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.

Interaction with avian cells and colonisation of specific pathogen free chicks by Shiga-toxin negative Escherichia coli O157:H7 (NCTC 12900)

The prevalence of Escherichia coli O157:H7 infection in birds is low but several deliberate inoculation studies show that poultry are readily and persistently infected by this organism indicating a possible threat to public health. The mechanisms of colonisation of poultry are not understood and the aim is to establish models to study the interaction of E. coli O157:H7, at the cellular and whole animal levels. A non-toxigenic E. coli O157:H7 (NCTC 12900) was used in adherence assays with an avian epithelial cell line (Div-1) and used to inoculate 1-day-old SPF chicks. In vitro, NCTC 12900 induced micro-colonies associated with cytoskeletal arrangements and pedestal formation with intimate bacterial attachment. In the 1-day-old SPF chick, a dose of 1 x 10(5) cfu resulted in rapid and extensive colonisation of the gastrointestinal tract and transient colonisation of the liver and spleen. The number of E. coli O157:H7 organisms attained approximately 10(8) cfu/ml caecal homogenate 24 h after inoculation and approximately 10(7) cfu/ml caecal homogenate was still present at day 92. Faecal shedding persisted for 169 days, ceasing 9 days after the birds came into lay and 6% of eggs were contaminated on the eggshell. Histological analysis of tissue samples from birds dosed with 1 x 10(7) cfu gave evidence for E coli O157:H7 NCTC 12900 induced micro-colonies on the caecal mucosa, although evidence for attaching effacing lesions was equivocal. These models may be suitable to study those factors of E. coli O157:H7 that mediate persistent colonisation in avian species.

Land-atmosphere energy exchange in Arctic tundra and boreal forest: available data and feedbacks to climate

This paper summarizes and analyses available data on the surface energy balance of Arctic tundra and boreal forest. The complex interactions between ecosystems and their surface energy balance are also examined, including climatically induced shifts in ecosystem type that might amplify or reduce the effects of potential climatic change. High latitudes are characterized by large annual changes in solar input. Albedo decreases strongly from winter, when the surface is snow-covered, to summer, especially in nonforested regions such as Arctic tundra and boreal wetlands. Evapotranspiration (QE) of high-latitude ecosystems is less than from a freely evaporating surface and decreases late in the season, when soil moisture declines, indicating stomatal control over QE, particularly in evergreen forests. Evergreen conifer forests have a canopy conductance half that of deciduous forests and consequently lower QE and higher sensible heat flux (QH). There is a broad overlap in energy partitioning between Arctic and boreal ecosystems, although Arctic ecosystems and light taiga generally have higher ground heat flux because there is less leaf and stem area to shade the ground surface, and the thermal gradient from the surface to permafrost is steeper. Permafrost creates a strong heat sink in summer that reduces surface temperature and therefore heat flux to the atmosphere. Loss of permafrost would therefore amplify climatic warming. If warming caused an increase in productivity and leaf area, or fire caused a shift from evergreen to deciduous forest, this would increase QE and reduce QH. Potential future shifts in vegetation would have varying climate feedbacks, with largest effects caused by shifts from boreal conifer to shrubland or deciduous forest (or vice versa) and from Arctic coastal to wet tundra. An increase of logging activity in the boreal forests appears to reduce QE by roughly 50% with little change in QH, while the ground heat flux is strongly enhanced.

A microblock ionomer in proton exchange membrane electrolysis for the production of high purity hydrogen

Proton exchange membranes (PEM’s) are currently under investigation for membrane water electrolysis (PEMWE) to deliver efficient production of the high purity hydrogen needed to supply emerging clean-energy technologies such as hydrogen fuel cells. The microblock aromatic ionomer described in this work achieves high mechanical strength in an aqueous environment as a result of its designed, biphasic morphology and displays many of the qualities required in a PEM. The new ionomer membrane thus shows good proton conductivity (63 mS cm−1 at 80 °C and 100% RH), while retaining mechanical integrity under high temperature, hydrated conditions. Testing in electrolysis has shown good energy efficiency (1.67 V at 1 A cm−2 and 80 °C, corresponding to 4 kWh/Nm3 of H2), making this ionomer a potential candidate for commercial application in PEMWE.

The bile acid receptor TGR5 does not interact with β-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid, DCA, taurolithocholic acid, TLCA) and the selective agonists oleanolic acid (OA) and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide (CCDC) stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, assessed by confocal microscopy. DCA, TLCA and OA did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, determined by bioluminescence resonance energy transfer. CCDC stimulated a low level of TGR5 interaction with β-arrestin2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of extracellular signal regulated kinase (ERK1/2). BRET analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.

