Effects of addition of phenolic compounds on the acid gelation of milk

Tannic acid (0.1–1%, w/w) and gallic acid (0.3–1%, w/w) were added to skim milk prior to acidification with GDL. The acid gelation of tannic and gallic acid fortified milk had a faster gelation time in comparison with the control gel without phenolic compounds. The addition of tannic acid and gallic acid (up to 0.8%) to the milk resulted in a higher storage modulus (G′), decrease in the water mobility (T2 time) and had no significant effect on the syneresis index (SI). However, the inclusion of 1% gallic acid resulted in a significant decrease in G′, a significant increase in the SI and a wider T2 distribution. Lowering the temperature of the gels from 30 to 5 °C caused the G′ for the gels with gallic and tannic acid to increase significantly in comparison with the control, possibly due to increased hydrogen bonding in the presence of phenolic compounds

Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7

P>Type III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.

Identification of components of the Sigma B Regulon in Listeria monocytogenes that contribute to acid and salt tolerance

Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

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.

Effects of repeated cycles of acid challenge and growth on the phenotype and virulence of Salmonella enterica

Aims: The aim of the study was to investigate how stresses like low pH, which may be encountered in farms or food preparation premises, shape populations of Salmonella enterica by the selection of stress-resistant variants. Methods and Results: Stationary-phase cultures of S. enterica serovar Enteritidis and serovar Typhimurium (one strain of each) were exposed to pH 2Æ5 for up to 4 h, followed by growth at pH 7 for 48 h. This process was repeated 15 times in two separate experiments, which increased the acid resistance of the three out of four populations we obtained, by three- to fourfold. Sustainable variants derived from the populations showed changes in colony morphology, expression of SEF17 fimbriae, growth, increased heat resistance and reduced virulence. Conclusions: The study demonstrates that low pH environments can select for populations of S. enterica with persistent phenotypic changes such as increased acid resistance and occasionally increased SEF17 expression and lower virulence. Significance and Impact of the Study: There is a common belief that increased acid resistance coincides with increased virulence. This study demonstrates for the first time that increased acid resistance often impairs virulence and affects the general phenotype of S. enterica.

Salmon consumption during pregnancy alters fatty acid composition and secretory IgA concentration in human breast milk

Fish oil supplementation during pregnancy alters breast milk composition, but there is little information about the impact of oily fish consumption. We determined whether increased salmon consumption during pregnancy alters breast milk fatty acid composition and immune factors. Women (n = 123) who rarely ate oily fish were randomly assigned to consume their habitual diet or to consume 2 portions of farmed salmon per week from 20 wk of pregnancy until delivery. The salmon provided 3.45 g long-chain (LC) (n-3) PUFA/wk. Breast milk fatty acid composition and immune factors [soluble CD14, transforming growth factor-b (TGFb)1, TGFb2, and secretory IgA] were analyzed at 1, 5, 14, and 28 d postpartum (PP). Breast milk from the salmon group had higher proportions of EPA (80%), docosapentaenoic acid (30%), and DHA (90%) on d 5 PP compared with controls (P < 0.01). The LC (n-6) PUFA:LC (n-3) PUFA ratio was lower for the salmon group on all days of PP sampling (P < 0.004), although individual (n-6) PUFA proportions, including arachidonic acid, did not differ. All breast milk immune factors decreased between d 1 and 28 PP (P < 0.001). Breast milk secretory IgA (sIgA) was lower in the salmon group (d 1–28 PP; P = 0.006). Salmon consumption during pregnancy, at the current recommended intakes, increases the LC (n-3) PUFA concentration of breast milk in early lactation, thus improving the supply of these important fatty acids to the breast-fed neonate. The consequence of the lower breast milk concentration of sIgA in the salmon group is not clear.

Immune factors and fatty acid composition in human milk from river/lake, coastal and inland regions of China

Breast milk fatty acid composition may be affected by maternal diet during gestation and lactation. The influence of dietary and breast milk fatty acids on breast milk immune factors is poorly defined. We determined the fatty acid composition and immune factor concentrations of breast milk from women residing in river & lake, coastal, and inland regions of China, which differ in their consumption of lean fish and oily fish. Breast milk samples were collected on days 3 to 5 (colostrum), 14 and 28 post-partum and analysed for soluble CD14 (sCD14), transforming growth factor (TGF)-β1, TGF-β2, secretory immunoglobulin A (sIgA) and fatty acids. The fatty acid composition of breast milk differed between regions and with time post-partum. The concentrations of all four immune factors in breast milk decreased over time, with sCD14, sIgA and TGF-β1 being highest in colostrum in the river & lake region. Breast milk DHA and arachidonic acid (AA) were positively associated, and γ-linolenic acid and EPA negatively associated, with the concentrations of each of the four immune factors. In conclusion, breast milk fatty acids and immune factors differ between regions in China characterised by different patterns of fish consumption and change during the course of lactation. A higher breast milk DHA and AA concentration is associated with higher concentrations of immune factors in breast milk, suggesting a role for these fatty acids in promoting gastrointestinal and immune maturation of the infant.

