Multicompartmental LC-Q-TOF-based metabonomics as an exploratory tool to identify novel pathways affected by polyphenol-rich diets in mice

Metabonomics has recently been used to study the physiological response to a given nutritional intervention, but such studies have usually been restricted to changes in either plasma or urine. In the present study, we demonstrate that the use of LC-Q-TOF-based metabolome analyses (foodstuff, plasma, urine, and caecal content metabolomes) in mice offer higher order information, including intra- and intercompartment relationships. To illustrate this, we performed an intervention study with three different phenolic-rich extracts in mice over 3 weeks. Both unsupervised (PCA) and supervised (PLS-DA) multivariate analyses used for pattern recognition revealed marked effects of diet in each compartment (plasma, urine, and caecal contents). Specifically, dietary intake of phenolic-rich extract affects pathways such as bile acid and taurine metabolism. Q-TOF-based metabonomics demonstrated that the number of correlations is higher in caecal contents and urine than in plasma. Moreover, intercompartment correlations showed that caecal contents-plasma correlations are the most frequent in mice, followed by plasma-urine ones. The number of inter- and intracompartment correlations is significantly affected by diet. These analyses reveal the complexity of interorgan metabolic relationships and their sensitivity to dietary changes.     

Human Health Risks Derived from Dietary Exposure to Toxic Metals in Catalonia,Spain: Temporal Trend

The present study was aimed at estimating the current (2012) dietary intake of arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) by the population of Catalonia, Spain. The temporal trends with respect to previous surveys, performed in 2000, 2005, and 2008, were also determined. For that purpose, metal concentrations were analyzed in a number of widely consumed foodstuffs. A speciation study was also conducted by experimentally determining the levels of inorganic As (InAs) and methylmercury (MeHg) in the same food items. Furthermore, the dietary intake of those metals and species was calculated both deterministically and probabilistically by considering two food consumption surveys: ENCAT and ENIDE, representative of the Catalan and Spanish populations, respectively. An important temporal decrease of the dietary intake was noted for most elements, irrespective of the age-gender population group. Considering data for a male adult, the current dietary intake of As, InAs, Cd, Hg, MeHg, and Pb was estimated in 216, 2.6, 8.7, 10, 7.3, and 8.4 μg/day, respectively, being these values lower than the respective provisional tolerable weekly intakes (PTWIs) or benchmark dose lower confidence limits (BMDLs). Moreover, new calculations by means of ENIDE survey indicated similar results to those previously obtained by ENCAT, with the exception of MeHg, whose intake exceeded the maximum recommended values for some part of the population. Although our data are similar to those frequently found in other European countries, the important intake of MeHg, which is linked to the high consumption of fish and shellfish, deserves further investigation.     

A theoretical approach to spot active regions in antimicrobial proteins

Background  

Much effort goes into identifying new antimicrobial compounds able to evade the increasing resistance of microorganisms to antibiotics. One strategy relies on antimicrobial peptides, either derived from fragments released by proteolytic cleavage of proteins or designed from known antimicrobial protein regions.     

Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein

Eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are proteins of the ribonuclease A (RNase A) superfamily that have developed biological properties related to the function of eosinophils. ECP is a potent cytotoxic molecule, and although the mechanism is still unknown this cytotoxic activity has been associated with its highly cationic character. Using liposome vesicles as a model, we have demonstrated that ECP tends to disrupt preferentially acidic membranes. On the basis of structure analysis, ECP variants modified at basic and hydrophobic residues have been constructed. Changes in the leakage of liposome vesicles by these ECP variants have indicated the role of both aromatic and basic specific amino acids in cellular membrane disruption. This is the case with the two tryptophans at positions 10 and 35, but not phenylalanine 76, and the two arginines 101 and 104. The bactericidal activity of both native ECP and point-mutated variants, tested against Escherichia coli and Staphylococcus aureus, suggests that basic amino acids play, in addition to the effect on the disruption of the cellular membrane, other roles such as specific binding on the surface of the bacteria cell.     

Rapid clearance of a recombinant Salmonella vaccine carrier prevents enhanced antigen-specific CD8 T-cell responses after oral boost immunizations

The type III secretion system of Salmonella enterica serovar Typhimurium can be used to target heterologous antigens directly into the cytosol of antigen-presenting cells. Our laboratory has previously reported that the single oral immunization of mice with a recombinant Salmonella strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal wild-type Listeria challenge infection. In the present study, we investigated whether these antigen-specific cytotoxic T lymphocytes induced by the prime immunization contribute to a more rapid clearance of the vaccine carrier after subsequent boost immunizations and whether oral boost immunizations lead to an augmented p60-specific CD8 T-cell response. We found that the ability of recombinant Salmonella strains to colonize the intestine, mesenteric lymph nodes, and spleen was markedly impaired after boost immunizations but that this effect was independent of existing CD8 T cells reactive with p60(217-225). A significant elevation of antigen-specific CD8 T cells could not be detected by enzyme-linked immunospot assay after the second or the third oral immunization, possibly due to the rapid clearance of the bacterial vaccine carrier from lymphatic organs.     

Biology of Triatoma vitticeps (Stal, 1859) under laboratory conditions (Hemiptera: Reduviidae: Triatominae). II. Resistance to fasting

Following the study on the biology of Triatoma vitticeps (Gon?alves et al., 1988) observations have been made on its resistance to starvation. Of the 286 eggs obtained only 201 hatched and reached the intended stage for observation. The others did not eclode, neither reached the ecdisis nor died, without explanation. The nymphs were kept, separately, in Borrel flasks and properly listed. The blood-meal was performed in mice, although the insects were kept without feeding as soon as moulted. The starvation was evaluated in two ways: the time-lapse in days between the last meal/death and between moult/death. The starvation was directly related with the developmental stage. In relation to the parameters last meal/death and moult/death, both sexes were less resistant than 3rd and 2nd stage, respectively. The experiment have been carried out for 15 months and by this time the average minimum and maximum temperatures and the humidity were 25 +/- 2 degrees C - 28 +/- 2 degrees C and 81 +/- 3% UR, respectively. The material spent belongs to the triatomine colony of the Oswaldo Cruz Institute Department of Entomology.     

Pre-existing anti-Salmonella vector immunity prevents the development of protective antigen-specific CD8 T-cell frequencies against murine listeriosis

Our laboratory has focused its research on the use of the type III secretion system of Salmonella enterica serovar Typhimurium to translocate heterologous antigens directly into the cytosol of antigen-presenting cells. We have previously reported that the single oral immunization of mice with a recombinant Salmonella aroA/sptP mutant strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes in a type III-mediated fashion results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal Listeria challenge infection. In the present study, we determined whether pre-existing anti-Salmonella vector immunity influences the induction of p60-specific CD8 T cells and modulates protective immunity against listeriosis after oral vaccination with recombinant Salmonella. After single oral immunization, the Salmonella aroA/sptP double mutant strain was found to colonize spleens of mice for 21days. In contrast, the period of colonization was significantly shortened to 6days due to anti-Salmonella vector immunity after second oral immunization. The latter scenario led to the induction of low-level frequencies of antigen-specific CD8 T cells. Compared to the significantly higher numbers of p60-specific T lymphocytes elicited after single oral immunization, the low amount of Listeria-specific CD8 T cells did not confer protection against listeriosis.     

Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase

Objective

Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes.

Methods

ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR.

Results

ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG.

Conclusions

ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.