Predicting microbial pollution concentrations in UK rivers in response to land use change

The Water Framework Directive has caused a paradigm shift towards the integrated management of recreational water quality through the development of drainage basin-wide programmes of measures. This has increased the need for a cost-effective diagnostic tool capable of accurately predicting riverine faecal indicator organism (FIO) concentrations. This paper outlines the application of models developed to fulfil this need, which represent the first transferrable generic FIO models to be developed for the UK to incorporate direct measures of key FIO sources (namely human and livestock population data) as predictor variables. We apply a recently developed transfer methodology, which enables the quantification of geometric mean presumptive faecal coliforms and presumptive intestinal enterococci concentrations for base- and high-flow during the summer bathing season in unmonitored UK watercourses, to predict FIO concentrations in the Humber river basin district. Because the FIO models incorporate explanatory variables which allow the effects of policy measures which influence livestock stocking rates to be assessed, we carry out empirical analysis of the differential effects of seven land use management and policy instruments (fiscal constraint, production constraint, cost intervention, area intervention, demand-side constraint, input constraint, and micro-level land use management) all of which can be used to reduce riverine FIO concentrations. This research provides insights into FIO source apportionment, explores a selection of pollution remediation strategies and the spatial differentiation of land use policies which could be implemented to deliver river quality improvements. All of the policy tools we model reduce FIO concentrations in rivers but our research suggests that the installation of streamside fencing in intensive milk producing areas may be the single most effective land management strategy to reduce riverine microbial pollution.

Eicosapentaenoic acid and docosahexaenoic acid from fish oils: differential associations with lipid responses

Fish-oil supplementation can reduce circulating triacylglycerol (TG) levels and cardiovascular risk. This study aimed to assess independent associations between changes in platelet eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and fasting and postprandial (PP) lipoprotein concentrations and LDL oxidation status, following fish-oil intervention. Fiftyfive mildly hypertriacylglycerolaemic (TG 1·5–4·0 mmol/l) men completed a double-blind placebo controlled cross over study, where individuals consumed 6 g fish oil (3 g EPA � DHA) or 6 g olive oil (placebo)/d for two 6-week intervention periods, with a 12-week wash-out period in between. Fish-oil intervention resulted in a significant increase in the platelet phospholipid EPA (+491 %, P,0·001) and DHA (+44 %, P,0·001) content and a significant decrease in the arachidonic acid (210 %, P,0·001) and g-linolenic acid (224 %, P,0·001) levels. A 30% increase in ex vivo LDL oxidation (P,0·001) was observed. In addition, fish oil resulted in a significant decrease in fasting and PP TG levels (P,0·001), PP non-esterified fatty acid (NEFA) levels, and in the percentage LDL as LDL-3 (P�0·040), and an increase in LDLcholesterol (P�0·027). In multivariate analysis, changes in platelet phospholipid DHA emerged as being independently associated with the rise in LDL-cholesterol, accounting for 16% of the variability in this outcome measure (P�0·030). In contrast, increases in platelet EPA were independently associated with the reductions in fasting (P�0·046) and PP TG (P�0·023), and PP NEFA (P�0·015), explaining 15–20% and 25% of the variability in response respectively. Increases in platelet EPA � DHA were independently and positively associated with the increase in LDL oxidation (P�0·011). EPA and DHA may have differential effects on plasma lipids in mildly hypertriacylglycerolaemic men.

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification

Objectives: The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-ToIC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide. Methods: The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT-PCR, ability to form a biofilm and motility assays. Results: Despite important differences in the triclosan-dependent transcriptomes of the two species, increased expression of efflux pump component genes was seen in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent. Conclusions: These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents.

p90 ribosomal S6 kinases play a significant role in early gene regulation in the cardiomyocyte response to Gq protein-coupled receptor stimuli, endothelin-1 and α1-adrenergic receptor agonists