Eddy-driven exchange between the open ocean and a sub-ice shelf cavity

The exchange between the open ocean and sub-ice shelf cavities is important to both water mass transformations and ice shelf melting. Here we use a high-resolution (500 m) numerical model to investigate to which degree eddies produced by frontal instability at the edge of a polynya are capable of transporting dense High Salinity Shelf Water (HSSW) underneath an ice shelf. The applied surface buoyancy flux and ice shelf geometry is based on Ronne Ice Shelf in the southern Weddell Sea, an area of intense wintertime sea ice production where a flow of HSSW into the cavity has been observed. Results show that eddies are able to enter the cavity at the southwestern corner of the polynya where an anticyclonic rim current intersects the ice shelf front. The size and time scale of simulated eddies are in agreement with observations close to the Ronne Ice Front. The properties and strength of the inflow are sensitive to the prescribed total ice production, flushing the ice shelf cavity at a rate of 0.2–0.4 × 106 m3 s−1 depending on polynya size and magnitude of surface buoyancy flux. Eddy-driven HSSW transport into the cavity is reduced by about 50% if the model grid resolution is decreased to 2-5 km and eddies are not properly resolved.

APOA5 genotype influences the association between 25-hydroxyvitamin D and high density lipoprotein cholesterol

OBJECTIVE: Circulating levels of 25-hydroxyvitamin D (25OHD) are positively associated with high density lipoprotein (HDL) cholesterol. We sought to replicate a previously reported interaction between APOA5 genotype and vitamin D, and to examine whether HDL-associated genetic loci modify the association between serum 25OHD and HDL cholesterol. METHODS: We examined whether 42 single nucleotide polymorphisms (SNPs) modify the association between serum 25OHD and HDL cholesterol in the 1958 British Birth cohort (aged 45 years, n = 4978). RESULTS: We identified a borderline interaction between the SNP rs12272004 (near the APOA5) and serum 25OHD on HDL cholesterol (P(interaction) = 0.05). The interaction was particularly prominent among the samples collected during winter (P(interaction) = 0.001). None of the other loci showed an interaction with serum 25OHD concentrations on HDL cholesterol. CONCLUSIONS: Our study in 4978 British Whites provides further support that APOA5 genotype modifies the association between vitamin D metabolites and HDL cholesterol.

Genetic association analysis of vitamin D pathway with obesity traits

Objective:Observational studies have examined the link between vitamin D deficiency and obesity traits. Some studies have reported associations between vitamin D pathway genes such as VDR, GC and CYP27B1 with body mass index (BMI) and waist circumference (WC); however, the findings have been inconsistent. Therefore, we investigated the involvement of vitamin D metabolic pathway genes in obesity-related traits in a large population-based study.Methods:We undertook a comprehensive analysis between 100 tagging single nucleotide polymorphisms (tagSNPs) in genes encoding for DHCR7, CYP2R1, VDBP, CYP27B1, CYP27A1, CYP24A1, VDR and RXRG, and obesity traits in 5224 participants (aged 45 years) in the 1958 British birth cohort (1958BC). We further extended our analyses to investigate the associations between SNPs and obesity traits using the summary statistics from the GIANT (Genetic Investigation of Anthropometric Traits) consortium (n=123 865).Results:In the 1958BC (n=5224), after Bonferroni correction, none of the tagSNPs were associated with obesity traits except for one tagSNP from CYP24A1 that was associated with waist-hip ratio (WHR) (rs2296239, P=0.001). However, the CYP24A1 SNP was not associated with BMI-adjusted WHR (WHRadj) in the 1958BC (rs2296239, P=1.00) and GIANT results (n=123 865, P=0.18). There was also no evidence for an interaction between the tagSNPs and obesity on BMI, WC, WHR and WHRadj in the 1958BC. In the GIANT consortium, none of the tagSNPs were associated with obesity traits.Conclusions:Despite a very large study, our findings suggest that the vitamin D pathway genes are unlikely to have a major role in obesity-related traits in the general population.

Association between FTO variant and change in body weight and its interaction with dietary factors: the DiOGenes study

Although FTO is an established obesity-susceptibility locus, it remains unknown whether it influences weight change in adult life and whether diet attenuates this association. Therefore, we investigated the association of FTO-rs9939609 with changes in weight and waist circumference (WC) during 6.8 years follow-up in a large-scale prospective study and examined whether these associations were modified by dietary energy percentage from fat, protein, carbohydrate, or glycemic index (GI). This study comprised data from five countries of European Prospective Investigation into Cancer and Nutrition (EPIC) and was designed as a case-cohort study for weight gain. Analyses included 11,091 individuals, of whom 5,584 were cases (age (SD), 47.6 (7.5) years), defined as those with the greatest unexplained annual weight gain during follow-up and 5,507 were noncases (48.0 (7.3) years), who were compared in our case-noncase (CNC) analyses. Furthermore, 6,566 individuals (47.9 (7.3) years) selected from the total sample (all noncases and 1,059 cases) formed the random subcohort (RSC), used for continuous trait analyses. Interactions were tested by including interaction terms in the models. In the RSC-analyses, FTO-rs9939609 was associated with BMI (β (SE), 0.17 (0.08) kg·m(-2)/allele; P = 0.034) and WC (0.47 (0.21) cm/allele; P = 0.026) at baseline, but not with weight change (5.55 (12.5) g·year(-1)/allele; P = 0.66) during follow up. In the CNC-analysis, FTO-rs9939609 was associated with increased risk of being a weight-gainer (OR: 1.1; P = 0.045). We observed no interaction between FTO-rs9939609 and dietary fat, protein and carbohydrate, and GI on BMI and WC at baseline or on change in weight and WC. FTO-rs9939609 is associated with BMI and WC at baseline, but association with weight gain is weak and only observed for extreme gain. Dietary factors did not influence the associations.

