GDF‐15 is a better complimentary marker for risk stratification of arrhythmic death in non‐ischaemic,dilated cardiomyopathy than soluble ST2

Growth differentiation factor (GDF)‐15 and soluble ST2 (sST2) are established prognostic markers in acute and chronic heart failure. Assessment of these biomarkers might improve arrhythmic risk stratification of patients with non‐ischaemic, dilated cardiomyopathy (DCM) based on left ventricular ejection fraction (LVEF). We studied the prognostic value of GDF‐15 and sST2 for prediction of arrhythmic death (AD) and all‐cause mortality in patients with DCM. We prospectively enrolled 52 patients with DCM and LVEF ≤ 50%. Primary end‐points were time to AD or resuscitated cardiac arrest (RCA), and secondary end‐point was all‐cause mortality. The median follow‐up time was 7 years. A cardiac death was observed in 20 patients, where 10 patients had an AD and 2 patients had a RCA. One patient died a non‐cardiac death. GDF‐15, but not sST2, was associated with increased risk of the AD/RCA with a hazard ratio (HR) of 2.1 (95% CI = 1.1‐4.3; P = .031). GDF‐15 remained an independent predictor of AD/RCA after adjustment for LVEF with adjusted HR of 2.2 (95% CI = 1.1‐4.5; P = .028). Both GDF‐15 and sST2 were independent predictors of all‐cause mortality (adjusted HR = 2.4; 95% CI = 1.4‐4.2; P = .003 vs HR = 1.6; 95% CI = 1.05‐2.7; P = .030). In a model including GDF‐15, sST2, LVEF and NYHA functional class, only GDF‐15 was significantly associated with the secondary end‐point (adjusted HR = 2.2; 95% CI = 1.05‐5.2; P = .038). GDF‐15 is superior to sST2 in prediction of fatal arrhythmic events and all‐cause mortality in DCM. Assessment of GDF‐15 could provide additional information on top of LVEF and help identifying patients at risk of arrhythmic death.     

TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

The oncotropism of Minute Virus of Mice (MVMp) is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs) in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR) involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs) as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN) of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9^+/+), our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal sensing of the parvovirus genomes by TLR-9.     

Wnt signaling is required for early development of zebrafish swimbladder

BACKGROUND: Wnt signaling plays critical roles in mammalian lung development. However, Wnt signaling in the development of the zebrafish swimbladder, which is considered as a counterpart of mammalian lungs, have not been explored. To investigate the potential conservation of signaling events in early development of the lung and swimbladder, we wish to address the question whether Wnt signaling plays a role in swimbladder development. METHODOLOGY/PRINCIPAL FINDINGS: For analysis of zebrafish swimbladder development, we first identified, by whole-mount in situ hybridization (WISH), has2 as a mesenchymal marker, sox2 as the earliest epithelial marker, as well as hprt1l and elovl1a as the earliest mesothelial markers. We also demonstrated that genes encoding Wnt signaling members Wnt5b, Fz2, Fz7b, Lef1, Tcf3 were expressed in different layers of swimbladder. Then we utilized the heat-shock inducible transgenic lines hs:Dkk1-GFP and hs:ΔTcf-GFP to temporarily block canonical Wnt signaling. Inhibition of canonical Wnt signaling at various time points disturbed precursor cells specification, organization, anterioposterior patterning, and smooth muscle differentiation in all three tissue layers of swimbladder. These observations were also confirmed by using a chemical inhibitor (IWR-1) of Wnt signaling. In addition, we found that Hedgehog (Hh) signaling was activated by canonical Wnt signaling and imposed a negative feedback on the latter. SIGNIFICANCE/CONCLUSION: We first provided a new set of gene markers for the three tissue layers of swimbladder in zebrafish and demonstrated the expression of several key genes of Wnt signaling pathway in developing swimbladder. Our functional analysis data indicated that Wnt/β-catenin signaling is required for swimbladder early development and we also provided evidence for the crosstalk between Wnt and Hh signaling in early swimbladder development.     

