Thrombin Generation in the Glasgow Myocardial Infarction Study

Background

Thrombin is a key protease in coagulation also implicated in complex pathology including atherosclerosis. To address the role of thrombin in relation to myocardial infarction (MI) we explored thrombin generation analysis in plasma from patients and controls that had participated in the Glasgow MI Study (GLAMIS).

Methods

Thrombin generation at 1 and 2 pM TF and with and without thrombomodulin (TM) was performed on plasmas from 356 subjects (171 cases, 185 age and sex matched controls) from GLAMIS collected between 3 and 9 months after the MI event.

Results

Although thrombin generation was slightly delayed in cases (lag time increased from 3.3 to 3.6 min) at the highest trigger, the overall potential to generate thrombin was increased by 7% for the ETP and by 15% for the peak height (both at the 1 pM TF trigger) in cases. Addition of TM did not reveal differences. Furthermore, an increased thrombin generation was associated with MI [normalized ETP: adjusted OR for the highest percentile = 2.4 (95% CI 1.3–4.5) and normalized peak height: adjusted OR = 2.6 (1.3–5.0)] at the lowest trigger; normalized ETP and peak height being 2.1 (1.1–3.8) and 2.0 (1.0–4.1) at the higher 2 pM trigger.

Conclusion

In GLAMIS, patients with a previous MI had an increased thrombin generation compared to controls. The absence of a clear difference in TM reduction suggests an unaltered anticoagulant activity in these patients. Further research is needed in order to unravel the underlying mechanisms of enhanced thrombin generation after MI.     

Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G₁ phase to G₂ and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G₁ cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.     

Morphology and systematic significance of sclerolepidia in the weevils (Coleoptera: Curculionoidea)

The Russian sturgeon, Acipenser gueldenstaedtii, is closely related to three other sturgeon species (A persicus, A. naccarii and A baerii), with populations in the Caspian Sea containing a cryptic lineage with an A. baerii‐like mtDNA profile. Using morphological evidence (morphometrics, meristics) and additional genetic analysis (cytochrome b gene and control region sequencing), cryptic lineages within the Russian sturgeon and their relation to other closely related species of sturgeons are further examined. These data indicate that three genetic forms exist within what is presently known as A. gueldenstaedtii. These forms include the pure A. gueldenstaedtii and A. baerii‐like individuals plus a third rare genetic form whose mtDNA is similar to the mtDNA of the Adriatic sturgeon, A. naccarii. Morphological comparison of the three forms and the Yenisei River A baerii indicates that although the three forms of A. gueldenstaedtii are not different from one another, all three significantly differ from the Yenisei River A. baerii. Competing explanations, including translocation and centre of origin hypotheses are considered. The three geneticforms of A. gueldenstaedtii likely colonized different geographic areas during different geological periods, and subsequently evolved in these regions independently into the species currently recognized as A. gueldenstaedtii, A. naccarii and A. baerii.     

Recruitment of human cord blood-derived endothelial colony-forming cells to sites of tumor angiogenesis

Background aimsEndothelial progenitor cells (EPCs) specifically home to sites of malignant growth, rendering them attractive for anti-cancer therapies. Data are conflicting on the phenotype and quantitative contribution toward tumor angiogenesis based on differing culture assays to outgrow EPCs. To evaluate the origin and early phenotype of EPCs and to define a population with enhanced tumor-targeting capacity, we evaluated a hierarchy of cord blood-derived EPCs modeling the multi-step nature of tumor homing.MethodsCD34⁺ mononuclear cells were isolated from fresh cord blood and cultured to derive endothelial colony-forming cells (ECFCs). Human umbilical vein endothelial cells (HUVECs) served as control. Using intra-vital microscopy, the recruitment was analyzed in mice bearing C6 xenografts. Adhesion, migration, transmigration and differentiation were further addressed.ResultsWithin the primary passage, ECFCs underwent a rapid maturation from a CD45⁺ and CD31⁺ phenotype to a CD45^? and endothelial marker positive phenotype. Assessing in vivo tumor recruitment, ECFCs had the highest activity in all steps analyzed. In vitro, ECFCs demonstrated significantly higher adhesion under static and flow conditions. Similarly, ECFCs exhibited highest migratory and trans-migratory activity toward tumor-conditioned medium. On subcutaneous implantation, only ECFCs formed blood vessels covered with perivascular cells, similar to HUVECs.ConclusionsOur study indicates that ECFCs emerge from a CD45⁺ and CD31⁺ progenitor and rapidly mature in culture. ECFCs have a significantly higher potential for tumor targeting than non-cultured CD34⁺ cells and HUVECs. They are ideal candidates for future cell-based anti-cancer therapies.     

