these agents often exhibit complex structures, consist of multiple components, or lack of a chromophore, traditional means of characterization are often not feasible. Furthermore, substances of small molecular weight or strong polar character generally exhibit poor chromatographic properties, thus, conventional procedures such as high-performance liquid chromatography are often not applicable. Instead, quantitative nuclear magnetic resonance (qNMR) spectroscopy has emerged as an alternative or orthogonal method in drug analysis. In this review, we elaborate on the application of qNMR to three important classes of biological substances, namely polysaccharides, amino acids, and lipids, and demonstrate the benefits of this modern tool in contrast to traditional techniques.     

Artemisinin biosynthesis in growing plants of Artemisia annua. A 13CO2 study

Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is still unknown whether the isoprenoid precursors units are obtained by the mevalonate pathway or the more recently discovered non-mevalonate pathway. In order to address that question, a plant of A. annua was grown in an atmosphere containing 700 ppm of ¹³CO₂ for 100 min. Following a chase period of 10 days, artemisinin was isolated and analyzed by ¹³C NMR spectroscopy. The isotopologue pattern shows that artemisinin was predominantly biosynthesized from (E,E)-farnesyl diphosphate (FPP) whose central isoprenoid unit had been obtained via the non-mevalonate pathway. The isotopologue data confirm the previously proposed mechanisms for the cyclization of (E,E)-FPP to amorphadiene and its oxidative conversion to artemisinin. They also support deprotonation of a terminal allyl cation intermediate as the final step in the enzymatic conversion of FPP to amorphadiene and show that either of the two methyl groups can undergo deprotonation.     

Genetic fine-mapping of <Emphasis Type="Italic">DIPLOSPOROUS</Emphasis> in <Emphasis Type="Italic">Taraxacum</Emphasis> (dandelion; Asteraceae) indicates a duplicated <Emphasis Type="Italic">DIP</Emphasis>-gene

Background  

DIPLOSPOROUS (DIP) is the locus for diplospory in Taraxacum, associated to unreduced female gamete formation in apomicts. Apomicts reproduce clonally through seeds, including apomeiosis, parthenogenesis, and autonomous or pseudogamous endosperm formation. In Taraxacum, diplospory results in first division restitution (FDR) nuclei, and inherits as a dominant, monogenic trait, independent from the other apomixis elements. A preliminary genetic linkage map indicated that the DIP-locus lacks suppression of recombination, which is unique among all other map-based cloning efforts of apomeiosis to date. FDR as well as apomixis as a whole are of interest in plant breeding, allowing for polyploidization and fixation of hybrid vigor, respectively. No dominant FDR or apomixis genes have yet been isolated. Here, we zoom-in to the DIP-locus by largely extending our initial mapping population, and by analyzing (local) suppression of recombination and allele sequence divergence (ASD).     

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.Phosphorylation of proteins is an integral part of the signal transduction of eukaryotic cells as it modulates the activity of complex protein networks. Although Western blot- and immunoprecipitation-based MS approaches (1, 2) can lead to detailed insights into these processes, most of the integrated approaches only allow a static view of protein phosphorylation because they are not suitable for the screening of hundreds of samples. Either planar or bead array-based sandwich immunoassays can be used to analyze the quantity and activation state of signaling molecules in multiplex, enabling the systematic profiling of protein abundance and post-translational modifications (3–6) in hundreds of samples. However, multiplex immunoassays are only suitable for the simultaneous analysis of a limited number of proteins. The detection of comprehensive phosphorylation patterns is difficult as this involves assay systems that are incompatible with multiplexing.In principle, two sandwich immunoassay setups are possible for probing the phosphorylation state of a protein. The first setup applies a capture antibody specific for a non-modified part of the protein and uses a phosphorylation state-specific detection antibody. When applied to an array-based format, however, this setup does not allow for the simultaneous measurement of the abundance and the degree of phosphorylation (3, 4). A mixture of detection antibodies, one specific for the phosphorylation site and one specific for the non-modified site of the protein, would bind simultaneously to the two different epitopes, and assay signals could not be further deconvoluted by the spatial or color code of the array. The second sandwich immunoassay setup for the analysis of protein phosphorylation applies a phosphorylation state-specific capture antibody and a protein-specific detection antibody. In such a setup, an anti-phosphotyrosine antibody (e.g. mAb 4G10) cannot be applied as a capture antibody because a huge variety of tyrosine phosphorylated proteins would be captured, and specific signals could rarely be deconvoluted. Using capture antibodies that bind to phosphorylated epitopes in the context of their flanking amino acids is not a problem until a multiplex readout is desired. If one antibody specific for the phosphosite and one antibody specific for the abundance of a protein are used together in a multiplex assay panel they might compete for their analyte. The situation becomes even more complex if the protein of interest contains various phosphorylation sites such as e.g. the epidermal growth factor receptor. Several capture antibodies target different epitopes of the same protein and therefore compete for the overall amount of targeted protein in the sample, thus making a valid simultaneous measurement problematic.Although different ways of tackling the problem of assay multiplexing are in use, we demonstrated the feasibility to sequentially perform such incompatible assays from the same sample using a magnetic particle handler that moves particles through the samples and reagents (Fig. 1). Using a model assay, we confirmed that suspension bead array-based immunoassays work under ambient analyte conditions. As described by Roger Ekins (7), decreasing of the amount of capture antibody in a sandwich immunoassay setup from a macrospot (e.g. a microtiter plate assay) to a microspot generates a scenario where only a tiny fraction of the present target analytes is captured on the microspot. Therefore, the overall concentration of the analyte molecules in the sample does not change significantly even in the case of low target concentrations and high affinity binding reactions. Furthermore, as the initial concentration of the analyte is not significantly changed when performing a miniaturized sandwich immunoassay, multiple post-translational modifications within the same protein can be measured either in sequence or in parallel in the same multiplex panel.Open in a separate windowFig. 1.Sequential multiplex analyte capturing. Magnetic suspension bead array assays can be performed sequentially, reusing the same sample material (indicated by the blue arrow). The use of a magnetic particle handler enables the quantitative transfer (black arrow) of the magnetic beads from the sample well into the wells containing washing solutions or other assay reagents. Magnetic beads from the first bead array panel are incubated with the samples to capture their respective analyte. Then the magnetic beads are subjected to washing and detection steps and are finally transferred into the readout plate (first row). After retracting the magnetic suspension bead array of the first assay panel from the sample, a bead array from the second assay panel is added and processed as described above but using different detection antibodies (second row). A third bead array assay panel can be applied after removing the second panel (third row) and so on.By probing tumor cell lines for the abundance of seven different receptor tyrosine kinases and their generic tyrosine phosphorylation, we generated complex phosphorylation patterns and thereby demonstrated the potential of this approach. More importantly, demonstrating ambient analyte conditions allowed the parallel detection of phosphorylation at different sites of the EGFR1 using phosphorylation site-specific antibodies as capture molecules with one assay panel. Phosphorylation of eight different sites and the abundance of the EGFR could be quantified relative to one another without any interference of the different immunoassays during multiplexing because competition for the analyte can be prevented by running the assays under ambient analyte conditions.