Parallel development and characterisation of an anti-oxidant stent coating and an in vitro biological model for qualitative assessment

Restenosis is a major cause of coronary artery stent failure and is linked to vascular endothelium damage with resultant oxidative and inflammatory stress. Drug eluting stents (DES) have failed to eliminate this risk; particularly for diabetic patients. Here, we have assessed the potential therapeutic effects of a novel antioxidant stent coating using (i) a common chemical assay, DPPH (2,2-diphenyl-1-picrylhydrazyl) and (ii) superoxide scavenging ability using NBT (nitroblue tetrazolium) reduction and have identified high antioxidant potential that is not dependent on drug incorporation within the coating. To assess the biological effect of this novel antioxidant coating, we have initially used human umbilical vein endothelial cells (HUVECs) treated with pro-inflammatory cytokines to mimic the inflammation stress encountered post-stent placement. Pro-inflammatory signalling was assessed by measuring phospho-P65 (pP65) expression using quantitative western blotting and oxidative stress assessed by measuring reactive oxygen species (ROS) generation using DCFDA. In addition, we have specifically examined expression and activation (via oxidation) of an enzyme called Ca2+/Calmodulin-dependent protein kinase II-delta (CaMKIIδ) that is known to be a central component of vascular pathology during acute and chronic inflammation and oxidative stress. HUVECs were stimulated with pro-inflammatory cytokines, Tissue Necrotic Factor alpha (TNFα) and Interleukin 1-beta (IL-1β). Stimulation with IL-1β (10 ng/ml) for 1h resulted in the highest overall level of P65 phosphorylation (7.80 ± 1.41 (fold increase ± S.E.M. in pP65 expression in IL-1β -stimulated cells c.f. unstimulated controls, n=4, p<0.05). Conversely, stimulation with TNFα (10 ng/ml) for 6h led to the highest overall increase in ROS (5.02 ± 0.54, fold-stimulation ± S.E.M. in ROS in TNFα -stimulated cells c.f. unstimulated controls, n=3, p<0.05). We have shown that CaMKIIδ is highly expressed in HUVECs and that stimulation with IL-1β (10 ng/ml) for 3h significantly induced CaMKII oxidation (1.56 ± 0.06 (fold-increase ± S.E.M. in oxCaMKII in IL-1β stimulated cells c.f. unstimulated controls, n=4, p<0.05). Preventing the oxidation-induced activation of CaMKII may present a therapeutic mechanism by which antioxidant stents can improve endothelial recovery and future work will examine the reduction or reversal of this effect in the presence of our novel stent coating. In conclusion, we have developed a novel anti-oxidant stent coating and established an in vitro system to test biological activity. This approach will now be applied to more physiologically relevant cell types before examining the novel coating’s efficacy

Characterising endothelial CaMKII oxidation in a cellular model of inflammation to investigate the therapeutic potential of a novel anti-oxidant stent coating

Drug eluting stent use is widespread but restenosis due to endothelial dysfunction remains a challenge. Ca2+/Calmodulin-dependent protein kinase II-delta (CaMKIIδ) is implicated as a key modulator of cardiovascular pathology and hyper-activation of CaMKIIδ promotes endothelial inflammation and oxidative stress [1]. This study has characterised different endothelial cell responses for the purpose of testing a novel antioxidant stent coating. Interleukin 1-beta (IL-1β) or tissue necrosis factor-alpha (TNFα) have been used to mimic pro-inflammatory signalling and CaMKII activation observed following endothelial stress. Human umbilical vein and coronary artery endothelial cells (HUVECs and HCAECs) were treated with IL-1β or TNFα (both 10ng/ml) up to 6 h. Quantitative immunoblotting was used to assess pro-inflammatory signalling, phospho-P65 (pP65; Cell Signalling) and CaMKII activation, phospho-CaMKII (pCaMKII; Thermo Fisher) and oxidised-CaMKII (oxCaMKII; GeneTex) expression. Increased pP65 expression was observed following 30 min stimulation with either IL-1β or TNFα in HUVECs[2]. Similarly, significant increases in pP65 expression were observed in HCAECs following stimulation with both cytokines (IL-1β: 1.93±0.29 and TNFα: 1.54±0.15 mean fold-change c.f. control ±S.E.M; n=4, p<0.05). This indicates both cell types produce similar pro-inflammatory responses following cytokine stimulation. In contrast, CaMKII activation in response to cytokine stimulation was different across cell types. Significant activation (via oxidation) of CaMKII was observed in HUVECs only after IL-1β stimulation[2]. In HCAECs however, activation of CaMKII was observed only after TNFα stimulation, via both oxidation (TNFα (6h): 1.50±0.12, mean fold-change c.f. control ±S.E.M; n=4, p<0.05), and phosphorylation (TNFα (30min): 1.22±0.05, mean fold-change c.f. control ±S.E.M; n=4, p<0.05).This work highlights the differences in responses of HUVECs and HCAECs to stimulation by inflammatory cytokines. Results demonstrate the importance of considering the endothelial model used for research into therapeutic intervention

Scientists' warning to humanity on illegal or unsustainable wildlife trade

Illegal or unsustainable wildlife trade is growing at a global level, threatening the traded species and coexisting biota, and promoting the spread of invasive species. From the loss of ecosystem services to diseases transmitted from wildlife to humans, or connections with major organized crime networks and disruption of local to global economies, its ramifications are pervading our daily lives and perniciously affecting our well-being. Here we build on the manifesto 'World Scientists' Warning to Humanity, issued by the Alliance of World Scientists. As a group of researchers deeply concerned about the consequences of illegal or unsustainable wildlife trade, we review and highlight how these can negatively impact species, ecosystems, and society. We appeal for urgent action to close key knowledge gaps and regulate wildlife trade more stringently.Peer reviewe

Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags (‘barcodes’). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three ‘bait’ sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species ‘barcodes’ that currently use the cox1 gene only

Developing Feature Types and Related Catalogues for the Marine Community - Lessons from the MOTIIVE project.

MOTIIVE (Marine Overlays on Topography for annex II Valuation and Exploitation) is a project funded as a Specific Support Action (SSA) under the European Commission Framework Programme 6 (FP6) Aeronautics and Space Programme. The project started in September 2005 and finished in October 2007. The objective of MOTIIVE was to examine the methodology and cost benefit of using non-proprietary data standards. Specifically it considered the harmonisation requirements between the INSPIRE data component ‘elevation’ (terrestrial, bathymetric and coastal) and INSPIRE marine thematic data for ‘sea regions’, ‘oceanic spatial features’ and ‘coastal zone management areas’. This was examined in context of the requirements for interoperable information systems as required to realise the objectives of GMES for ‘global services’. The work draws particular conclusions on the realisation of Feature Types (ISO 19109) and Feature Type Catalogues (ISO 19110) in this respect. More information on MOTIIVE can be found at www.motiive.net