M.A. Prieto

A Mutation in Flavobacterium psychrophilum tlpB Inhibits Gliding Motility and Induces Biofilm Formation

ABSTRACT Flavobacterium psychrophilum is a psychrotrophic, fish-pathogenic bacterium belonging to the Cytophaga-Flavobacterium-Bacteroides group. Tn4351-induced mutants deficient in gliding motility, growth on iron-depleted media, and extracellular proteolytic activity were isolated. Some of these mutants were affected in only one of these characteristics, whereas others had defects in two or more. FP523, a mutant deficient in all of these properties, was studied further. FP523 had a Tn4351 insertion in tlpB (thiol oxidoreductase-like protein gene), which encodes a 41.4-kDa protein whose sequence does not exhibit high levels of similar to the sequences of proteins having known functions. TlpB has two domains; the N-terminal domains has five transmembrane regions, whereas the C-terminal domains has the Cys-X-X-Cys motif and other conserved motifs characteristic of thiol:disulfide oxidoreductases. Quantitative analysis of the thiol groups of periplasmic proteins revealed that TlpB is required for reduction of these groups. The tlpB gene is part of the fpt (F. psychrophilum thiol oxidoreductase) operon that contains two other genes, tlpA and tpiA, which encode a thiol:disulfide oxidoreductase and a triosephosphate isomerase, respectively. FP523 exhibited enhanced biofilm formation and decreased virulence and cytotoxicity. Complementation with the tlpB loci restored the wild-type phenotype. Gliding motility and biofilm formation appear to be antagonistic properties, which are both affected by TlpB.

Optimization of ultrasound-assisted extraction to obtain mycosterols from Agaricus bisporus L. by response surface methodology and comparison with conventional Soxhlet extraction.

Ergosterol, a molecule with high commercial value, is the most abundant mycosterol in Agaricus bisporus L. To replace common conventional extraction techniques (e.g. Soxhlet), the present study reports the optimal ultrasound-assisted extraction conditions for ergosterol. After preliminary tests, the results showed that solvents, time and ultrasound power altered the extraction efficiency. Using response surface methodology, models were developed to investigate the favourable experimental conditions that maximize the extraction efficiency. All statistical criteria demonstrated the validity of the proposed models. Overall, ultrasound-assisted extraction with ethanol at 375 W during 15 min proved to be as efficient as the Soxhlet extraction, yielding 671.5 ± 0.5mg ergosterol/100 g dw. However, with n-hexane extracts with higher purity (mg ergosterol/g extract) were obtained. Finally, it was proposed for the removal of the saponification step, which simplifies the extraction process and makes it more feasible for its industrial transference.

β-Carotene Assay Revisited. Application To Characterize and Quantify Antioxidant and Prooxidant Activities in a Microplate

The β-carotene bleaching assay, a common method for evaluating antioxidant activity, has been widely criticized due to its low reproducibility, problematic quantification, complex reagent preparation, and interference of different factors (temperature, pH, solvents, and metals). In this work we have examined the effects of these factors and developed a highly reproducible procedure for microplate assay, evaluated the critical points of the method, and proposed a kinetic model for quantifying both antioxidant and prooxidant activities. The application of these tools produced very consistent results, which provide robust and meaningful criteria to compare in detail the characteristics of several well-known commercial antioxidants, as well as several predictable prooxidants, and can be easily applied to natural extracts, food samples, and many other type of compounds. As an example, we have tested a set of commercial antioxidants and some typical lipophilic prooxidants. The activity of the tested antioxidants decreased in the following order: ethoxyquin ≫ α-tocopherol > butylhydroxyanisole > butylhydroxytoluene ≫ propyl gallate. On the other hand, hemoglobin and Fe(2+), Fe(3+), Co(2+), and Cu(2+) showed a strong prooxidant effect, and the activity was null in Cd(2+), Ni(2+), and Sr(2+), slightly antioxidant in Mg(2+), and strongly antioxidant in Zn(2+) and Mn(2+).

