Novel in vitro assays for the characterization of EMT in tumourigenesis

Background Two novel assays quantifying Epithelial to Mesenchymal Transition (EMT) were compared to traditional motility and migration assays. TGF-ß1 treatment of AY-27 rat bladder cancer cells acted as a model of EMT in tumourigenesis. Methods AY-27 rat bladder cancer cells incubated with 3ng/ml TGF-ß1 or control media for 24 or 48h were assessed using novel and traditional assays. The Spindle Index, a novel measure of spindle phenotype, was derived from the ratio of maximum length to maximum width of cells. The area covered by cells which migrated from a fixed coverslip towards supplemented agarose was measured in a novel chemoattractant assay. Motility, migration and immunoreactivity for E-cadherin, Vimentin and cytokeratin were assessed. Results TGF-ß1 treated cells had increased “spindle” phenotype together with decreased E-cadherin, decreased Cytokeratin-18 and increased Vimentin immunoreactivity. After 48h, the mean Spindle Index of TGF-ß1 treated cells was significantly higher than Mock (p=0.02 Bonferroni test) and there were significant differences in migration across treatment groups measured using the novel chemoattractant assay (p = 0.02, Chi-Square). TGF-ß1 significantly increased matrigel invasion. Conclusion The Spindle Index and the novel chemoattractant assay are valuable adjunctive assays for objective characterization of EMT changes during tumourigenesis.

Impaired Immune Tolerance to Porphyromonas gingivalis Lipopolysaccharide Promotes Neutrophil Migration and Decreased Apoptosis.

Periodontitis, a chronic inflammatory disease of the tissues supporting the teeth, is characterized by an exaggerated host immune and inflammatory response to periopathogenic bacteria. Toll-like receptor activation, cytokine network induction, and accumulation of neutrophils at the site of inflammation are important in the host defense against infection. At the same time, induction of immune tolerance and the clearance of neutrophils from the site of infection are essential in the control of the immune response, resolution of inflammation, and prevention of tissue destruction. Using a human monocytic cell line, we demonstrate that Porphyromonas gingivalis lipopolysaccharide (LPS), which is a major etiological factor in periodontal disease, induces only partial immune tolerance, with continued high production of interleukin-8 (IL-8) but diminished secretion of tumor necrosis factor alpha (TNF-) after repeated challenge. This cytokine response has functional consequences for other immune cells involved in the response to infection. Primary human neutrophils incubated with P. gingivalis LPS-treated naïve monocyte supernatant displayed a high migration index and increased apoptosis. In contrast, neutrophils treated with P. gingivalis LPS-tolerized monocyte supernatant showed a high migration index but significantly decreased apoptosis. Overall, these findings suggest that induction of an imbalanced immune tolerance in monocytes by P. gingivalis LPS, which favors continued secretion of IL-8 but decreased TNF- production, may be associated with enhanced migration of neutrophils to the site of infection but also with decreased apoptosis and may play a role in the chronic inflammatory state seen in periodontal disease.

Antibody Targeting of Cathepsin S Inhibits Angiogenesis and Synergistically Enhances Anti-VEGF

BACKGROUND: Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development.

METHODOLOGY/PRINCIPAL FINDINGS: Cathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed.

CONCLUSIONS/SIGNIFICANCE: Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.

Local, European and global: An exploration of migration patterns of social workers into Ireland

Whilst the project to increase workforce mobility is part of a common labour force policy across the European Union, for some social workers, it has also been coupled with an aspiration to develop a pan-European identity within the profession (Lorenz, 1994, 2000; Frost, 2008). In this article, taking the island of Ireland as an example, we examine empirical data on the movement of social workers in recent years, both within Ireland and inward from outside the island, and consider the challenges and opportunities this has presented for the profession, also drawing on the British experience. From this analysis, some tentative conclusions are drawn about what is happening, what is possible and what is desirable in balancing aspirations for increased mobility within the European Union and a pan-European social work identity against the needs and interests of local, European and global communities.

