An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157: H7 with bovine intestinal epithelium

Enterohaemorrhagic Escherichia coli O157 : H7 is a bacterial pathogen that can cause haemorrhagic colitis and haemolytic uremic syndrome. In the primary reservoir host, cattle, the terminal rectum is the principal site of E. coli O157 colonization. In this study, bovine terminal rectal primary epithelial cells were used to examine the role of H7 flagella in epithelial adherence. Binding of a fliC(H7) mutant O157 strain to rectal epithelium was significantly reduced as was binding of the flagellated wild-type strain following incubation with H7-specific antibodies. Complementation of fliC(H7) mutant O157 strain with fliC(H7) restored the adherence to wild-type levels; however, complementation with fliC(H6) did not restore it. High-resolution ultrastructural and imunofluorescence studies demonstrated the presence of abundant flagella forming physical contact points with the rectal epithelium. Binding to terminal rectal epithelium was specific to H7 by comparison with other flagellin types tested. In-cell Western assays confirmed temporal expression of flagella during O157 interaction with epithelium, early expression was suppressed during the later stages of microcolony and attaching and effacing lesion formation. H7 flagella are expressed in vivo by individual bacteria in contact with rectal mucosa. Our data demonstrate that the H7 flagellum acts as an adhesin to bovine intestinal epithelium and its involvement in this crucial initiating step for colonization indicates that H7 flagella could be an important target in intervention strategies.

Mozzarella-type curd made from buffalo, cows’ and ultrafiltered cows’ milk. 2. Physicochemical properties, curd yield and quality, casein fractions & micelles size

Buffalo milk contains (40–60 %) more protein, fat and calcium than cows’ milk. These constituents were enhanced by ultrafiltration (UF) of cows’ milk to give a product with similar levels to those found in the buffalo milk. Mozzarella-type curd was made from buffalo, cows’ and UF cows’ milk to compare the overall curd yield and quality. The curd yield on both dry and wet weight basis, curd moisture content and overall curd fat retention were found to be higher in the UF cows’ milk than for either the buffalo or the cows’ milk preparations. The minimum whey fat losses occurred in the UF cows’ curd when compared to the cows’ and the buffalo curd. The whey protein losses were found to be higher in the UF cows’ curd than those for the buffalo and the cows’ curds. The total mineral content of the curd was also higher in the UF cows’ milk than that found in either the buffalo or the cows’ milk. SEM micrographs showed that casein micelles sizes were different in the two different types of milk. Casein micelles were also observed to be deformed in the UF cows’ milk samples. UF cows’ milk contained higher amounts of both the αs1- and αs2-casein moieties than either the buffalo or the cows’ milk. Buffalo milk was found to contain a higher concentration of β-casein than either the UF cows’ or untreated cows’ milk samples. Gel strength was found to be higher in the resultant buffalo curd than for curds made from either native cows’ milk or those made from UF cows’ milk. The mineral distribution was also different in the three different types of bovine milk, measured by energy-dispersive X-ray (EDX) analysis. Differences in the curd quality observed between the buffalo and the cows’ milk appear to result from the differences in casein composition and overall micelle structure, rather than casein concentration alone.

Characterisation of the intracellular-glutamate decarboxylase system (GAD): analysis of its function, transcription and role in the acid resistance of various strains of Listeria monocytogenes

The glutamate decarboxylase (GAD) system is important for the acid resistance of Listeria monocytogenes. We previously showed that under acidic conditions, glutamate (Glt)/γ-aminobutyrate (GABA) antiport is impaired in minimal media but not in rich ones, like brain heart infusion. Here we demonstrate that this behavior is more complex and it is subject to strain and medium variation. Despite the impaired Glt/GABA antiport, cells accumulate intracellular GABA (GABA(i)) as a standard response against acid in any medium, and this occurs in all strains tested. Since these systems can occur independently of one another, we refer to them as the extracellular (GAD(e)) and intracellular (GAD(i)) systems. We show here that GAD(i) contributes to acid resistance since in a ΔgadD1D2 mutant, reduced GABA(i) accumulation coincided with a 3.2-log-unit reduction in survival at pH 3.0 compared to that of wild-type strain LO28. Among 20 different strains, the GAD(i) system was found to remove 23.11% ± 18.87% of the protons removed by the overall GAD system. Furthermore, the GAD(i) system is activated at milder pH values (4.5 to 5.0) than the GAD(e) system (pH 4.0 to 4.5), suggesting that GAD(i) is the more responsive of the two and the first line of defense against acid. Through functional genomics, we found a major role for GadD2 in the function of GAD(i), while that of GadD1 was minor. Furthermore, the transcription of the gad genes in three common reference strains (10403S, LO28, and EGD-e) during an acid challenge correlated well with their relative acid sensitivity. No transcriptional upregulation of the gadT2D2 operon, which is the most important component of the GAD system, was observed, while gadD3 transcription was the highest among all gad genes in all strains. In this study, we present a revised model for the function of the GAD system and highlight the important role of GAD(i) in the acid resistance of L. monocytogenes.