Extracellular signal-regulated kinases 1/2 (ERK1/2) and their substrates, p90 ribosomal S6 kinases (RSKs), phosphorylate different transcription factors, contributing differentially to transcriptomic profiles. In cardiomyocytes, ERK1/2 are required for >70% of the transcriptomic response to endothelin-1. Here, we investigated the role of RSKs in the transcriptomic responses to Gq protein-coupled receptor agonists, endothelin-1, phenylephrine (generic α1-adrenergic receptor agonist) and A61603 (α1A-adrenergic receptor selective). Phospho-ERK1/2 and phospho-RSKs appeared in cardiomyocyte nuclei within 2-3 min of stimulation (endothelin-1>a61603≈phenylephrine). All agonists increased nuclear RSK2, but only endothelin-1 increased nuclear RSK1 content. PD184352 (inhibits ERK1/2 activation) and BI-D1870 (inhibits RSKs) were used to dissect the contribution of RSKs to the endothelin-1-responsive transcriptome. Of 213 RNAs upregulated at 1 h, 51% required RSKs for upregulation whereas 29% required ERK1/2 but not RSKs. The transcriptomic response to phenylephrine overlapped with, but was not identical to, endothelin-1. As with endothelin-1, PD184352 inhibited upregulation of most phenylephrine-responsive transcripts, but the greater variation in effects of BI-D1870 suggests that differential RSK signalling influences global gene expression. A61603 induced similar changes in RNA expression in cardiomyocytes as phenylephrine, indicating that the signal was mediated largely through α1A-adrenergic receptors. A61603 also increased expression of immediate early genes in perfused adult rat hearts and, as in cardiomyocytes, upregulation of the majority of genes was inhibited by PD184352. PD184352 or BI-D1870 prevented the increased surface area induced by endothelin-1 in cardiomyocytes. Thus, RSKs play a significant role in regulating cardiomyocyte gene expression and hypertrophy in response to Gq protein-coupled receptor stimulation.

Pharmacometabonomic characterization of xenobiotic and endogenous metabolic phenotypes that account for inter-individual variation in isoniazid-induced toxicological response

An NMR-based pharmacometabonomic approach was applied to investigate inter-animal variation in response to isoniazid (INH; 200 and 400 mg/kg) in male Sprague-Dawley rats, alongside complementary clinical chemistry and histopathological analysis. Marked inter-animal variability in central nervous system (CNS) toxicity was identified following administration of a high dose of INH, which enabled characterization of CNS responders and CNS non-responders. High-resolution post-dose urinary (1)H NMR spectra were modeled both by their xenobiotic and endogenous metabolic information sets, enabling simultaneous identification of the differential metabolic fate of INH and its associated endogenous metabolic consequences in CNS responders and CNS non-responders. A characteristic xenobiotic metabolic profile was observed for CNS responders, which revealed higher urinary levels of pyruvate isonicotinylhydrazone and β-glucosyl isonicotinylhydrazide and lower levels of acetylisoniazid compared to CNS non-responders. This suggested that the capacity for acetylation of INH was lower in CNS responders, leading to increased metabolism via conjugation with pyruvate and glucose. In addition, the endogenous metabolic profile of CNS responders revealed higher urinary levels of lactate and glucose, in comparison to CNS non-responders. Pharmacometabonomic analysis of the pre-dose (1)H NMR urinary spectra identified a metabolic signature that correlated with the development of INH-induced adverse CNS effects and may represent a means of predicting adverse events and acetylation capacity when challenged with high dose INH. Given the widespread use of INH for the treatment of tuberculosis, this pharmacometabonomic screening approach may have translational potential for patient stratification to minimize adverse events.

Differential effect of beetroot bread on postprandial DBP according to Glu298Asp polymorphism in the eNOS gene: a pilot study.

Our objective was to investigate whether the presence of Glu298Asp polymorphism in the endothelial NO synthase (eNOS) gene differentially affects the postprandial blood pressure response to dietary nitrate-rich beetroot bread. A randomised, single-blind, controlled, crossover acute pilot study was performed in 14 healthy men (mean age: 34±9 years) who were retrospectively genotyped for Glu298Asp polymorphism (7GG; T carriers 7). Volunteers were randomised to receive 200 g beetroot-enriched bread (1.1 mmol nitrate) or control bread (no beetroot; 0.01 mmol nitrate) on two separate occasions 10 days apart. Baseline and incremental area under the curve of blood pressure and NOx (nitrate/nitrite) were measured for a 6-h postprandial period. A treatment × genotype interaction was observed for diastolic blood pressure (P<0.02), which was significantly lower in T carriers (P<0.01) after consumption of beetroot bread compared with control bread. No significant differences were observed in the GG group. The beneficial diastolic blood pressure reduction was observed only in the T carriers of the Glu298Asp polymorphism in the eNOS gene after consumption of nitrate-rich beetroot bread. These data require confirmation in a larger population group.

Differential brain activity during emotional versus nonemotional reversal learning

The ability to change an established stimulus–behavior association based on feedback is critical for adaptive social behaviors. This ability has been examined in reversal learning tasks, where participants first learn a stimulus–response association (e.g., select a particular object to get a reward) and then need to alter their response when reinforcement contingencies change. Although substantial evidence demonstrates that the OFC is a critical region for reversal learning, previous studies have not distinguished reversal learning for emotional associations from neutral associations. The current study examined whether OFC plays similar roles in emotional versus neutral reversal learning. The OFC showed greater activity during reversals of stimulus–outcome associations for negative outcomes than for neutral outcomes. Similar OFC activity was also observed during reversals involving positive outcomes. Furthermore, OFC activity is more inversely correlated with amygdala activity during negative reversals than during neutral reversals. Overall, our results indicate that the OFC is more activated by emotional than neutral reversal learning and that OFC's interactions with the amygdala are greater for negative than neutral reversal learning.