Getting off the escalator? A study of Scots out-migration from a global city region

Something new is happening to reverse the historical trend of skilled Scots moving to London for career progression. The Scottish population of London and the South East is falling and this despite Scots enjoying continued occupational success within the South East labour market. The authors ask why Scots are leaving the UK's main escalator region and then investigate how these migration changes can best be theorised relative to literature on the mobility of the 'new service class'. Building on Fielding's escalator region hypothesis, the authors report on recent research on longer distance flows out of the UK's main escalator region. They advance the critique of the escalator region hypothesis set out by Findlay et al and ask why people would leave a global city offering good opportunities for occupational mobility. Demographic regime change provides only a partial answer. Other explanations can be found in the changing mobilities of the new service class as they engage in what Smith has defined as 'translocal' and 'transnational' urbanism. The authors argue that Scotland's changing relationship with London and the South East may be representative of a wider set of changes in migration linkages between regional economies and global cities.

Computer simulations of seasonal outbreak and diurnal vertical migration of cyanobacteria

Algal blooms caused by cyanobacteria are characterized by two features with different time scales: one is seasonal outbreak and collapse of a bloom and the other is diurnal vertical migration. Our two-component mathematical model can simulate both phenomena, in which the state variables are nutrients and cyanobacteria. The model is a set of one-dimensional reaction-advection-diffusion equations, and temporal changes of these two variables are regulated by the following five factors: (1) annual variation of light intensity, (2) diurnal variation of light intensity, (3) annual variation of water temperature, (4) thermal stratification within a water column and (5) the buoyancy regulation mechanism. The seasonal change of cyanobacteria biomass is mainly controlled by factors, (1), (3) and (4), among which annual variations of light intensity and water temperature directly affect the maximum growth rate of cyanobacteria. The latter also contributes to formation of the thermocline during the summer season. Thermal stratification causes a reduction in vertical diffusion and largely prevents mixing of both nutrients and cyanobacteria between the epilimnion and the hypolimnion. Meanwhile, the other two factors, (2) and (5), play a significant role in diurnal vertical migration of cyanobacteria. A key mechanism of vertical migration is buoyancy regulation due to gas-vesicle synthesis and ballast formation, by which a quick reversal between floating and sinking becomes possible within a water column. The mechanism of bloom formation controlled by these five factors is integrated into the one-dimensional model consisting of two reaction-advection-diffusion equations.

First records of oceanic dive profiles for leatherback turtles, Dermochelys coriacea, indicate behavioural plasticity associated with long-distance migration

We used Satellite Relay Data Loggers to obtain the first dive profiles for critically endangered leatherback turtles outside the nesting season. As individuals moved from the Caribbean out into the Atlantic, key aspects of their diving behaviour changed markedly, in line with theoretical predictions for how dive duration should vary with foraging success. In particular, in the Atlantic, where foraging success is expected to be higher, dives became much longer than in the Caribbean. The deepest-ever dive profile recorded for a reptile was obtained in the oceanic Atlantic, with a 54-min dive to 626 m on 26 August 2002. However, dives were typically much shallower (generally

Modulational instability of dust acoustic waves in dusty plasmas: Modulation obliqueness, background ion nonthermality, and dust charging effects

The oblique modulational instability of dust acoustic (DA) waves in an unmagnetized warm dusty plasma with nonthermal ions, taking into account dust grain charge variation (charging), is investigated. A nonlinear Schrodinger-type equation governing the slow modulation of the wave amplitude is derived. The effects of dust temperature, dust charge variation, ion deviation from Maxwellian equilibrium (nonthermality) and constituent species' concentration on the modulational instability of DA waves are examined. It is found that these parameters modify significantly the oblique modulational instability domain in the k-theta plane. Explicit expressions for the instability rate and threshold have been obtained in terms of the dispersion laws of the system. The possibility and conditions for the existence of different types of localized excitations are also discussed. The findings of this investigation may be useful in understanding the stable electrostatic wave packet acceleration mechanisms close to the Moon, and also enhances our knowledge on the occurrence of instability associated to pickup ions around unmagnetized bodies, such as comets, Mars, and Venus.