Expression of the guanine nucleotide exchange factor, mr-gef, is regulated during the differentiation of specific subsets of telencephalic neurons

We have performed a screen combining subtractive hybridization with PCR to isolate genes that are regulated when neuroepithelial (NE) cells differentiate into neurons. From this screen, we have isolated a number of known genes that have not previously been associated with neurogenesis, together with several novel genes. Here we report that one of these genes, encoding a guanine nucleotide exchange factor (GEF), is regulated during the differentiation of distinct neuronal populations. We have cloned both rat and mouse GEF genes and shown that they are orthologs of the human gene, MR-GEF, which encodes a GEF that specifically activates the small GTPase, Rap1. We have therefore named the rat gene rat mr-gef (rmr-gef) and the mouse gene mouse mr-gef (mmr-gef). Here, we will collectively refer to these two rodent genes as mr-gef. Expression studies show that mr-gef is expressed by young neurons of the developing rodent CNS but not by progenitor cells in the ventricular zone (VZ). The expression pattern of mr-gef during early telencephalic neurogenesis is strikingly similar to that of GABA and the LIM homeobox gene Lhx6, a transcription factor expressed by GABAergic interneurons generated in the ventral telencephalon, some of which migrate into the cortex during development. These observations suggest that mr-gef encodes a protein that is part of a signaling pathway involved in telencephalic neurogenesis; particularly in the development of GABAergic interneurons.

Dimer formation by symbiotic donor–acceptor interaction between two molecules of a specially designed dioxomolybdenum(VI) complex containing both donor and acceptor centers – A structural, spectroscopic and DFT study

This work presents a model study for the formation of a dimeric dioxomolybdenum(VI) complex [MoO2L]2, generated by simultaneous satisfaction of acceptor and donor character existing in the corresponding monomeric Mo(VI) complex MoO2L. This mononuclear complex is specially designed to contain a coordinatively unsaturated Mo(VI) acceptor centre and a free donor group, (e.g. –NH2 group) strategically placed in the ligand skeleton [H2L = 2-hydroxyacetophenonehydrazone of 2-aminobenzoylhydrazine]. Apart from the dimer [MoO2L]2, complexes of the type MoO2L·B (where B = CH3OH, γ-picoline and imidazole) are also reported. All the complexes are characterized by elemental analysis, spectroscopic (UV–Vis, IR, 1H NMR) techniques and cyclic voltammetry. Single crystal X-ray structures of [MoO2L]2 (1), MoO2L·CH3OH (2), and MoO2L.(γ-pic) (3) have been determined and discussed. DFT calculation on these complexes corroborates experimental data and provides clue for the facile formation of this type of dimer not reported previously. The process of dimer formation may also be viewed as an interaction between two molecules of a specially designed complex acting as a monodentate ligand. This work is expected to open up a new field of design and synthesis of dimeric complexes through the process of symbiotic donor–acceptor (acid–base) interaction between two molecules of a specially designed monomer.

Hydrophilic interaction chromatography–mass spectrometry for anionic metabolic profiling of urine from antibiotic-treated rats

Hydrophilic interaction chromatography–mass spectrometry (HILIC–MS) was used for anionic metabolic profiling of urine from antibiotic-treated rats to study microbial–host co-metabolism. Rats were treated with the antibiotics penicillin G and streptomycin sulfate for four or eight days and compared to a control group. Urine samples were collected at day zero, four and eight, and analyzed by HILIC–MS. Multivariate data analysis was applied to the urinary metabolic profiles to identify biochemical variation between the treatment groups. Principal component analysis found a clear distinction between those animals receiving antibiotics and the control animals, with twenty-nine discriminatory compounds of which twenty were down-regulated and nine up-regulated upon treatment. In the treatment group receiving antibiotics for four days, a recovery effect was observed for seven compounds after cessation of antibiotic administration. Thirteen discriminatory compounds could be putatively identified based on their accurate mass, including aconitic acid, benzenediol sulfate, ferulic acid sulfate, hippuric acid, indoxyl sulfate, penicillin G, phenol and vanillin 4-sulfate. The rat urine samples had previously been analyzed by capillary electrophoresis (CE) with MS detection and proton nuclear magnetic resonance (1H NMR) spectroscopy. Using CE–MS and 1H NMR spectroscopy seventeen and twenty-five discriminatory compounds were found, respectively. Both hippuric acid and indoxyl sulfate were detected across all three platforms. Additionally, eight compounds were observed with both HILIC–MS and CE–MS. Overall, HILIC–MS appears to be highly complementary to CE–MS and 1H NMR spectroscopy, identifying additional compounds that discriminate the urine samples from antibiotic-treated and control rats.

