The Mode of Death of Pig Kidney Cells Infected with Cowpox Virus Is Governed by the Expression of thecrmAGene

AbstractPig kidney cells (LLC-PK1) were infected with one of three viruses: wild-type cowpox virus (Brighton red strain) expressing thecrmAgene; recombinant cowpox virus A602, lacking thecrmAgene; or cowpox virus A604, a revertant of virus A602, expressing thecrmAgene. The wild-type virus and virus A604 produced identical cytopathic effects consistent with death by necrosis. In these cells, the structural features of the plasma membrane, the nuclear membrane, and the chromatin were maintained until lysis of the cells. In contrast, cowpox virus A602 produced cytopathic effects consistent with death by apoptosis. These effects included loss of microvilli on the cell surface, margination and condensation of the chromatin, progressive convolution of the nuclear membrane, release of dense chromatin masses on disintegration of the nucleus, fragmentation of the DNA, and the generation of apoptotic bodies. These results suggest that thecrmAgene is necessary to inhibit processes of apoptosis induced in LLC-PK1cells by infection with cowpox virus. Thus in cells of certain types, thecrmAgene can act with other viral genes to control the mode of death of the virus-infected cell. This capability may be advantageous to virus replicationin vivo,potentially facilitating both virus trafficking and interference with antiviral immune defenses

The structure of calcium metaphosphate glass obtained from x-ray and neutron diffraction and reverse Monte Carlo modelling

The short range structure of (CaO)(0.5)(P2O5)(0.5) glass has been studied using x-ray and neutron diffraction and modelled using the reverse Monte Carlo method. Using this combination of techniques has allowed six interatomic correlations to be distinguished and fitted to obtain a set of bond lengths and coordination numbers that describe the structure of the glass. The glass consists of metaphosphate chains of phosphate tetrahedra and each phosphate unit has two non-bridging oxygen atoms available for coordination with Ca. The Ca-O correlation was fitted with two peaks at 2.35 and 2.86 angstrom, representing a broad distribution of bond lengths. The total Ca-O coordination is 6.9 and is consistent with distorted polyhedral units such as capped octahedra or capped trigonal prisms. It is found that most non-bridging oxygen atoms are bonded to two calcium atoms. All of these observations are consistent with Hoppe's model for phosphate glasses. Furthermore, the medium range order is revealed to consist of phosphate chains intertwined with apparently elongated clusters of Ca ions, and the Ca-O and Ca-P correlations contributed significantly to the first sharp diffraction peak in x-ray diffraction

Production of prostaglandin E2 in response to infection with modified vaccinia Ankara virus

AbstractProstaglandin E2 (PGE2) is an arachidonic acid (AA)-derived signaling molecule that can influence host immune responses to infection or vaccination. In this study, we investigated PGE2 production in vitro by cells infected with the poxvirus vaccine strain, modified vaccinia Ankara virus (MVA). Human THP-1 cells, murine bone marrow-derived dendritic cells, and murine C3HA fibroblasts all accumulated PGE2 to high levels in culture supernatants upon infection with MVA. We also demonstrated that MVA induced the release of AA from infected cells, and this was, most unusually, independent of host cytosolic phospholipase A2 activity. The accumulation of AA and PGE2 was dependent on viral gene expression, but independent of canonical NF-κB signaling via p65/RelA. The production of PGE2 required host cyclooxygenase-2 (COX-2) activity, and COX-2 protein accumulated during MVA infection. The results of this study provide insight into a novel aspect of MVA biology that may affect the efficacy of MVA-based vaccines

A 43-Nucleotide RNACis-Acting Element Governs the Site-Specific Formation of the 3′ End of a Poxvirus Late mRNA

AbstractThe 3′ ends of late mRNAs of theatigene, encoding the major component of the A-type inclusions, are generated by endoribonucleolytic cleavage at a specific site in the primary transcript [Antczaket al.,(1992),Proc. Natl. Acad. Sci. USA89, 12033–12037]. In this study, sequence analysis of cDNAs of the 3′ ends ofatimRNAs showed these mRNAs are 3′ polyadenylated at the RNA cleavage site. This suggests thatatimRNA 3′ end formation involves cleavage of a late transcript, with subsequent 3′ polyadenylation of the 5′ cleavage product. The RNAcis-acting element, the AX element, directing orientation-dependent formation of these mRNA 3′ ends, was mapped to a 345-bpAluI–XbaI fragment. Deletion analyses of this fragment showed that the boundaries of the AX element are within −5 and +38 of the RNA cleavage site. Scanning mutagenesis showed that the AX element contains at least two subelements: subelement I, 5′-UUUAU↓CCGAUAAUUC-3′, containing the cleavage site (↓), separated from the downstream subelement II, 5′-AAUUUCGGAUUUGAAUGC-3′, by a 10-nucleotide region, whose composition may be altered without effect on RNA 3′ end formation. These features, which differ from those of other elements controlling RNA processing, suggest that the AX element is a component of a novel mechanism of RNA 3′ end formation