Microbial Recognition Via Toll-Like Receptor-Dependent and -Independent Pathways Determines the Cytokine Response of Murine Dendritic Cell Subsets to CD40 Triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

Differential roles of the PKC novel isoforms, PKCδ and PKCε, in mouse and human platelets

Background Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation. Methodology/Principal Findings In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor. Conclusions These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.

Differential roles for the adapters Gads and LAT in platelet activation by GPVI and CLEC-2.

The results demonstrate that Gads plays a key role in linking the adapter LAT to SLP-76 in response to weak activation of GPVI and CLEC-2 whereas LAT is required for full activation over a wider range of agonist concentrations. These results reveal the presence of a Gads-independent pathway of platelet activation downstream of LAT.

Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts.

The translocation of protein kinase C (PKC) isoforms PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from soluble to particulate fractions was studied in ventricular cardiomyocytes cultured from neonatal rats. Endothelin-1 (ET-1) caused a rapid ETA receptor-mediated translocation of PKC-delta and PKC-epsilon (complete in 0.5-1 min). By 3-5 min, both isoforms were returning to the soluble fraction, but a greater proportion of PKC-epsilon remained associated with the particulate fraction. The EC50 of translocation for PKC-delta was 11-15 nM ET-1 whereas that for PKC-epsilon was 1.4-1.7 nM. Phenylephrine caused a rapid translocation of PKC-epsilon (EC50 = 0.9 microM) but the proportion lost from the soluble fraction was less than with ET-1. Translocation of PKC-delta was barely detectable with phenylephrine. Neither agonist caused any consistent translocation of PKC-alpha or PKC-zeta. Activation of p42 and p44 mitogen-activated protein kinase (MAPK) by ET-1 or phenylephrine followed more slowly (complete in 3-5 min). Phosphorylation of p42-MAPK occurred simultaneously with its activation. The proportion of the total p42-MAPK pool phosphorylated in response to ET-1 (50%) was greater than with phenylephrine (20%). In addition to activation of MAPK, an unidentified p85 protein kinase was activated by ET-1 in the soluble fraction whereas an unidentified p58 protein kinase was activated in the particulate fraction.

Differential interaction of estrogen receptor and thyroid hormone receptor isoforms on the rat oxytocin receptor promoter leads to differences in transcriptional regulation

Both the estrogen receptor (ER) and thyroid hormone receptor (TR) are members of the nuclear receptor superfamily. Two isoforms of the ER, alpha and beta, exist. The TRalpha and beta isoforms are products of two distinct genes that are further differentially spliced to give TRalpha1 and alpha2, TRbeta1 and beta2. The TRs have been shown to interfere with ER-mediated transcription from both the consensus estrogen response element (ERE) and the rat preproenkephalin (PPE) promoter, possibly by competing with ER binding to the ERE or by squelching coactivators essential for ER-mediated transcription. The rat oxytocin receptor (OTR) gene is thought to be involved in several facets of reproductive and affiliative behaviors. 17beta-Estradiol-bound ERs upregulate the OTR gene in the ventromedial hypothalamus, a region critical for the induction of lordosis behavior in several species. We investigated the effects of the ligand-binding TR isoforms on the ER-mediated transcription from a physiological promoter of a behaviorally relevant gene such as the OTR. Only ERalpha could induce the OTR gene in two cell lines tested, the CV-1 and the SK-N-BE2C neuroblastoma cell lines. ERbeta was incapable of inducing the gene in either cell line. ERalpha is therefore not equivalent to ERbeta on this physiological promoter. Indeed, in the neural cell line, ERbeta can inhibit ERalpha-mediated induction from the OTR promoter. While the TRalpha1 isoform inhibited ERalpha-mediated induction in the neural cell line, the TRbeta1 isoform stimulated induction, thus demonstrating isoform specificity in the interaction. The use of a DNA-binding mutant, the TR P box mutant, showed that inhibition of ERalpha-mediated induction of the rat OTR gene promoter by the TRalpha1 isoform does not require DNA-binding ability. SRC-1 overexpression relieved TRalpha1-mediated inhibition in both cell lines, suggesting that squelching for coactivators is an important molecular mechanism in TRalpha-mediated inhibition. Such interactions between TR and ER isoforms on the rat OTR promoter provide a mechanism to achieve neuroendocrine integration.