Propagation of the Madden-Julian Oscillation through the Maritime Continent and scale interaction with the diurnal cycle of precipitation

The convectively active part of the Madden-Julian Oscillation (MJO) propagates eastward through the warm pool, from the Indian Ocean through the Maritime Continent (the Indonesian archipelago) to the western Pacific. The Maritime Continent's complex topography means the exact nature of the MJO propagation through this region is unclear. Model simulations of the MJO are often poor over the region, leading to local errors in latent heat release and global errors in medium-range weather prediction and climate simulation. Using 14 northern winters of TRMM satellite data it is shown that, where the mean diurnal cycle of precipitation is strong, 80% of the MJO precipitation signal in the Maritime Continent is accounted for by changes in the amplitude of the diurnal cycle. Additionally, the relationship between outgoing long-wave radiation (OLR) and precipitation is weakened here, such that OLR is no longer a reliable proxy for precipitation. The canonical view of the MJO as the smooth eastward propagation of a large-scale precipitation envelope also breaks down over the islands of the Maritime Continent. Instead, a vanguard of precipitation (anomalies of 2.5 mm day^-1 over 10^6 km^2) jumps ahead of the main body by approximately 6 days or 2000 km. Hence, there can be enhanced precipitation over Sumatra, Borneo or New Guinea when the large-scale MJO envelope over the surrounding ocean is one of suppressed precipitation. This behaviour can be accommodated into existing MJO theories. Frictional and topographic moisture convergence and relatively clear skies ahead of the main convective envelope combine with the low thermal inertia of the islands, to allow a rapid response in the diurnal cycle which rectifies onto the lower-frequency MJO. Hence, accurate representations of the diurnal cycle and its scale interaction appear to be necessary for models to simulate the MJO successfully.

Mechanisms of motivation-cognition interaction: challenges and opportunities

There has been a recent rejuvenation of interest in studies of motivation-cognition interactions arising from many different areas of psychology and neuroscience. The current issue of Cognitive, Affective, and Behavioral Neuroscience provides a sampling of some of the latest research from a number of these different areas. In this introductory paper, we provide an overview of the current state of the field, in terms of key research developments and candidate neural mechanisms receiving focused investigation as potential sources of motivation-cognition interaction. However, our primary goal is conceptual: to highlight the distinct perspectives taken by different research areas in terms of how motivation is defined, the relevant dimensions and dissociations that are emphasized, and the theoretical questions being targeted. Together, these distinctions present both challenges and opportunities for efforts aiming towards a more unified and cross-disciplinary approach. We identify a set of pressing research questions calling out for this sort of cross-disciplinary approach, with the explicit goal of encouraging integrative and collaborative investigations directed towards them.

Mixed layer lateral eddy fluxes mediated by air-sea interaction

The modulation of air–sea heat fluxes by geostrophic eddies due to the stirring of temperature at the sea surface is discussed and quantified. It is argued that the damping of eddy temperature variance by such air–sea fluxes enhances the dissipation of surface temperature fields. Depending on the time scale of damping relative to that of the eddying motions, surface eddy diffusivities can be significantly enhanced over interior values. The issues are explored and quantified in a controlled setting by driving a tracer field, a proxy for sea surface temperature, with surface altimetric observations in the Antarctic Circumpolar Current (ACC) of the Southern Ocean. A new, tracer-based diagnostic of eddy diffusivity is introduced, which is related to the Nakamura effective diffusivity. Using this, the mixed layer lateral eddy diffusivities associated with (i) eddy stirring and small-scale mixing and (ii) surface damping by air–sea interaction is quantified. In the ACC, a diffusivity associated with surface damping of a comparable magnitude to that associated with eddy stirring (;500 m2 s21) is found. In frontal regions prevalent in the ACC, an augmentation of surface lateral eddy diffusivities of this magnitude is equivalent to an air–sea flux of 100 W m22 acting over a mixed layer depth of 100 m, a very significant effect. Finally, the implications for other tracer fields such as salinity, dissolved gases, and chlorophyll are discussed. Different tracers are found to have surface eddy diffusivities that differ significantly in magnitude.