Bioactive metabolites from the endophytic fungus Ampelomyces sp. isolated from the medicinal plant Urospermum picroides

Extracts of cultures grown in liquid or on solid rice media of the fungal endophyte Ampelomyces sp. isolated from the medicinal plant Urospermum picroides exhibited considerable cytotoxic activity when tested in vitro against L5178Y cells. Chromatographic separation yielded 14 natural products that were unequivocally identified based on their ¹H and ¹³C NMR as well as mass spectra and comparison with previously published data. Six compounds (2, 4, 5, 7, 9 and 11) were natural products. Both fungal extracts differed considerably in their secondary metabolites. The extract obtained from liquid cultures afforded a pyrone (2) and sulfated anthraquinones (7 and 9) along with the known compounds 1, 3, 6 and 8. When grown on solid rice medium the fungus yielded three compounds 4, 5 and 11 in addition to several known metabolites including 6, 8, 10, 12, 13 and 14. Compounds 4, 8 and 10 showed the strongest cytotoxic activity against L5178Y cells with EC₅₀ values ranging from 0.2–7.3 μg/ml. Furthermore, 8 and 10 displayed antimicrobial activity against the Gram-positive pathogens, Staphylococcus aureus, S. epidermidis and Enterococcus faecalis at minimal inhibitory concentrations (MIC) of 12.5 μg/ml and 12.5–25 μg/ml, respectively. Interestingly, 6 and 8 were also identified as constituents of an extract derived from a healthy plant sample of the host plant U. picroides thereby indicating that the production of bioactive natural products by the endophyte proceeds also under in situ conditions within the host plant.     

A human corneal equivalent constructed from SV40-immortalised corneal cell lines

Within the last decade, extensive research in the field of tissue and organ engineering has focused on the development of in vitro models of the cornea. The use of organotypic, three-dimensional corneal equivalents has several advantages over simple monolayer cultures. The aim of this study was to develop a corneal equivalent model composed of the same cell types as in the natural human tissue, but by using immortalised cell lines to ensure reproducibility and to minimise product variation. We report our success in the establishment of an SV40-immortalised human corneal keratocyte cell line (designated HCK). A collagen matrix, built up with these cells, displayed the morphological characteristics of the human stromal tissue and served as a biomatrix for the immortalised human corneal epithelial and endothelial cells. Histological cross-sections of the whole-cornea equivalents resemble human corneas in tissue structure. This organotypic in vitro model may serve as a research tool for the ophthalmic science community, as well as a model system for testing for eye irritancy and drug efficacy.     

Insight into the expression of toll-like receptors 2 and 4 in patients with abdominal aortic aneurysm

Molecular Biology Reports - An abdominal aortic aneurysm (AAA) is a relatively common, life-threatening disease prevalent in persons over the age of 65. In recent years, an increasing number of...     

Co-Culture of Neural Crest Stem Cells (NCSC) and Insulin Producing Beta-TC6 Cells Results in Cadherin Junctions and Protection against Cytokine-Induced Beta-Cell Death

Purpose

Transplantation of pancreatic islets to Type 1 diabetes patients is hampered by inflammatory reactions at the transplantation site leading to dysfunction and death of insulin producing beta-cells. Recently we have shown that co-transplantation of neural crest stem cells (NCSCs) together with the islet cells improves transplantation outcome. The aim of the present investigation was to describe in vitro interactions between NCSCs and insulin producing beta-TC6 cells that may mediate protection against cytokine-induced beta-cell death.

Procedures

Beta-TC6 and NCSC cells were cultured either alone or together, and either with or without cell culture inserts. The cultures were then exposed to the pro-inflammatory cytokines IL-1β and IFN-γ for 48 hours followed by analysis of cell death rates (flow cytometry), nitrite production (Griess reagent), protein localization (immunofluorescence) and protein phosphorylation (flow cytometry).