Growth and metabolic features of lactic acid bacteria in media with hydrolysed fish viscera. An approach to bio-silage of fishing by-products

The reparameterization of two classic growth models (the logistic and Gompertz equations), and the dynamic modification of the integrated form of the first of these, was applied to the description of the kinetics and metabolic behaviour of six strains of lactic acid bacteria in four media: three of these from visceral waste from fishing products, and one commercial medium. The descriptions obtained -always consistent models and statistically significant parameters- provided a wide range of reliable numerical values on notable characteristics of microbial growth and bioproductions, which allowed the assessment of the individual systems by direct comparison, and also the suggesting of the potentially most suitable groups (of species of fish and of bacteria) for bio-silage processes.

Estimation of the dietary intake of 13 priority additives in France, Italy, the UK and Ireland as part of the FACET project

The aim of this study was to assess the dietary exposure of 13 priority additives in four European countries (France, Italy, the UK and Ireland) using the Flavourings, Additives and Contact Materials Exposure Task (FACET) software. The studied additives were benzoates (E210–213), nitrites (E249–250) and sulphites (E220–228), butylated hydroxytoluene (E321), polysorbates (E432–436), sucroses esters and sucroglycerides (E473–474), polyglycerol esters of fatty acids (E475), stearoyl-lactylates (E481–482), sorbitan esters (E493–494 and E491–495), phosphates (E338–343/E450–452), aspartame (E951) and acesulfame (E950). A conservative approach (based on individual consumption data combined with maximum permitted levels (Tier 2)) was compared with more refined estimates (using a fitted distribution of concentrations based on data provided by the food industry (Tier 3)). These calculations demonstrated that the estimated intake is below the acceptable daily intake (ADI) for nine of the studied additives. However, there was a potential theoretical exceedance of the ADI observed for four additives at Tier 3 for high consumers (97.5th percentile) among children: E220–228 in the UK and Ireland, E432–436 and E481–482 in Ireland, Italy and the UK, and E493–494 in all countries. The mean intake of E493–494 could potentially exceed the ADI for one age group of children (aged 1–4 years) in the UK. For adults, high consumers only in all countries had a potential intake higher than the ADI for E493–494 at Tier 3 (an additive mainly found in bakery wares). All other additives examined had an intake below the ADI. Further refined exposure assessments may be warranted to provide a more in-depth investigation for those additives that exceeded the ADIs in this paper. This refinement may be undertaken by the introduction of additive occurrence data, which take into account the actual presence of these additives in the different food groups. Graphical Abstract

Comparison of several mathematical models for describing the joint effect of temperature and ph on glucanex activity

The aim of the present work was to evaluate with different statistical criteria the suitability of nine equations for describing and optimizing the simultaneous effect of temperature and pH on glucanex activity using two characteristic polysaccharides (curdlan and laminarin) as substrates. The most satisfactory solutions were found with an empirical equation constituted with parameters of practical interest (Rosso model), and a hybrid model between the Arrhenius equation and the mathematical expression generated by the protonation‐hydroxylation mechanism (Tijskens model). The joint optimal values of pH and temperature calculated with the Rosso model were obtained at 4.64 and 50°C with curdlan and 4.64 and 48°C using laminarin as substrate.

Evaluation of toxic effects of several carboxylic acids on bacterial growth by toxicodynamic modelling

BackgroundEffects of organic acids on microbial fermentation are commonly tested in investigations about metabolic behaviour of bacteria. However, they typically provide only descriptive information without modelling the influence of acid concentrations on bacterial kinetics.ResultsWe developed and applied a mathematical model (secondary model) to capture the toxicological effects of those chemicals on kinetic parameters that define the growth of bacteria in batch cultures. Thus, dose-response kinetics were performed with different bacteria (Leuconostoc mesenteroides, Carnobacterium pisicola, Escherichia coli, Bacillus subtilis and Listonella anguillarum) exposed at increasing concentrations of individual carboxylic acids (formic, acetic, propionic, butyric and lactic). In all bioassays the acids affected the maximum bacterial load (Xm) and the maximum growth rate (vm) but only in specific cases the lag phase (λ) was modified. Significance of the parameters was always high and in all fermentations the toxicodynamic equation was statistically consistent and had good predictability. The differences between D and L-lactic acid effects were significant for the growth of E. coli, L. mesenteroides and C. piscicola. In addition, a global parameter (EC50,τ) was used to compare toxic effects and provided a realistic characterization of antimicrobial agents using a single value.ConclusionsThe effect of several organic acids on the growth of different bacteria was accurately studied and perfectly characterized by a bivariate equation which combines the basis of dose-response theory with microbial growth kinetics (secondary model). The toxicity of carboxylic acids was lower with the increase of the molecular weight of these chemicals.

