Excision of anabaena PCC 7120 nifD element in escherichia coli: growth kinetics and RecA regulated xisA expression and DNA rearrangement

Anabaena PCC 7120 nifHDK operon is interrupted by an 11 kb DNA element which is excised during the development of heterocysts by Excisase A, encoded by the xisA gene residing on the element. The excision is a site-specific recombination event that occurs at the I I base pair direct repeats flanking the element. Earlier work showed the excision of the I I kb element in Escherichia coli at a frequency 0.3%. We report here the excision of this element at 1.1% and 1.98% in E. coli DH5 alpha, and 1.9% and 10.9% in E. coli JM 101 when grown on Luria broth and minimal media, respectively. Excision of nifD element in isogenic recA(-) (RK1) and recA(+) (RK2) E. coli JM101 P1 transductants, showed similar results to that of E. coli JM101 and DH5 alpha, respectively. A plasmid pMX32, carrying a xisA defective 11 kb element, showed no excision in E. coli RK2 strain. In contrast to Anabaena PCC 7120, excision of nifD element did not increase in E. call DH5 alpha grown in iron-deficient conditions. A PxisA::lacZ transcriptional fusion, used to detect the expression of elusive xisA gene, showed maximal beta-galactosidase activity in the stationary phase. The results suggest that the excision event in E. coli may involve additional factors, such as RecA and that the physiological status can influence the excision of nifD element. (C) 2007 Elsevier Ltd. All rights reserved.

Evidence for complex multigenic inheritance of radiation AML susceptibility in mice revealed using a surrogate phenotypic assay

The mapping of genes which affect individual cancer risk is an important but complex challenge. A surrogate assay of susceptibility to radiation-induced acute myeloid leukaemia (AML) in the mouse based on chromosomal radiosensitivity has been developed and validated. This assay was applied to the mapping of radiation-induced AML risk modifier loci by association with microsatellite markers. A region on chromosome (chr) 18 with strong association is identified and confirmed by backcross analysis. Additional loci on chrs 8 and 13 show significant association. A key candidate gene Rbbp8 on chr18 is identified. Rbbp8 is shown to be upregulated in response to X-irradiation in the AML sensitive CBA strain but not AML resistant C57BL/6 strain. This study demonstrates the strength of utilizing surrogate endpoints of cancer susceptibility in the mapping of mouse loci and identifies additional loci that may affect radiation cancer risk.

First 5f-element complexes with the tetradentate BTBP ligand. Synthesis and crystal structure of uranyl(VI) compounds with CyMe4BTBP

Treatment of [UO2(OTf)(2)] or [UO2I2(thf)(3)] with 1 equiv. of CyMe4BTBP in anhydrous acetonitrile led to the formation of [UO2(CyMe4BTBP)(OTf)(2)] (1) and [UO2(CyMe4BTBP)I-2] (2) which crystallized as the cationic forms [UO2(CyMe4BTBP)(py)][OTf](2) (3) and [UO2I(CyMe4BTBP)][I] (4) in pyridine and acetonitrile, respectively. These compounds are unique examples of structurally characterized actinide complexes with a BTBP molecule; this ligand adopts a planar conformation in the equatorial plane of the {UO2}(2+) ion. In pyridine, 1 is dissociated into [UO2(OTf)(2)(PY)(3)] and free CyMe4BTBP and the thermodynamic parameters (K, Delta H, Delta S) of this equilibrium have been determined by H-1 NMR spectroscopy. The ethoxide derivative [UO2(OEt)(CyMe4BTBP)][OTf] (5) crystallized from a solution of I in a mixture of ethanol and acetone under air, and the dinuclear mu-oxo complex [{UO2(CyMe4BTBP)}(2)(mu-O)][I](2) (6) was obtained from [UO2I(thf)(2.7)] and CyMe4BTBP. The crystal structures of 6 and of the analogous derivatives [{UO2(py)(4)}(2)(mu-O)][I](2)(7) and [{UO2(TPTZ)(py)}(2)(mu-O)][I-3](2)(8) exhibit a flexible [{UO2}-O-{UO2}](2+) moiety.

Designs for an adaptive tuned vibration absorber with variable shape stiffness element

An adaptive tuned vibration absorber (ATVA) with a smart variable stiffness element is capable of retuning itself in response to a time-varying excitation frequency., enabling effective vibration control over a range of frequencies. This paper discusses novel methods of achieving variable stiffness in an ATVA by changing shape, as inspired by biological paradigms. It is shown that considerable variation in the tuned frequency can be achieved by actuating a shape change, provided that this is within the limits of the actuator. A feasible design for such an ATVA is one in which the device offers low resistance to the required shape change actuation while not being restricted to low values of the effective stiffness of the vibration absorber. Three such original designs are identified: (i) A pinned-pinned arch beam with fixed profile of slight curvature and variable preload through an adjustable natural curvature; (ii) a vibration absorber with a stiffness element formed from parallel curved beams of adjustable curvature vibrating longitudinally; (iii) a vibration absorber with a variable geometry linkage as stiffness element. The experimental results from demonstrators based on two of these designs show good correlation with the theory.