Somatic point mutations in mtDNA control region are influenced by genetic background and associated with healthy aging: a GEHA stud

Tissue specific somatic mutations occurring in the mtDNA control region have been proposed to provide a survival advantage. Data on twins and on relatives of long-lived subjects suggested that the occurrence/accumulation of these mutations may be genetically influenced. To further investigate control region somatic heteroplasmy in the elderly, we analyzed the segment surrounding the nt 150 position (previously reported as specific of Leukocytes) in various types of leukocytes obtained from 195 ultra-nonagenarians sib-pairs of Italian or Finnish origin collected in the frame of the GEHA Project. We found a significant correlation of the mtDNA control region heteroplasmy between sibs, confirming a genetic influence on this phenomenon. Furthermore, many subjects showed heteroplasmy due to mutations different from the C150T transition. In these cases heteroplasmy was correlated within sibpairs in Finnish and northern Italian samples, but not in southern Italians. This suggested that the genetic contribution to control region mutations may be population specific. Finally, we observed a possible correlation between heteroplasmy and Hand Grip strength, one of the best markers of physical performance and of mortality risk in the elderly. Our study provides new evidence on the relevance of mtDNA somatic mutations in aging and longevity and confirms that the occurrence of specific point mutations in the mtDNA control region may represent a strategy for the age-related remodelling of organismal functions.

The Pleiotropic Effects of Simvastatin on Retinal Microvascular Endothelium Has Important Implications for Ischaemic Retinopathies

Background: Current guidelines encourage the use of statins to reduce the risk of cardiovascular disease in diabetic patients; however the impact of these drugs on diabetic retinopathy is not well defined. Moreover, pleiotropic effects of statins on the highly specialised retinal microvascular endothelium remain largely unknown. The objective of this study was to investigate the effects of clinically relevant concentrations of simvastatin on retinal endothelium in vitro and in vivo.

Methods and Findings: Retinal microvascular endothelial cells (RMECs) were treated with 0.01–10 µM simvastatin and a biphasic dose-related response was observed. Low concentrations enhanced microvascular repair with 0.1 µM simvastatin significantly increasing proliferation (p<0.05), and 0.01 µM simvastatin significantly promoting migration (p<0.05), sprouting (p<0.001), and tubulogenesis (p<0.001). High concentration of simvastatin (10 µM) had the opposite effect, significantly inhibiting proliferation (p<0.01), migration (p<0.01), sprouting (p<0.001), and tubulogenesis (p<0.05). Furthermore, simvastatin concentrations higher than 1 µM induced cell death. The mouse model of oxygen-induced retinopathy was used to investigate the possible effects of simvastatin treatment on ischaemic retinopathy. Low dose simvastatin(0.2 mg/Kg) promoted retinal microvascular repair in response to ischaemia by promoting intra-retinal re-vascularisation (p<0.01). By contrast, high dose simvastatin(20 mg/Kg) significantly prevented re-vascularisation (p<0.01) and concomitantly increased pathological neovascularisation (p<0.01). We also demonstrated that the pro-vascular repair mechanism of simvastatin involves VEGF stimulation, Akt phosphorylation, and nitric oxide production; and the anti-vascular repair mechanism is driven by marked intracellular cholesterol depletion and related disorganisation of key intracellular structures.

Conclusions: A beneficial effect of low-dose simvastatin on ischaemic retinopathy is linked to angiogenic repair reducing ischaemia, thereby preventing pathological neovascularisation. High-dose simvastatin may be harmful by inhibiting reparative processes and inducing premature death of retinal microvascular endothelium which increases ischaemia-induced neovascular pathology. Statin dosage should be judiciously monitored in patients who are diabetic or are at risk of developing other forms of proliferative retinopathy.