An in-vitro model for studying the interaction of Escherichia coli O157:H7 and other enteropathogens with bovine primary cell cultures

Sections of kidney, trachea, ileum, colon, rectum and rumen were removed at post mortem from a neonatal calf and, with the exception of the rumen, primary cell lines were established for each of the cell types. The adherence of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, enteropathogenic E. coli (EPEC) serotype O111, E. coli K12 (a laboratory adapted non-pathogenic strain) and Salmonella enterica serotype Typhimurium was assayed on each cell type. For all adherence assays on all cell lines, EHEC O157:H7 adhered to a significantly greater extent than the other bacteria. S. Typhimurium and EPEC O111 adhered to a similar extent to one another, whereas E. coli K12 was significantly less adherent by 100-fold. In all cell types, > 10% of adherent S. Typhimurium bacteria invaded, whereas c. 0.01-0.1% of adherent EHEC O157:H7 and EPEC O111 bacteria invaded, although they are regarded as non-invasive. EHEC O157 generated actin re-arrangements in all cell types as demonstrated by fluorescent actin staining (FAS) under densely packed bacterial micro-colonies. EPEC O111 readily generated the localised adherent phenotype on bovine cells but generated only densely packed micro-colonies on HEp-2 cells. The intensity of actin re-arrangements induced in bovine cells by EPEC O111 was less than that induced by EHEC O157:H7. The intimate attachment on all cell types by both EHEC O157:H7 and EPEC O111 was clearly demonstrated by scanning electron microscopy.

Membrane ruffling and invasion of human and avian cell lines is reduced for aflagellate mutants of Salmonella enterica serotype Enteritidis

Independent studies have demonstrated that flagella are associated with the invasive process of Salmonella enterica serotypes, and aflagellate derivatives of Salmonella enterica serotype Enteritidis are attenuated in murine and avian models of infection. One widely held view is that the motility afforded by flagella, probably aided by chemotactic responses, mediates the initial interaction between bacterium and host cell. The adherence and invasion properties of two S. Enteritidis wild-type strains and isogenic aflagellate mutants were assessed on HEp-2 and Div-1 cells that are of human and avian epithelial origin, respectively. Both aflagellate derivatives showed a significant reduction of invasion compared with wild type over the three hours of the assays. Complementation of the defective fliC allele recovered partially the wild-type phenotype. Examination of the bacterium-host cell interaction by electron and confocal microscopy approaches showed that wild-type bacteria induced ruffle formation and significant cytoskeletal rearrangements on HEp-2 cells within 5 minutes of contact. The aflagellate derivatives induced fewer ruffles than wild type. Ruffle formation on the Div-1 cell line was less pronounced than for HEp-2 cells for wild-type S. Enteritidis. Collectively, these data support the hypothesis that flagella play an active role in the early events of the invasive process.

A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in an hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main precursor of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homestasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

Regulation of cell migration in cancer: investigation into the regulation of cancer cell blebbing in the extracellular matrix

Cell migration is a highly coordinated process and any aberration in the regulatory mechanisms could result in pathological conditions such as cancer. The ability of cancer cells to disseminate to distant sites within the body has made it difficult to treat. Cancer cells also exhibit plasticity that makes them able to interconvert from an elongated, mesenchymal morphology to an amoeboid blebbing form under different physiological conditions. Blebs are spherical membrane protrusions formed by actomyosin-mediated contractility of cortical actin resulting in increased hydrostatic pressure and subsequent detachment of the membrane from the cortex. Tumour cells use blebbing as an alternative mode of migration by squeezing through preexisting gaps in the ECM, and bleb formation is believed to be mediated by the Rho-ROCK signaling pathway. However, the involvement of transmembrane water and ion channels in cell blebbing has not been examined. In the present study, the role of the transmembrane water channels, aquaporins, transmembrane ion transporters and lipid signaling enzymes in the regulation of blebbing was investigated. Using 3D matrigel matrix as an in vitro model to mimic normal extracellular matrix, and a combination of confocal and time-lapse microscopy, it was found that AQP1 knockdown by siRNA ablated blebbing of HT1080 and ACHN cells, and overexpression of AQP1-GFP not only significantly increased bleb size with a corresponding decrease in bleb numbers, but also induced bleb formation in non-blebbing cell lines. Importantly, AQP1 overexpression reduces bleb lifespan due to faster bleb retraction. This novel finding of AQP1-facilitated bleb retraction requires the activity of the Na+/H+ pump as inhibition of the ion transporter, which was found localized to intracellular vesicles, blocked bleb retraction in both cell lines. This study also demonstrated that a differential regulation of cell blebbing by AQP isoforms exists as knockdown of AQP5 had no effect on bleb formation. Data from this study also demonstrates that the lipid signaling PLD2 signals through PA in the LPA-LPAR-Rho-ROCK axis to positively regulate bleb formation in both cell lines. Taken together, this work provides a novel role of AQP1 and Na+/H+ pump in regulation of cell blebbing, and this could be exploited in the development of new therapy to treat cancer.

Comparison of terrain following and cut cell grids using a non-hydrostatic model

Terrain following coordinates are widely used in operational models but the cut cell method has been proposed as an alternative that can more accurately represent atmospheric dynamics over steep orography. Because the type of grid is usually chosen during model implementation, it becomes necessary to use different models to compare the accuracy of different grids. In contrast, here a C-grid finite volume model enables a like-for-like comparison of terrain following and cut cell grids. A series of standard two-dimensional tests using idealised terrain are performed: tracer advection in a prescribed horizontal velocity field, a test starting from resting initial conditions, and orographically induced gravity waves described by nonhydrostatic dynamics. In addition, three new tests are formulated: a more challenging resting atmosphere case, and two new advection tests having a velocity field that is everywhere tangential to the terrain following coordinate surfaces. These new tests present a challenge on cut cell grids. The results of the advection tests demonstrate that accuracy depends primarily upon alignment of the flow with the grid rather than grid orthogonality. A resting atmosphere is well-maintained on all grids. In the gravity waves test, results on all grids are in good agreement with existing results from the literature, although terrain following velocity fields lead to errors on cut cell grids. Due to semi-implicit timestepping and an upwind-biased, explicit advection scheme, there are no timestep restrictions associated with small cut cells. We do not find the significant advantages of cut cells or smoothed coordinates that other authors find.