Finite element aided design evolution of composite leaf spring

This paper shows the process of the virtual production development of the mechanical connection between the top leaf of a dual composite leaf spring system to a shackle using finite element methods. The commercial FEA package MSC/MARC has been used for the analysis. In the original design the joint was based on a closed eye-end. Full scale testing results showed that this configuration achieved the vertical proof load of 150 kN and 1 million cycles of fatigue load. However, a problem with delamination occurred at the interface between the fibres going around the eye and the main leaf body. To overcome this problem, a second design was tried using transverse bandages of woven glass fibre reinforced tape to wrap the section that is prone to delaminate. In this case, the maximum interlaminar shear stress was reduced by a certain amount but it was still higher than the material’s shear strength. Based on the fact that, even with delamination, the top leaf spring still sustained the maximum static and fatigue loads required, the third design was proposed with an open eye-end, eliminating altogether the interface where the maximum shear stress occurs. The maximum shear stress predicted by FEA is reduced significantly and a safety factor of around 2 has been obtained. Thus, a successful and safe design has been achieved.

(-)Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons

Emerging evidence suggests that the cellular actions of flavonoids relate not simply to their antioxidant potential but also to the modulation of protein kinase signalling pathways. We investigated in primary cortical neurons, the ability of the flavan-3-ol, (-)epicatechin, and its human metabolites at physiologically relevant concentrations, to stimulate phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity. (-)Epicatechin at 100-300 nmol/L stimulated a rapid, extracellular signal-regulated kinase (ERK)- and PI3K-dependent, increase in CREB phosphorylation. At micromolar concentrations, stimulation was no longer apparent and at the highest concentration tested (30 mu mol/L) (-)epicatechin was inhibitory. (-)Epicatechin also stimulated ERK and Akt phosphorylation with similar bell-shaped concentration-response characteristics. The human metabolite 3 '-O-methyl-(-)epicatechin was as effective as (-)epicatechin at stimulating ERK phosphorylation, but (-)epicatechin glucuronide was inactive. (-)Epicatechin and 3 '-O-methyl-(-)epicatechin treatments (100 nmol/L) increased CRE-luciferase activity in cortical neurons in a partially ERK-dependent manner, suggesting the potential to increase CREB-mediated gene expression. mRNA levels of the glutamate receptor subunit GluR2 increased by 60%, measured 18 h after a 15 min exposure to (-)epicatechin and this translated into an increase in GluR2 protein. Thus, (-)epicatechin has the potential to increase CREB-regulated gene expression and increase GluR2 levels and thus modulate neurotransmission, plasticity and synaptogenesis.

Plant- and marine-derived n-3 polyunsaturated fatty acids have differential effects on fasting and postprandial blood lipid concentrations and on the susceptibility of LDL to oxidative modification in moderately hyperlipidemic subjects

Background: Dietary a-linolenic acid (ALA) can be converted to long-chain n-3 polyunsaturated fatty acids (PUFAs) in humans and may reproduce some of the beneficial effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on cardiovascular disease risk factors. Objective: This study aimed to compare the effects of increased dietary intakes of ALA and EPA+DHA on a range of atherogenic risk factors. Design: This was a placebo-controlled, parallel study involving 150 moderately hyperlipidemic subjects randomly assigned to 1 of 5 interventions: 0.8 or 1.7 g EPA+DHA/d, 4.5 or 9.5 g ALA/d, or an n-6 PUFA control for 6 mo. Fatty acids were incorporated into 25 g of fat spread and 3 capsules to be consumed daily. Results: The change in fasting or postprandial lipid, glucose, or insulin concentrations or in blood pressure was not significantly different after any of the n-3 PUFA interventions compared with the n-6 PUFA control. The mean (+/-SEM) change in fasting triacylglycerols after the 1.7-g/d EPA+DHA intervention (-7.7 +/- 4.99%) was significantly (P < 0.05) different from the change after the 9.5-g/d ALA intervention (10.9 +/- 4.5%). The ex vivo susceptibility of LDL to oxidation was higher after the 1.7-g/d EPA+DHA intervention than after the control and ALA interventions (P < 0.05). There was no significant change in plasma a-tocopherol concentrations or in whole plasma antioxidant status in any of the groups. Conclusion: At estimated biologically equivalent intakes, dietary ALA and EPA+DHA have different physiologic effects.