Glu298Asp polymorphism influences the beneficial effects of fish oil fatty acids on postprandial vascular function

Our objective was to determine whether the endothelial nitric oxide synthase (eNOS) Glu298Asp polymorphism influences vascular response to raised NEFA enriched with saturated fatty acids (SFA) or long-chain (LC) n-3 polyunsaturated fatty acids (PUFA). Subjects were prospectively recruited for genotype (Glu298, n = 30 and Asp298, n = 29; balanced for age and gender) consumed SFA on two occasions, with and without the substitution of 0.07 g fat/kg body weight with LC n-3 PUFA, and with heparin infusion to elevate NEFA. Endothelial function was measured before and after NEFA elevation (240 min), with blood samples taken every 30 min. Flow-mediated dilation (FMD) decreased following SFA alone and increased following SFA+LC n-3 PUFA. There were 2-fold differences in the change in FMD response to the different fat loads between the Asp298 and Glu298 genotypes (P = 0.002) and between genders (P < 0.02). Sodium nitroprusside-induced reactivity, measured by laser Doppler imaging with iontophoresis, was significantly greater with SFA+LC n-3 PUFA in all female subjects (P < 0.001) but not in males. Elevated NEFA influences both endothelial-dependent and endothelial-independent vasodilation during the postprandial phase. Effects of fat composition appear to be genotype and gender dependent, with the greatest difference in vasodilatory response to the two fat loads seen in the Asp298 females.

Influence of sugar type on the bioavailability of cocoa flavanols.

The beneficial effects of cocoa on vascular function are mediated by the absorption of monomeric flavanols into the circulation from the small intestine. As such, an understanding of the impact of the food matrix on the delivery of flavanols to the circulation is critical in assessing the potential vascular impact of a food. In the present study, we investigated the impact of carbohydrate type on flavanol absorption and metabolism from chocolate. A randomised, double-blind, three-arm cross-over study was conducted, where fifteen volunteers were randomly assigned to either a high-flavanol (266 mg) chocolate containing maltitol, a high-flavanol (251 mg) chocolate with sucrose or a low-flavanol (48 mg) chocolate with sucrose. Test chocolates were matched for micro- and macronutrients, including the alkaloids theobromine and caffeine, and were similar in taste and appearance. Total flavanol absorption was lower after consumption of the maltitol-containing test chocolate compared with following consumption of its sucrose-containing equivalent (P = 0·002). Although the O-methylation pattern observed for absorbed flavanols was unaffected by sugar type, individual levels of unmethylated ( - )-epicatechin metabolites, 3'-O-methyl-epicatechin and 4'-O-methyl-epicatechin metabolites were lower for the maltitol-containing test chocolate compared with the sucrose-containing equivalent. Despite a reduction in the total plasma pool of flavanols, the maximum time (T max) was unaffected. The present data indicate that full assessment of intervention treatments is vital in future intervention trials with flavanols and that carbohydrate content is an important determinant for the optimal delivery of flavanols to the circulation.

Efficient calculation of the green function for acoustic propagation above a homogeneous impedance plane

This paper is concerned with the problem of propagation from a monofrequency coherent line source above a plane of homogeneous surface impedance. The solution of this problem occurs in the kernel of certain boundary integral equation formulations of acoustic propagation above an impedance boundary, and the discussion of the paper is motivated by this application. The paper starts by deriving representations, as Laplace-type integrals, of the solution and its first partial derivatives. The evaluation of these integral representations by Gauss-Laguerre quadrature is discussed, and theoretical bounds on the truncation error are obtained. Specific approximations are proposed which are shown to be accurate except in the very near field, for all angles of incidence and a wide range of values of surface impedance. The paper finishes with derivations of partial results and analogous Laplace-type integral representations for the case of a point source.