The New Caledonian genus Caledonotrichia Sykora (Trichoptera,Insecta) reviewed,with?descriptions?of?6?new species

The New Caledonian endemic hydroptilid genus Caledonotrichia Sykora (Trichoptera) is reviewed and 6 new species are described: Caledonotrichia bifida, Caledonotrichia capensis, Caledonotrichia minuta, Caledonotrichia ouinnica, Caledonotrichia sykorai and Caledonotrichia vexilla. Together with the established species for which revised diagnoses are given, these raise to 11 the number of species known in this genus. The new species, females of 3 species, and several unusual larval cases are examined and described for further insight into relationships of this enigmatic genus. A key to species is provided.     

The transcription factor Grainyhead like 3 (GRHL3) affects endothelial cell apoptosis and migration in a NO-dependent manner

Migratory capacity and resistance to apoptosis are crucial for proper endothelial function. In a screen for anti-apoptotic genes in a breast cancer cell line, we identified Grainyhead like 3 (GRHL3). Therefore, the aim of our study was to investigate whether GRHL3 is expressed in endothelial cells and moreover, to determine its role in migration, apoptosis and senescence. GRHL3 is expressed in human endothelial cells. GRHL3 is required for endothelial cell migration. The underlying mechanism is independent of vascular endothelial growth factor. GRHL3 induces Akt and endothelial nitric oxide synthase phosphorylation and its expression is increased by physiological concentrations of nitric oxide. Nitric oxide dependent migration is completely dependent on GRHL3 expression. Moreover, GRHL3 inhibits apoptosis of endothelial cells in an eNOS-dependent manner. Thus, loss of GRHL3 may result in endothelial dysfunction in vivo. One may consider new therapeutic strategies with the aim to conserve GRHL3 expression in the vasculature.     

Palaeoecology in a mud-dominated epicontinental sea: A case study of the Ordovician Elnes Formation,southern Norway

The Ordovician (Darriwilian to locally Sandbien) Elnes Formation of the Oslo Region, Norway, is dominated by dark grey, often marly and partly graptolite bearing mudstones. These were formed in a mid- to outer shelf environment at water-depths from perhaps less than 50 to over 200 m. More than 23,000 fossils have been systematically collected from three sections through the formation and seven fossil associations are recognised comprising the Endoceratid, Plectorthid–Diplotrypa, Asaphus–orthid, Asaphid–trinucleid (including the Raphiophorid–nileid and Alwynella–trinucleid sub-associations), Cathrynia–lingulid, Alwynella–lingulid and the Graptolite–lingulid associations. These correlate with specific lithofacies and reflect a depth transect. The ecological preferences inferred for each of the faunal groups agree well with studies of other Ordovician faunas, clearly supporting the note of a general similarity in the eco-faunal composition on a global scale. Changes in palaeo-depth during deposition of the Elnes Formation are to some extent out-of-phase with the eustatic sea level changes inferred for this time interval, probably reflecting ongoing local tectonic processes in the Oslo area. This is ascribed to the development of distal foreland conditions in the Oslo Region, heralding the Caledonian Orogeny.     

Binding of albumin promotes bacterial survival at the epithelial surface

Human serum albumin (HSA) is the dominating protein in human plasma. Many bacterial species, especially streptococci, express surface proteins that bind HSA with high specificity and affinity, but the biological consequences of these protein-protein interactions are poorly understood. Group G streptococci (GGS), carrying the HSA-binding protein G, colonize the skin and the mucosa of the upper respiratory tract, mostly without causing disease. In the case of bacterial invasion, pro-inflammatory cytokines are released that activate the epithelium to produce antibacterial peptides, in particular the chemokine MIG/CXCL9. In addition, the inflammation causes capillary leakage and extravasation of HSA and other plasma proteins, environmental changes at the epithelial surface to which the bacteria need to respond. In this study, we found that GGS adsorbed HSA from both saliva and plasma via binding to protein G and that HSA bound to protein G bound and inactivated the antibacterial MIG/CXCL9 peptide. Another surface protein of GGS, FOG, was found to mediate adherence of the bacteria to pharyngeal epithelial cells through interaction with glycosaminoglycans. This adherence was not affected by activation of the epithelium with a combination of IFN-γ and TNF-α, leading to the production of MIG/CXCL9. However, at the activated epithelial surface, adherent GGS were protected against killing by MIG/CXCL9 through protein G-dependent HSA coating. The findings identify a previously unknown bacterial survival strategy that helps to explain the evolution of HSA-binding proteins among bacterial species of the normal human microbiota.     

Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-TolC

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.