Effect of a 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in the pig

Objectives: There are concerns that the use of enrofloxacin in livestock production may contribute to the development of fluoroquinolone resistance in zoonotic bacteria. The objective of our study was to investigate the effect of a single 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in a pig model. Results: Our results showed that a single treatment failed to eradicate S. Typhimurium DT104, which continued to be isolated up to 35 days after treatment. We also provide evidence that treatment positively selects for S. Typhimurium DT104 strains that are already nalidixic acid resistant (gyrA Asn-87) or cyclohexane resistant, the latter being indicative of an up-regulated efflux pump. Emergence of fluoroquinolone resistance was not detected during treatment or post-treatment in any of the Salmonella strains monitored. However, the effect of enrofloxacin on the nalidixic acid-resistant and cyclohexane-resistant S. Typhimurium DT104 outlasted the current withdrawal time of 10 days for Baytril (commercial veterinary formulation of enrofloxacin). Conclusions: In conclusion, our study has provided direct evidence that enrofloxacin-treated pigs could be entering abattoirs with higher numbers of quinolone-resistant zoonotic bacteria than untreated pigs, increasing the risk of these entering the food chain.

Epoxide ring opening in a zinc(II) complex in water without any Lewis acid catalyst: formation of only one diastereomer out of 2

The epoxide ring in 5,6-dihydro-5,6-epoxy-1,10-phenanthroline (L) opens up in its reaction with 4-methylaniline and 4-methoxyaniline in water in equimolar proportion at room temperature without any Lewis acid catalyst to give a monohydrate of 6-(4-methyl-phenylamino)-5,6-dihydro-1,10-phenanthrolin-5-ol (L′·H2O) and 6-(4-methoxyphenyl-amino)-5,6-dihydro-1,10-phenanthrolin-5-ol (L″) respectively. Reaction time decreases from 72 to 14 h in boiling water. But the yields become less. Reaction of L with Zn(ClO4)2·6H2O in methanol in 3:1 molar ratio at room temperature affords white [ZnL3](ClO4)2·H2O. The X-ray crystal structure of the acetonitrile solvate [ZnL3](ClO4)2·MeCN has been determined which shows that the metal has a distorted octahedral N6 coordination sphere. [ZnL3](ClO4)2·2H2O reacts with 4-methylaniline and 4-methoxyaniline in boiling water in 1:3 molar proportion in the absence of any Lewis acid catalyst to produce [ZnL′3](ClO4)2·4H2O and [ZnL″3](ClO4)2·H2O, respectively in 1–4 h time in somewhat low yield. In the 1H NMR spectra of [ZnL′3](ClO4)2·4H2O and [ZnL″3](ClO4)2·H2O, only one sharp methyl signal is observed implicating that only one diastereomer out of the 23 possibilities is formed. The same diastereomers are obtained when L′·H2O and L″ are reacted directly with Zn(ClO4)2·6H2O in tetrahydrofuran at room temperature in very good yields. Reactions of L′·H2O and L″ with Ru(phen)2Cl2·2H2O (phen = 1,10-phenanthroline) in equimolar proportion in methanol–water mixture under refluxing condition lead to the isolation of two diastereomers of [Ru(phen)2L′](ClO4)2·2H2O and [Ru(phen)2L″](ClO4)2·2H2O.

Is fatty acid intake a predictor of arterial stiffness and blood pressure in men? Evidence from the Caerphilly Prospective Study

Background and aims: Arterial stiffness is an independent predictor of cardiovascular disease (CVD) events and all-cause mortality and may be differentially affected by dietary fatty acid (FA) intake. The aim of this study was to investigate the relationship between FA consumption and arterial stiffness and blood pressure in a community-based population. Methods and results: The Caerphilly Prospective Study recruited 2398 men, aged 45-59 years, who were followed up at 5-year intervals for a mean of 17.8-years (n 787). A semi-quantitative food frequency questionnaire estimated intakes of total, saturated, mono- and poly-unsaturated fatty acids (SFA, MUFA, PUFA). Multiple regression models investigated associations between intakes of FA at baseline with aortic pulse wave velocity (aPWV), augmentation index (AIx), systolic and diastolic blood pressure (SBP, DBP) and pulse pressure after a 17.8-year follow-up - as well as cross-sectional relationships with metabolic markers. After adjustment, higher SFA consumption at baseline was associated with higher SBP (P = 0.043) and DBP (P = 0.002) and after a 17.8-year follow-up was associated with a 0.51 m/s higher aPWV (P = 0.006). After adjustment, higher PUFA consumption at baseline was associated with lower SBP (P = 0.022) and DBP (P = 0.036) and after a 17.8-year follow-up was associated with a 0.63 m/s lower aPWV (P = 0.007). Conclusion: This study suggests that consumption of SFA and PUFA have opposing effects on arterial stiffness and blood pressure. Importantly, this study suggests that consumption of FA is an important risk factor for arterial stiffness and CVD.