NOEC and LOEC as merely concessive expedients: two unambiguous alternatives and some criteria to maximize the efficiency of dose-response experimental designs.

NOEC and LOEC (no and lowest observed effect concentrations, respectively) are toxicological concepts derived from analysis of variance (ANOVA), a not very sensitive method that produces ambiguous results and does not provide confidence intervals (CI) of its estimates. For a long time, despite the abundant criticism that such concepts have raised, the field of the ecotoxicology is reticent to abandon them (two possible reasons will be discussed), adducing the difficulty of clear alternatives. However, this work proves that a debugged dose-response (DR) modeling, through explicit algebraic equations, enables two simple options to accurately calculate the CI of substantially lower doses than NOEC. Both ANOVA and DR analyses are affected by the experimental error, response profile, number of observations and experimental design. The study of these effects--analytically complex and experimentally unfeasible--was carried out using systematic simulations with realistic data, including different error levels. Results revealed the weakness of NOEC and LOEC notions, confirmed the feasibility of the proposed alternatives and allowed to discuss the--often violated--conditions that minimize the CI of the parametric estimates from DR assays. In addition, a table was developed providing the experimental design that minimizes the parametric CI for a given set of working conditions. This makes possible to reduce the experimental effort and to avoid the inconclusive results that are frequently obtained from intuitive experimental plans.

Prevalence, Diagnosis, Treatment, and Control of Hypertension in Spain. Results of the Di@bet.es Study.

INTRODUCTION AND OBJECTIVES Di@bet.es is a national study designed to estimate the prevalence of diabetes mellitus and other cardiovascular risk factors in the Spanish adult population. The prevalence of hypertension and the degree to which it is recognized, treated, and controlled are described. METHODS The study included a sample of the Spanish population with 5048 adults aged ≥ 18 years. Patients were questioned and examined, with 3 blood pressure readings while seated and at rest to calculate the mean of the 3 readings. Hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg and/or prescription for antihypertensive drug therapy. RESULTS Hypertension was found in 42.6% of the Spanish adult population aged ≥ 18 years and was more common among men (49.9%) than women (37.1%). The prevalence was higher among prediabetics (67.9%) and diabetics (79.4%). Undiagnosed hypertension was identified in 37.4% of patients and was more common in men (43.3%) than in women (31.5%). Among patients with known hypertension, 88.3% were receiving drug therapy. Well-controlled blood pressure was found in only 30% and was more common among women (24.9%) than men (16%). CONCLUSIONS The prevalence of hypertension in Spain is high, and a considerable percentage of hypertensive patients have still not been diagnosed. Hypertension is associated with diabetes and prediabetes, and although drug therapy is increasingly common, the degree of control has not improved and remains low. Population campaigns should be developed and promoted for hypertension prevention, detection, and treatment.

Crocin bleaching antioxidant assay revisited: application to microplate to analyse antioxidant and pro-oxidant activities.

The crocin bleaching assay (CBA) is a common method for evaluating the antioxidant activity of hydrosoluble samples. It is criticised due to its low reproducibility, problematic quantification of results, differences in reagent preparation, doubtful need for a preheating phase and sensitivity to factors such as temperature, pH, solvents and metals. Here, the critical points of the method were extensively revised, and a highly reproducible procedure for microplate readers redeveloped. The problems of using quantification procedures, disregarding kinetic considerations, are discussed in detail and a model is proposed for quantifying simultaneously anti- and pro-oxidant activities as function of concentration and time. Thus, the combined use of a reproducible procedure and robust mathematical modeling produced consistent and meaningful criteria for comparative characterization of any oxidation modifier, taking into account the dose-time-dependent behaviour. The method was verified by characterising several commercial antioxidants and some metal compounds using the parametric values of the proposed models. The activity of the tested antioxidants decreased in the order ETX>TR>PG>AA>TBHQ>BHA. Others, such as the lipophilic antioxidants of BHT and α-Tocopherol did not show any activity. Interference from metals were for Fe(2+), Fe(3+), Cd(2+), Ni(2+), Mg(2+), Zn(2+) and Sr(2+), slightly antioxidant for Cu(1+) and Cu(2+), and strongly antioxidant for Mn(2+). None of the tested metals showed a pro-oxidant activity.