Dietary flavonoid (-)epicatechin stimulates phosphatidylinositol 3-kinase-dependent anti-oxidant response element activity and up-regulates glutathione in cortical astrocytes

Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 mu M) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

A moving-mesh finite element method and its application to the numerical solution of phase-change problems

A distributed Lagrangian moving-mesh finite element method is applied to problems involving changes of phase. The algorithm uses a distributed conservation principle to determine nodal mesh velocities, which are then used to move the nodes. The nodal values are obtained from an ALE (Arbitrary Lagrangian-Eulerian) equation, which represents a generalization of the original algorithm presented in Applied Numerical Mathematics, 54:450--469 (2005). Having described the details of the generalized algorithm it is validated on two test cases from the original paper and is then applied to one-phase and, for the first time, two-phase Stefan problems in one and two space dimensions, paying particular attention to the implementation of the interface boundary conditions. Results are presented to demonstrate the accuracy and the effectiveness of the method, including comparisons against analytical solutions where available.

Changes in race-specific virulence in pseudomonas syringae pv. phaseolicola are Associated with a chimeric transposable element and rare deletion events in a plasmid-borne pathogenicity island

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF, confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS1492 from Pseudomonas putida and IS1090 from Ralstonia eutropha. The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF. However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS1090 homolog in the two deletion types and in the other 47 insertions of the IS1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF.

Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence

Objectives: To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics. Methods: Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry. Results: Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB-TolC efflux system after PFD exposure. Conclusions: Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB-TolC efflux system, but that single exposure was insufficient to select for MAR strains.

Sulphate and sulphurous acid alter the relative susceptibility of wheat to Phaeosphaeria nodorum and Mycosphaerella graminicola

The relative abundances of DNA of Mycosphaerella graminicola and Phaeosphaeria nodorum in archived wheat samples are closely correlated with UK anthropogenic emissions of oxidized sulphur over the last 160 years. To test whether this could be a causal relationship, possible modes of action of sulphur on the two fungi were examined. Mycelial growth of the two fungi in solutions of sulphurous acid was similar. Sulphurous acid at pH 4 reduced percentage germination of P. nodorum conidia more strongly than M. graminicola conidia. In spray inoculations of wheat cv. Squarehead’s Master, Cappelle Desprez and Riband with water or sulphurous acid (pH 4), the ratio of leaves infected by P. nodorum to leaves infected by M. graminicola was increased by factors of 2.5, 2.1 and 0.6, respectively at pH 4. The same three cultivars of wheat were grown in sand and vermiculite and fertilized with nutrient solution containing 2.5 or 0.5 mM sulphate. Both pathogens infected less frequently at 2.5 mM sulphate, by a factor of about 2. The severity of infection by M. graminicola was reduced on all three cultivars by a factor of about 4-5 at 2.5mM sulphate, but severity of P. nodorum was reduced only by a factor of about 2. Both elevated free sulphate concentrations in soil and sulphite in rainwater could therefore increase the prevalence of P. nodorum relative to M. graminicola, which is consistent with the historical changes in abundance

A high frequency $hp$ boundary element method for scattering by convex polygons

In this paper we propose and analyze a hybrid $hp$ boundary element method for the solution of problems of high frequency acoustic scattering by sound-soft convex polygons, in which the approximation space is enriched with oscillatory basis functions which efficiently capture the high frequency asymptotics of the solution. We demonstrate, both theoretically and via numerical examples, exponential convergence with respect to the order of the polynomials, moreover providing rigorous error estimates for our approximations to the solution and to the far field pattern, in which the dependence on the frequency of all constants is explicit. Importantly, these estimates prove that, to achieve any desired accuracy in the computation of these quantities, it is sufficient to increase the number of degrees of freedom in proportion to the logarithm of the frequency as the frequency increases, in contrast to the at least linear growth required by conventional methods.

A finite element-spherical harmonics model for radiative transfer in inhomogeneous clouds. Part II. some applications

EVENT has been used to examine the effects of 3D cloud structure, distribution, and inhomogeneity on the scattering of visible solar radiation and the resulting 3D radiation field. Large eddy simulation and aircraft measurements are used to create realistic cloud fields which are continuous or broken with smooth or uneven tops. The values, patterns and variance in the resulting downwelling and upwelling radiation from incident visible solar radiation at different angles are then examined and compared to measurements. The results from EVENT confirm that 3D cloud structure is important in determining the visible radiation field, and that these results are strongly influenced by the solar zenith angle. The results match those from other models using visible solar radiation, and are supported by aircraft measurements of visible radiation, providing confidence in the new model.