Using synoptic type analysis to understand New Zealand climate during the Mid-Holocene

Diagnosing the climate of New Zealand from low-resolution General Circulation Models (GCMs) is notoriously difficult due to the interaction of the complex topography and the Southern Hemisphere (SH) mid-latitude westerly winds. Therefore, methods of downscaling synoptic scale model data for New Zealand are useful to help understand past climate. New Zealand also has a wealth of palaeoclimate-proxy data to which the downscaled model output can be compared, and to provide a qualitative method of assessing the capability of GCMs to represent, in this case, the climate 6000 yr ago in the Mid-Holocene. In this paper, a synoptic weather and climate regime classification system using Empirical Orthogonal Function (EOF) analysis of GCM and reanalysis data was used. The climate regimes are associated with surface air temperature and precipitation anomalies over New Zealand. From the analysis in this study, we find at 6000 BP that increased trough activity in summer and autumn led to increased precipitation, with an increased north-south pressure gradient ("zonal events") in winter and spring leading to drier conditions. Opposing effects of increased (decreased) temperature are also seen in spring (autumn) in the South Island, which are associated with the increased zonal (trough) events; however, the circulation induced changes in temperature are likely to have been of secondary importance to the insolation induced changes. Evidence from the palaeoclimate-proxy data suggests that the Mid-Holocene was characterized by increased westerly wind events in New Zealand, which agrees with the preference for trough and zonal regimes in the models.

Influence of dietary protein intake and glycemic index on the association between TCF7L2 HapA and weight gain

BACKGROUND: Genetic polymorphisms of transcription factor 7-like 2 (TCF7L2) have been associated with type 2 diabetes and BMI. OBJECTIVE: The objective was to investigate whether TCF7L2 HapA is associated with weight development and whether such an association is modulated by protein intake or by the glycemic index (GI). DESIGN: The investigation was based on prospective data from 5 cohort studies nested within the European Prospective Investigation into Cancer and Nutrition. Weight change was followed up for a mean (±SD) of 6.8 ± 2.5 y. TCF7L2 rs7903146 and rs10885406 were successfully genotyped in 11,069 individuals and used to derive HapA. Multiple logistic and linear regression analysis was applied to test for the main effect of HapA and its interaction with dietary protein or GI. Analyses from the cohorts were combined by random-effects meta-analysis. RESULTS: HapA was associated neither with baseline BMI (0.03 ± 0.07 BMI units per allele; P = 0.6) nor with annual weight change (8.8 ± 11.7 g/y per allele; P = 0.5). However, a previously shown positive association between intake of protein, particularly of animal origin, and subsequent weight change in this population proved to be attenuated by TCF7L2 HapA (P-interaction = 0.01). We showed that weight gain becomes independent of protein intake with an increasing number of HapA alleles. Substitution of protein with either fat or carbohydrates showed the same effects. No interaction with GI was observed. CONCLUSION: TCF7L2 HapA attenuates the positive association between animal protein intake and long-term body weight change in middle-aged Europeans but does not interact with the GI of the diet.

Endocannabinoids in neuroendopsychology: multiphasic control of mitochondrial function

The endocannabinoid system (ECS) is a construct based on the discovery of receptors that are modulated by the plant compound tetrahydrocannabinol and the subsequent identification of a family of nascent ligands, the 'endocannabinoids'. The function of the ECS is thus defined by modulation of these receptors-in particular, by two of the best-described ligands (2-arachidonyl glycerol and anandamide), and by their metabolic pathways. Endocannabinoids are released by cell stress, and promote both cell survival and death according to concentration. The ECS appears to shift the immune system towards a type 2 response, while maintaining a positive energy balance and reducing anxiety. It may therefore be important in resolution of injury and inflammation. Data suggest that the ECS could potentially modulate mitochondrial function by several different pathways; this may help explain its actions in the central nervous system. Dose-related control of mitochondrial function could therefore provide an insight into its role in health and disease, and why it might have its own pathology, and possibly, new therapeutic directions.

