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.     

Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa

The gene coding for the aminoglycoside adenylyltransferase (aadA6) from a clinical isolate of Pseudomonas aeruginosa was cloned and expressed in Escherichia coli strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5 mM MgCl₂ for normal Michaelis–Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1 mM MgCl₂. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100 μM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin.     

Unikaryon phyllotretae sp. n. (Protista,Microspora), a new microsporidian pathogen of Phyllotreta undulata (Coleoptera; Chrysomelidae)

The microsporidium Unikaryon phyllotretae sp. n., a new pathogen of Phyllotreta undulata, is described based on light microscopic and ultrastructural characteristics. Microscopic examination of parasitized individuals revealed two types of spores. The majority of the spores were of the first type, which are oval and measured 2.74±0.17×1.93±0.17 μm when fresh. Fresh spores of the second type (very rare) are elongated and measured 4.39±0.18×1.61±0.20 μm. All life stages have single nuclei. Sporogony ends with uninucleate single sporoblasts and spores. The spores were only observed in Malpighian tubules. The isofilar polar filament of the parasite has six to eight coils, and a well-developed polaroplast was of the lamellated type, with closely packed anterior lamellae and loosely packed posterior lamellae.     

Quantitative Site-specific Phosphorylation Dynamics of Human Protein Kinases during Mitotic Progression

Reversible protein phosphorylation is a key regulatory mechanism of mitotic progression. Importantly, protein kinases themselves are also regulated by phosphorylation-dephosphorylation processes; hence, phosphorylation dynamics of kinases hold a wealth of information about phosphorylation networks. Here, we investigated the site-specific phosphorylation dynamics of human kinases during mitosis using synchronization of HeLa suspension cells, kinase enrichment, and high resolution mass spectrometry. In biological triplicate analyses, we identified 206 protein kinases and more than 900 protein kinase phosphorylation sites, including 61 phosphorylation sites on activation segments, and quantified their relative abundances across three specific mitotic stages. Around 25% of the kinase phosphorylation site ratios were found to be changed by at least 50% during mitotic progression. Further network analysis of jointly regulated kinase groups suggested that Cyclin-dependent kinase- and mitogen-activated kinase-centered interaction networks are coordinately down- and up-regulated in late mitosis, respectively. Importantly, our data cover most of the already known mitotic kinases and, moreover, identify attractive candidates for future studies of phosphorylation-based mitotic signaling. Thus, the results of this study provide a valuable resource for cell biologists and provide insight into the system properties of the mitotic phosphokinome.Reversible phosphorylation is a ubiquitous posttranslational protein modification that is involved in the regulation of almost all biological processes (1–3). In human, 518 protein kinases have been identified in the genome that phosphorylate the majority of cellular proteins and increase the diversity of the proteome by severalfold (4). Addition of a phosphate group to a protein can alter its structural, catalytic, and functional properties; hence, kinases require tight regulation to avoid unspecific phosphorylation, which can be deleterious to cells (5–7). As a result, cells use a variety of mechanisms to ensure proper regulation of kinase activities (8). Importantly, most kinases are also in turn regulated through autophosphorylation and phosphorylation by other kinases, thus generating complex phosphorylation networks. In particular, phosphorylation on activation segments is a common mechanism to modulate kinase activities (9–11), but additional phosphorylation sites are also frequently required for fine tuning of kinase localizations and functions (12). Some kinases contain phosphopeptide binding domains that recognize prephosphorylated sites on other kinases, resulting in processive phosphorylation and/or targeting of kinases to distinct cellular locations (13–16). Because such priming phosphorylation events depend on the activities of the priming kinases, these motifs act as conditional docking sites and restrict the interaction with docking kinases to a particular point in time and physiological state. In addition, phosphorylation sites may act through combinatorial mechanisms or through cross-talk with other posttranslational modifications (PTMs)¹ (17, 18), thus further increasing the complexity of kinase regulatory networks.Regulation of kinases is of particular interest in mitosis as most of the mitotic events are regulated by reversible protein phosphorylation (19). During mitosis, error-free segregation of sister chromatids into the two daughter cells is essential to ensure genomic stability. Physically, this process is carried out by the mitotic spindle, a highly dynamic microtubule-based structure. After entry into mitosis, the major microtubule-organizing centers in animal cells, the centrosomes, start to increase microtubule nucleation and move to opposite poles of the cell. Throughout prometaphase, microtubules emanating from centrosomes are captured by kinetochores, protein complexes assembled on centromeric chromosomal DNA. This eventually leads to the alignment of all chromosomes in a metaphase plate. Because proper bipolar attachment of chromosomes to spindle microtubules is essential for the correct segregation of chromosomes, this critical step is monitored by a signaling pathway known as the spindle assembly checkpoint (SAC) (20). This checkpoint is silenced only after all chromosomes have attached to the spindle in a bioriented fashion, resulting in the synchronous segregation of sister chromatids during anaphase. Simultaneously, a so-called central spindle is formed between the separating chromatids, and the formation of a contractile ring initiates cytokinesis. Finally, in telophase, the chromosomes decondense and reassemble into nuclei, whereas remnants of the central spindle form the midbody, marking the site of abscission. Cyclin-dependent kinase 1 (Cdk1), an evolutionarily conserved master mitotic kinase, is activated prior to mitosis and initiates most of the mitotic events. Cdk1 works in close association with other essential mitotic kinases such as Plk1, Aurora A, and Aurora B for the regulation of mitotic progression (19, 21–24). Plk1 and Aurora kinases dynamically localize to different subcellular locations to perform multiple functions during mitosis and are phosphorylated at several conserved sites. Although little is known about the precise roles of these phosphorylation sites, emerging data indicate that they are involved in regulating localization-specific functions (25, 26). Furthermore, the kinases Bub1, BubR1, and TTK (Mps1) and kinases of the Nek family play important roles in maintaining the fidelity and robustness of mitosis (19). Recently, a genome-wide RNA-mediated interference screen identified M phase phenotypes for many kinases that have not previously been implicated in cell cycle functions, indicating that additional kinases have important mitotic functions (27).Although protein phosphorylation plays a pivotal role in the regulation of cellular networks, many phosphorylation events remain undiscovered mainly because of technical limitations (28). The advent of mass spectrometry-based proteomics along with developments in phosphopeptide enrichment methods has enabled large scale global phosphoproteomics studies (29, 30). However, the number of phosphorylation sites identified on kinases is limited compared with other proteins because of their frequently low expression levels. To overcome this problem, small inhibitor-based kinase enrichment strategies were developed, resulting in the identification of more than 200 kinases from HeLa cell lysates (31, 32). This method was also used recently to compare the phosphokinomes during S phase and M phase of the cell cycle, resulting in the identification of several hundreds of M phase-specific kinase phosphorylation sites (31). In the present study, we address the dynamics of the phosphokinome during mitotic progression using large scale cell synchronization at three distinct mitotic stages, small inhibitor-based kinase enrichment, and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry. Thus, we determined the mitotic phosphorylation dynamics of more than 900 kinase phosphorylation sites and identified distinctly regulated kinase interaction networks. Our results provide a valuable resource for the dynamics of the kinome during mitotic progression and give insight into the system properties of kinase interaction networks.     