Results

We observed that beta-TC6 cells co-cultured with NCSCs were protected against cytokine-induced cell death, but not when separated by cell culture inserts. This occurred in parallel with (i) augmented production of nitrite from beta-TC6 cells, indicating that increased cell survival allows a sustained production of nitric oxide; (ii) NCSC-derived laminin production; (iii) decreased phospho-FAK staining in beta-TC6 cell focal adhesions, and (iv) decreased beta-TC6 cell phosphorylation of ERK(T202/Y204), FAK(Y397) and FAK(Y576). Furthermore, co-culture also resulted in cadherin and beta-catenin accumulations at the NCSC/beta-TC6 cell junctions. Finally, the gap junction inhibitor carbenoxolone did not affect cytokine-induced beta-cell death during co-culture with NCSCs.

Conclusion

In summary, direct contacts, but not soluble factors, promote improved beta-TC6 viability when co-cultured with NCSCs. We hypothesize that cadherin junctions between NCSC and beta-TC6 cells promote powerful signals that maintain beta-cell survival even though ERK and FAK signaling are suppressed. It may be that future strategies to improve islet transplantation outcome may benefit from attempts to increase beta-cell cadherin junctions to neighboring cells.     

DNA repair of clustered lesions in mammalian cells: involvement of non-homologous end-joining

Clustered lesions are defined as ≥two lesions within 20 bps and are generated in DNA by ionizing radiation. In vitro studies and work in bacteria have shown that attempted repair of two closely opposed lesions can result in the formation of double strand breaks (DSBs). Since mammalian cells can repair DSBs by non-homologous end-joining (NHEJ), we hypothesized that NHEJ would repair DSBs formed during the removal of clustered tetrahydrofurans (furans). However, two opposing furans situated 2, 5 or 12 bps apart in a firefly luciferase reporter plasmid caused a decrease in luciferase activity in wild-type, Ku80 or DNA-PKcs-deficient cells, indicating the generation of DSBs. Loss of luciferase activity was maximal at 5 bps apart and studies using siRNA implicate the major AP endonuclease in the initial cleavage. Since NHEJ-deficient cells had equivalent luciferase activity to their isogenic wild-type cells, NHEJ was not involved in accurate repair of clustered lesions. However, quantitation and examination of re-isolated DNA showed that damage-containing plasmids were inaccurately repaired by Ku80-dependent, as well as Ku80-independent mechanisms. This work indicates that not even NHEJ can completely prevent the conversion of clustered lesions to potentially lethal DSBs, so demonstrating the biological relevance of ionizing radiation-induced clustered damage.     

Evidence for plant viruses in the region of Argentina Islands, Antarctica

This work focused on the assessment of plant virus occurrence among primitive and higher plants in the Antarctic region. Sampling occurred during two seasons (2004/5 and 2005/6) at the Ukrainian Antarctic Station 'Academician Vernadskiy' positioned on Argentina Islands. Collected plant samples of four moss genera (Polytrichum, Plagiatecium, Sanionia and Barbilophozia) and one higher monocot plant species, Deschampsia antarctica, were further subjected to enzyme-linked immunosorbent assay to test for the presence of common plant viruses. Surprisingly, samples of Barbilophozia and Polytrichum mosses were found to contain antigens of viruses from the genus Tobamovirus, Tobacco mosaic virus and Cucumber green mottle mosaic virus, which normally parasitize angiosperms. By contrast, samples of the monocot Deschampsia antarctica were positive for viruses typically infecting dicots: Cucumber green mottle mosaic virus, Cucumber mosaic virus and Tomato spotted wilt virus. Serological data for Deschampsia antarctica were supported in part by transmission electron microscopy observations and bioassay results. The results demonstrate comparatively high diversity of plant viruses detected in Antarctica; the results also raise questions of virus specificity and host susceptibility, as the detected viruses normally infect dicotyledonous plants. However, the means of plant virus emergence in the region remain elusive and are discussed.