Polysialic acid as a drug carrier: evaluation of a new polysialic acid–epirubicin conjugate and its comparison against established drug carriers

This paper explores the potential of polysialic acid (PSA) as a carrier for low molecular weight anticancer drugs. A PSA–epirubicin (Epi) conjugate was synthesized and compared against Epi conjugates containing established carriers, namely: N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, poly(ethylene glycol) (PEG) and polyglutamic acid (PGA). Biological assessments in the breast cancer cell line MCF-7 and in the anthracycline resistant MCF-7/DX showed that the PSA–Epi conjugate had the highest activity (40% and 30% cell death in the two cell lines at 1 mM Epi equiv., respectively). FACS studies confirmed internalization of all conjugates by cholesterol-dependent endocytosis. PSA–Epi showed release of Epi (40% at 5 h) when incubated with lysosome extracts. In vivo evaluation showed that all conjugates had a significantly longer half-life compared to free Epi. This study also allowed an investigation on the effect of the polymeric carrier on the biological activity of a conjugate, with the biodegradability of the carrier emerging as an important feature.

Incremental effect of a calcium salt of cis-monounsaturated fatty acids supplement on milk fatty acid composition in cows fed maize silage-based diets

In most Western countries, saturated fatty acid (SFA) intake exceeds recommended levels, which is considered a risk factor for cardiovascular disease (CVD). As milk and dairy products are major contributors to SFA intake in many countries, recent research has focused on sustainable methods of producing milk with a lower saturated fat concentration by altering dairy cow diets. Human intervention studies have shown that CVD risk can be reduced by consuming dairy products with reduced SFA and increased cis-monounsaturated fatty acid (MUFA) concentrations. This milk fatty acid profile can be achieved by supplementing dairy cow diets with cis-MUFA-rich unsaturated oils. However, rumen exposure of unsaturated oils also leads to enhanced milk trans fatty acid (TFA) concentrations. Because of concerns about the effects of TFA consumption on CVD, feeding strategies that increase MUFA concentrations in milk without concomitant increases in TFA concentration are preferred by milk processors. In an attempt to limit TFA production and increase the replacement of SFA by cis-MUFA, a preparation of rumen-protected unsaturated oils was developed using saponification with calcium salts. Four multiparous Holstein-Friesian cows in mid-late lactation were used in a 4 × 4 Latin square design with 21-d periods to investigate the effect of incremental dietary inclusion of a calcium salt of cis-MUFA product (Ca-MUFA; 20, 40, and 60 g/kg of dry matter of a maize silage-based diet), on milk production, composition, and fatty acid concentration. Increasing Ca-MUFA inclusion reduced dry matter intake linearly, but no change was observed in estimated ME intake. No change in milk yield was noted, but milk fat and protein concentrations were linearly reduced. Supplementation with Ca-MUFA resulted in a linear reduction in total SFA (from 71 to 52 g/100 g of fatty acids for control and 60 g/kg of dry matter diets, respectively). In addition, concentrations of both cis- and trans-MUFA were increased with Ca-MUFA inclusion, and increases in other biohydrogenation intermediates in milk fat were also observed. The Ca-MUFA supplement was very effective at reducing milk SFA concentration and increasing cis-MUFA concentrations without incurring any negative effects on milk and milk component yields. However, reduced milk fat and protein concentrations, together with increases in milk TFA concentrations, suggest partial dissociation of the calcium salts in the rumen

Seasonal variation in the fatty acid composition of milk available at retail in the United Kingdom and implications for dietary intake

Milk and dairy products are major sources of fat in the human diet, but there are few detailed reports on the fatty acid composition of retail milk, trans fatty acids in particular, and how these change throughout the year. Semi-skimmed milk was collected monthly for one year from five supermarkets and analysed for fatty acid composition. Relative to winter, milk sold in the summer contained lower total saturated fatty acid (SFA; 67 vs 72 g/100 g fatty acids) and higher cis-monounsaturated fatty acid (MUFA; 23 vs 21 g/100 g fatty acids) and total trans fatty acid (6.5 vs 4.5 g/100 g fatty acids) concentrations. Concentrations of most trans-18:1 and -18:2 isomers also exhibited seasonal variation. Results were applied to national dietary intakes, and indicated that monthly variation in the fatty acid composition of milk available at retail has limited influence on total dietary fatty acid consumption by UK adults.

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

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.