Pollen limitation meets resource allocation: towards a comprehensive methodology

The standard method of measuring pollen limitation is to add pollen to a number of flowers, preferably to a whole plant, and to compare fruit and seed set with that of naturally pollinated flowers on other plants. In 25 yr of research, this method has yielded valuable data, but it is difficult to use in large plants. This has caused a bias in the available data towards smaller, herbaceous plants with relatively few flowers. I argue that, in order to widen our knowledge of how pollen limitation affects plants, we should go beyond whole-plant pollen addition and change our concept of how a flowering plant functions. The traditional method does not take into account the variation in and dynamics of resource allocation and pollen availability. The concept of integrated physiological units (IPUs) does, but, although it has been applied to pollination biology, it has not received the attention it deserves. I use this article to present its merits again, to propose a step-by-step methodology for studying pollen limitation, and to examine factors influencing possible plant strategies.     

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.     

Tumor-derived chemokine MCP-1/CCL2 is sufficient for mediating tumor tropism of adoptively transferred T cells

To exert a therapeutic effect, adoptively transferred tumor-specific CTLs must traffic to sites of tumor burden, exit the circulation, and infiltrate the tumor microenvironment. In this study, we examine the ability of adoptively transferred human CTL to traffic to tumors with disparate chemokine secretion profiles independent of tumor Ag recognition. Using a combination of in vivo tumor tropism studies and in vitro biophotonic chemotaxis assays, we observed that cell lines derived from glioma, medulloblastoma, and renal cell carcinoma efficiently chemoattracted ex vivo-expanded primary human T cells. We compared the chemokines secreted by tumor cell lines with high chemotactic activity with those that failed to elicit T cell chemotaxis (Daudi lymphoma, 10HTB neuroblastoma, and A2058 melanoma cells) and found a correlation between tumor-derived production of MCP-1/CCL2 (> or =10 ng/ml) and T cell chemotaxis. Chemokine immunodepletion studies confirmed that tumor-derived MCP-1 elicits effector T cell chemotaxis. Moreover, MCP-1 is sufficient for in vivo T cell tumor tropism as evidenced by the selective accumulation of i.v. administered firefly luciferase-expressing T cells in intracerebral xenografts of tumor transfectants secreting MCP-1. These studies suggest that the capacity of adoptively transferred T cells to home to tumors may be, in part, dictated by the species and amounts of tumor-derived chemokines, in particular MCP-1.