The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition

The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1-31 of St I (StI1-31) or 1-30 of St II (StII1-30) and demonstrated that StII1-30 promotes erythrocyte lysis to a higher extent than StI1-31. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1-10 hydrophobic sequence in StII1-30 for displaying higher binding and activity, in spite of both peptides' abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities.     

Application of the modular approach to an in-house validation study of real-time PCR methods for the detection and serogroup determination of verocytotoxigenic Escherichia coli

European Commission regulation 2073/2005 on the microbiological criteria for food requires that Escherichia coli is monitored as an indicator of hygienic conditions. Since verocytotoxigenic E. coli (VTEC) strains often cause food-borne infections by the consumption of raw food, the Biological Hazards (BIOHAZ) panel of the European Food Safety Authority (EFSA) recommended their monitoring in food as well. In particular, VTEC strains belonging to serogroups such as O26, O103, O111, O145, and O157 are known causative agents of several human outbreaks. Eight real-time PCR methods for the detection of E. coli toxin genes and their variants (stx(1), stx(2)), the intimin gene (eae), and five serogroup-specific genes have been proposed by the European Reference Laboratory for VTEC (EURL-VTEC) as a technical specification to the European Normalization Committee (CEN TC275/WG6). Here we applied a "modular approach" to the in-house validation of these PCR methods. The modular approach subdivides an analytical process into separate parts called "modules," which are independently validated based on method performance criteria for a limited set of critical parameters. For the VTEC real-time PCR module, the following parameters are being assessed: specificity, dynamic range, PCR efficiency, and limit of detection (LOD). This study describes the modular approach for the validation of PCR methods to be used in food microbiology, using single-target plasmids as positive controls and showing their applicability with food matrices.     

Fast and easy colorimetric tests for single mismatch recognition by PNA-DNA duplexes with the diethylthiadicarbocyanine dye and succinyl-beta-cyclodextrin

The 3,3'-diethylthiacarbocyanine (DiSC(2)(5)) dye is able to aggregate on full matched PNA-DNA duplexes by changing its absorption properties, which are manifested by an instantaneous colour shift from blue to purple. However the spontaneous aggregation of the dye also on mismatched duplexes and even on free PNA strands makes the test quite aspecific. Here it is demonstrated that the addition of succinyl-beta-cyclodextrin (Succ-beta-CyD) to the solutions containing PNA-DNA duplexes and the dye strongly enhances the specificity of the colour shift, allowing for a fast, very specific and extremely sensitive visual recognition of mismatches in DNA strands by using PNA probes in combination with the DiSC(2)(5) dye. The phenomenon has been studied by CD and NMR spectroscopies. The method has been optimized and preliminarily applied for the recognition of an apoE gene mutation in human DNA samples.     

Testing and estimating the non-disjunction fraction in Meiosis I using reference priors

In this paper we analyze the fraction of non-disjunction in Meiosis I assuming reference (non-informative) priors. We consider Jeffreys's approach to built a non-informative prior (Jeffreys's prior) for the fraction of non-disjunction in Meiosis I. We prove that Jeffreys's prior is a proper distribution. We perform Monte Carlo studies in order to compare Bayes estimates obtained assuming Jeffreys's and uniform priors. We consider full Bayesian significance test (FBST) and Bayes factor (BF) for testing precise hypothesis on the fraction of non-disjunction in Meiosis I. The ultimate goal of this paper is to compare these two test procedures through simulation studies using both prior specifications. An application to Down Syndrome data is also presented.     

Evaluation of Lignans from Piper cubeba against Schistosoma mansoni Adult Worms: A Combined Experimental and Theoretical Study

Six dibenzylbutyrolactonic lignans ((?)‐hinokinin ( 1 ), (?)‐cubebin ( 2 ), (?)‐yatein ( 3 ), (?)‐5‐methoxyyatein ( 4 ), dihydrocubebin ( 5 ) and dihydroclusin ( 6 )) were isolated from Piper cubeba seed extract and evaluated against Schistosoma mansoni. All lignans, except 5 , were able to separate the adult worm pairs and reduce the egg numbers during 24 h of incubation. Lignans 1 , 3 and 4 (containing a lactone ring) were the most efficient concerning antiparasitary activity. Comparing structures 3 and 4 , the presence of the methoxy group at position 5 appears to be important for this activity. Considering 1 and 3 , it is possible to see that the substitution pattern change (methylenedioxy or methoxy groups) in positions 3′ and 4′ alter the biological response, with 1 being the second most active compound. Computational calculations suggest that the activity of compound 4 can be correlated with the largest lipophilicity value.     

Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains

Background

Enteroaggregative Haemorrhagic E. coli (EAHEC) is a new pathogenic group of E. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of Enteroaggregative E. coli (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992–2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available.