Differential crosstalk between estrogen receptor (ER) alpha and ER beta and the thyroid hormone receptor isoforms results in flexible regulation of the consensus ERE

Crosstalk between nuclear receptors is important for conversion of external and internal stimuli to a physiologically meaningful response by cells. Previous studies from this laboratory have demonstrated crosstalk between the estrogen (ER) and thyroid hormone receptors (TR) on two estrogen responsive physiological promoters, the preproenkephalin and oxytocin receptor gene promoter. Since ERa and ERb are isoforms possessing overlapping and distinct transactivation properties, we hypothesized that the interaction of ERa and b with the various TR isoforms would not be equivalent. To explore this hypothesis, the consensus estrogen response element (ERE)derived from the Xenopus vitellogenin gene is used to investigate the differences in interaction between ERa and b isoforms and the different TR isoforms in fibroblast cells. Both the ER isoforms transactivate from the consensus ERE, though ERa transactivates to a greater extent than ERb. Although neither of the TRb isoforms have an effect on ERa transactivation from the consensus ERE, the liganded TRa1 inhibits the ERa transactivation from the consensus ERE. In contrast, the liganded TRa1 facilitates ERb-mediated transactivation. The crosstalk between the TRb isoforms with the ERa isoform, on the consensus ERE, is different from that with the ERb isoform. The use of a TRa1 mutant, which is unable to bind DNA, abolishes the ability of the TRa1 isoform to interact with either of the ER isoforms. These differences in nuclear receptor crosstalk reveal an important functional difference between isoforms, which provides a novel mechanism for neuroendocrine integration.

Crosstalk between oestrogen receptors and thyroid hormone receptor isoforms results in differential regulation of the preproenkephalin gene

Nuclear receptors are ligand-activated transcription factors, which have the potential to integrate internal metabolic events in an organism, with consequences for control of behaviour. Previous studies from this laboratory have shown that thyroid hormone receptor (TR) isoforms can inhibit oestrogen receptor (ER)alpha-mediated induction of preproenkephalin (PPE) gene expression in the hypothalamus. Also, thyroid hormone administration inhibits lordosis, a behaviour facilitated by PPE expression. We have examined the effect of multiple ligand-binding TR isoforms on the ER-mediated induction of the PPE gene in transient transfection assays in CV-1 cells. On a natural PPE gene promoter fragment containing two putative oestrogen response elements (EREs), both ER alpha and beta isoforms mediate a four to five-fold induction by oestrogen. Cotransfection of TR alpha 1 along with ER alpha inhibited the ER alpha transactivation of PPE by approximately 50%. However, cotransfection with either TR beta 1 or TR beta 2 expression plasmids produced no effect on the ER alpha or ER beta mediated induction of PPE. Therefore, under these experimental conditions, interactions with a single ER isoform are specific to an individual TR isoform. Transfection with a TR alpha 1 DNA-binding mutant could also inhibit ER alpha transactivation, suggesting that competition for binding on the ERE may not be the exclusive mechanism for inhibition. Data with the coactivator, SRC-1, suggested that coactivator squelching may participate in the inhibition. In dramatic contrast, when ER beta is cotransfected, TR alpha 1 stimulated ER beta-mediated transactivation of PPE by approximately eight-fold over control levels. This is the first study revealing specific interactions among nuclear receptor isoforms on a neuroendocrine promoter. These data also suggest that the combinatorics of ER and TR isoforms allow multiple forms of flexible gene regulations in the service of neuroendocrine integration.

Differential gene expression analysis of iodide-treated rat thyroid follicular cell line PCCl3

The inhibitory effect of supraphysiological iodide concentrations on thyroid hormone synthesis (Wolff - Chaikoff effect) and on thyrocyte proliferation is largely known as iodine autoregulation. However, the molecular mechanisms by which iodide modulates thyroid function remain unclear. In this paper, we analyze the transcriptome profile of the rat follicular cell lineage PCCl3 under untreated and treated conditions with 10 (- 3) M sodium iodide (NaI). Serial analysis of gene expression (SAGE) revealed 84 transcripts differentially expressed in response to iodide (p <= 0.001). We also showed that iodide excess inhibits the expression of essential genes for thyroid differentiation: Tshr, Nis, Tg, and Tpo. Relative expression of 14 of 20 transcripts selected by SAGE was confirmed by real-time PCR. Considering the key role of iodide organification in thyroid physiology, we also observed that both the oxidized form of iodide and iodide per se are responsible for gene expression modulation in response to iodide excess. (c) 2008 Elsevier Inc. All rights reserved.