Endocannabinoids, FOXO and the metabolic syndrome: redox, function and tipping point--the view from two systems

The endocannabinoid system (ECS) was only 'discovered' in the 1990s. Since then, many new ligands have been identified, as well as many new intracellular targets--ranging from the PPARs, to mitochondria, to lipid rafts. It was thought that blocking the CB-1 receptor might reverse obesity and the metabolic syndrome. This was based on the idea that the ECS was dysfunctional in these conditions. This has met with limited success. The reason may be that the ECS is a homeostatic system, which integrates energy seeking and storage behaviour with resistance to oxidative stress. It could be viewed as having thrifty actions. Thriftiness is an innate property of life, which is programmed to a set point by both environment and genetics, resulting in an epigenotype perfectly adapted to its environment. This thrifty set point can be modulated by hormetic stimuli, such as exercise, cold and plant micronutrients. We have proposed that the physiological and protective insulin resistance that underlies thriftiness encapsulates something called 'redox thriftiness', whereby insulin resistance is determined by the ability to resist oxidative stress. Modern man has removed most hormetic stimuli and replaced them with a calorific sedentary lifestyle, leading to increased risk of metabolic inflexibility. We suggest that there is a tipping point where lipotoxicity in adipose and hepatic cells induces mild inflammation, which switches thrifty insulin resistance to inflammation-driven insulin resistance. To understand this, we propose that the metabolic syndrome could be seen from the viewpoint of the ECS, the mitochondrion and the FOXO group of transcription factors. FOXO has many thrifty actions, including increasing insulin resistance and appetite, suppressing oxidative stress and shifting the organism towards using fatty acids. In concert with factors such as PGC-1, they also modify mitochondrial function and biogenesis. Hence, the ECS and FOXO may interact at many points; one of which may be via intracellular redox signalling. As cannabinoids have been shown to modulate reactive oxygen species production, it is possible that they can upregulate anti-oxidant defences. This suggests they may have an 'endohormetic' signalling function. The tipping point into the metabolic syndrome may be the result of a chronic lack of hormetic stimuli (in particular, physical activity), and thus, stimulus for PGC-1, with a resultant reduction in mitochondrial function and a reduced lipid capacitance. This, in the context of a positive calorie environment, will result in increased visceral adipose tissue volume, abnormal ectopic fat content and systemic inflammation. This would worsen the inflammatory-driven pathological insulin resistance and inability to deal with lipids. The resultant oxidative stress may therefore drive a compensatory anti-oxidative response epitomised by the ECS and FOXO. Thus, although blocking the ECS (e.g. via rimonabant) may induce temporary weight loss, it may compromise long-term stress resistance. Clues about how to modulate the system more safely are emerging from observations that some polyphenols, such as resveratrol and possibly, some phytocannabinoids, can modulate mitochondrial function and might improve resistance to a modern lifestyle.

Acute glycaemic load breakfast manipulations do not attenuate cognitive impairments in adults with type 2 diabetes

Abnormalities in glucose tolerance such as type 2 diabetes can have demonstrable negative effects on a range of cognitive functions. However, there was no evidence that low GL breakfasts administered acutely could confer benefits for cognitive function (ClincalTrials.gov identifier, NCT01047813).

Replacement of saturated with unsaturated fats had no impact on vascular function but beneficial effects on lipid biomarkers, E-selectin and blood pressure: results from the randomized, controlled Dietary Intervention and VAScular function (DIVAS) study