Synthesis, characterisation and performance of (TiO2)(0.18)(SiO2)(0.82) xerogel catalysts

The synthesis of high surface area xerogels has been achieved using the sol-gel route. Heptane washing was used during the stages of drying to minimise capillary pressures and hence preserve pore structure and maximise the surface area. SAXS data have identified that heptane washing during drying, in general, results in a preservation of the pore structure and surface areas of up to 450 m(2) g(-1). O-17 NMR showed that Ti is fully mixed into the silica network in all of the samples. XANES data confirm that reversible 4-fold Ti sites are more prevalent in samples with high surface areas, as expected. The calcined xerogels were tested for their catalytic activity using the epoxidation of cyclohexene with tert-butyl hydroperoxide (TBHP) as a test reaction, with excellent selectivities and reasonable percentage conversions. FT-IR spectroscopy has revealed that the catalytic activity is correlated with the intensity of the Si-O-Ti signal, after accounting for variations in Si-OH and Si-O-Si. The most effective catalyst was produced with heptane washing, a calcination temperature of 500 degreesC, and a heating rate of 5 degreesC min(-1)

Span of control in supervision of rail track work

The supervision of engineering work on the railways has received relatively little examination despite being both safety-critical in its own right and having wider implications for the successful running of the railways. The present paper is concerned with understanding the factors that make different engineering works perceived as easier or harder to manage. We describe an approach building on notions of ‘span of control’, through which we developed the TOECAP inventory (Team, Organisation, Environment, Communication, Activity and Personal). This tool was validated through both interviews and questionnaires. As well as identifying the physical factors involved, the work also emphasised the importance of collaborative and attitudinal factors. We conclude by discussing limitations of the present work and future directions for development

Insulin pump therapy with automated insulin suspension in response to hypoglycemia: reduction in nocturnal hypoglycemia in those at greatest risk.

OBJECTIVE: To evaluate a sensor-augmented insulin pump with a low glucose suspend (LGS) feature that automatically suspends basal insulin delivery for up to 2 h in response to sensor-detected hypoglycemia. RESEARCH DESIGN AND METHODS: The LGS feature of the Paradigm Veo insulin pump (Medtronic, Inc., Northridge, CA) was tested for 3 weeks in 31 adults with type 1 diabetes. RESULTS: There were 166 episodes of LGS: 66% of daytime LGS episodes were terminated within 10 min, and 20 episodes lasted the maximum 2 h. LGS use was associated with reduced nocturnal duration ≤2.2 mmol/L in those in the highest quartile of nocturnal hypoglycemia at baseline (median 46.2 vs. 1.8 min/day, P = 0.02 [LGS-OFF vs. LGS-ON]). Median sensor glucose was 3.9 mmol/L after 2-h LGS and 8.2 mmol/L at 2 h after basal restart. CONCLUSIONS: Use of an insulin pump with LGS was associated with reduced nocturnal hypoglycemia in those at greatest risk and was well accepted by patients

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome

Bioactive sol-gel glasses at the atomic scale: the complementary use of advanced probe and computer modelling methods

Sol-gel synthesised bioactive glasses may be formed via a hydrolysis condensation reaction, silica being introduced in the form of tetraethyl orthosilicate (TEOS) and calcium is typically added in the form of calcium nitrate. The synthesis reaction proceeds in an aqueous environment; the resultant gel is dried, before stabilisation by heat treatment. These materials, being amorphous, are complex at the level of their atomic-scale structure, but their bulk properties may only be properly understood on the basis of that structural insight. Thus, a full understanding of their structure : property relationship may only be achieved through the application of a coherent suite of leading-edge experimental probes, coupled with the cogent use of advanced computer simulation methods. Using as an exemplar a calcia-silica sol-gel glass of the kind developed by Larry Hench, to whose memory this paper is dedicated, we illustrate the successful use of high-energy x-ray and neutron scattering (diffraction) methods, magic-angle spinning solid state NMR, and molecular dynamics simulation as components to a powerful methodology for the study of amorphous materials