Background: Public health strategies to lower cardiovascular disease (CVD) risk involve reducing dietary saturated fatty acid (SFA) intake to ≤10% of total energy (%TE). However, the optimal type of replacement fat is unclear. Objective: We investigated the substitution of 9.5-9.6%TE dietary SFA with either monounsaturated (MUFA) or n-6 polyunsaturated fatty acids (PUFA) on vascular function and other CVD risk factors. Design: Using a randomized, controlled, single-blind, parallel group dietary intervention, 195 men and women aged 21-60 y with moderate CVD risk (≥50% above the population mean) from the United Kingdom followed one of three 16-wk isoenergetic diets (%TE target compositions, total fat:SFA:MUFA:n-6 PUFA): SFA-rich (36:17:11:4, n = 65), MUFA-rich (36:9:19:4, n = 64) or n-6 PUFA-rich (36:9:13:10, n = 66). The primary outcome measure was flow-mediated dilatation (%FMD); secondary outcome measures included fasting serum lipids, microvascular reactivity, arterial stiffness, ambulatory blood pressure, and markers of insulin resistance, inflammation and endothelial activation. Results: Replacing SFA with MUFA or n-6 PUFA did not significantly impact on %FMD (primary endpoint) or other measures of vascular reactivity. Of the secondary outcome measures, substitution of SFA with MUFA attenuated the increase in night systolic blood pressure (-4.9 mm Hg, P = 0.019) and reduced E-selectin (-7.8%, P = 0.012). Replacement with MUFA or n-6 PUFA lowered fasting serum total cholesterol (TC; -8.4% and -9.2%, respectively), low-density lipoprotein cholesterol (-11.3% and -13.6%) and TC to high-density lipoprotein cholesterol ratio (-5.6% and -8.5%) (P ≤ 0.001). These changes in low-density lipoprotein cholesterol equate to an estimated 17-20% reduction in CVD mortality. Conclusions: Substitution of 9.5-9.6%TE dietary SFA with either MUFA or n-6 PUFA did not impact significantly on %FMD or other measures of vascular function. However, the beneficial effects on serum lipid biomarkers, blood pressure and E-selectin offer a potential public health strategy for CVD risk reduction.

Symmorphosis through dietary regulation: a combinatorial role for proteolysis, autophagy and protein synthesis in normalising muscle metabolism and function of hypertrophic mice after acute starvation

Animals are imbued with adaptive mechanisms spanning from the tissue/organ to the cellular scale which insure that processes of homeostasis are preserved in the landscape of size change. However we and others have postulated that the degree of adaptation is limited and that once outside the normal levels of size fluctuations, cells and tissues function in an aberant manner. In this study we examine the function of muscle in the myostatin null mouse which is an excellent model for hypertrophy beyond levels of normal growth and consequeces of acute starvation to restore mass. We show that muscle growth is sustained through protein synthesis driven by Serum/Glucocorticoid Kinase 1 (SGK1) rather than Akt1. Furthermore our metabonomic profiling of hypertrophic muscle shows that carbon from nutrient sources is being channelled for the production of biomass rather than ATP production. However the muscle displays elevated levels of autophagy and decreased levels of muscle tension. We demonstrate the myostatin null muscle is acutely sensitive to changes in diet and activates both the proteolytic and autophagy programmes and shutting down protein synthesis more extensively than is the case for wild-types. Poignantly we show that acute starvation which is detrimental to wild-type animals is beneficial in terms of metabolism and muscle function in the myostatin null mice by normalising tension production.

Fetal programming of adipose tissue function: an evolutionary perspective

Obesity is an escalating threat of pandemic proportions and has risen to such unrivaled prominence in such a short period of time that it has come to define a whole generation in many countries around the globe. The burden of obesity, however, is not equally shared among the population, with certain ethnicities being more prone to obesity than others, while some appear to be resistant to obesity altogether. The reasons behind this ethnic basis for obesity resistance and susceptibility, however, have remained largely elusive. In recent years, much evidence has shown that the level of brown adipose tissue thermogenesis, which augments energy expenditure and is negatively associated with obesity in both rodents and humans, varies greatly between ethnicities. Interestingly, the incidence of low birth weight, which is associated with an increased propensity for obesity and cardiovascular disease in later life, has also been shown to vary by ethnic background. This review serves to reconcile ethnic variations in BAT development and function with ethnic differences in birth weight outcomes to argue that the variation in obesity susceptibility between ethnic groups may have its origins in the in utero programming of BAT development and function as a result of evolutionary adaptation to cold environments.

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnA(loxP/loxP) females with GATA1-Cre males. FlnA(loxP/y) GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbalpha because they lack the normal cytoskeletal linkage of GPIbalpha to underlying actin filaments. This results in approximately 70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-like-mediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased alpha-granule secretion, integrin alphaIIbbeta3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase C-gamma2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnA-Syk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-like-containing receptor signaling and platelet function.