Results

The whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage {"type":"entrez-protein","attrs":{"text":"P13374","term_id":"138227","term_text":"P13374"}}P13374 present in the EAHEC O104:H4 strain isolated during the German outbreak 20 years later. Moreover, it was also almost identical to those of the other vtx2-phages of EAHEC O104:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.The comparison of the vtx2-phages sequences from EAHEC strains with those from the vtx-phages of typical Verocytotoxin-producing E. coli strains showed the presence of a 900 bp sequence uniquely associated with EAHEC phages and encoding a tail fiber.

Conclusions

At least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of EAHEC. The finding of an identical vtx2-phage in two EAggEC strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.The observation that different EAHEC infections have been traced back to countries where EAggEC infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the EAHEC pathotype. In these regions, EAggEC of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-574) contains supplementary material, which is available to authorized users.     

Confirmatory Factor Analysis of the WHO Violence Against Women Instrument in Pregnant Women: Results from the BRISA Prenatal Cohort

Background

Screening for violence during pregnancy is one of the strategies for the prevention of abuse against women. Since violence is difficult to measure, it is necessary to validate questionnaires that can provide a good measure of the phenomenon. The present study analyzed the psychometric properties of the World Health Organization Violence Against Women (WHO VAW) instrument for the measurement of violence against pregnant women.

Methods

Data from the Brazilian Ribeirão Preto and São Luís birth cohort studies (BRISA) were used. The sample consisted of 1,446 pregnant women from São Luís and 1,378 from Ribeirão Preto, interviewed in 2010 and 2011. Thirteen variables were selected from a self-applied questionnaire. Confirmatory factor analysis was used to investigate whether violence is a uni-or-multidimensional construct consisting of psychological, physical and sexual dimensions. The mean-and-variance-adjusted weighted least squares estimator was used. Models were fitted separately for each city and a third model combining data from the two settings was also tested. Models suggested from modification indices were tested to determine whether changes in the WHO VAW model would produce a better fit.

Results

The unidimensional model did not show good fit (Root mean square error of approximation [RMSEA]  = 0.060, p<0.001 for the combined model). The multidimensional WHO VAW model showed good fit (RMSEA = 0.036, p = 0.999 for the combined model) and standardized factor loadings higher than 0.70, except for the sexual dimension for SL (0.65). The models suggested by the modification indices with cross loadings measuring simultaneously physical and psychological violence showed a significantly better fit compared to the original WHO model (p<0.001 for the difference between the model chi-squares).

Conclusions

Violence is a multidimensional second-order construct consisting of psychological, physical and sexual dimensions. The WHO VAW model and the modified models are suitable for measuring violence against pregnant women.     

Selection of a Streptomyces strain able to produce cell wall degrading enzymes and active against Sclerotinia sclerotiorum

Control of plant pathogen Sclerotinia sclerotiorum is an ongoing challenge because of its wide host range and the persistence of its sclerotia in soil. Fungicides are the most commonly used method to control this fungus but these can have ecotoxicity impacts. Chitinolytic Streptomyces strains isolated from Brazilian tropical soils were capable of inhibiting S. sclerotiorum growth in vitro, offering new possibilities for integrated pest management and biocontrol, with a new approach to dealing with an old problem. Strain Streptomyces sp. 80 was capable of irreversibly inhibiting fungal growth. Compared to other strains, its crude enzymes had the highest chitinolytic levels when measured at 25°C and strongly inhibited sclerotia from S. sclerotiorum. It produced four hydrolytic enzymes involved in fungal cell wall degradation when cultured in presence of the fungal mycelium. The best production, obtained after three days, was 0.75 U/ml for exochitinase, 0.9 U/ml for endochitinase, 0.16 U/ml for glucanase, and 1.78 U/ml for peptidase. Zymogram analysis confirmed two hydrolytic bands of chitinolytic activity with apparent molecular masses of 45.8 and 206.8 kDa. One glucanase activity with an apparent molecular mass of 55 kDa was also recorded, as well as seven bands of peptidase activity with apparent molecular masses ranging from 15.5 to 108.4 kDa. Differential interference contrast microscopy also showed alterations of hyphal morphology after co-culture. Streptomyces sp. 80 seems to be promising as a biocontrol agent against S. sclerotiorum, contributing to the development of new methods for controlling plant diseases and reducing the negative impact of using fungicides.     

HIV-1 tropism and CD4 T lymphocyte recovery in a prospective cohort of patients initiating HAART in Ribeirão Preto, Brazil

While human immunodeficiency virus (HIV)-1 chemokine co-receptors 5 tropism and the GWGR motif in the envelope third variable region (V3 loop) have been associated with a slower disease progression, their influence on antiretroviral response remains unclear. The impact of baseline V3 characteristics on treatment response was evaluated in a randomised, double blind, prospective cohort study with patients initiating highly active antiretroviral therapy with lopinavir or efavirenz plus azithothymidine/3TC (1:1) over 48 weeks. Similar virological and immunological responses were observed for both treatment regimens. The 43 individuals had a mean baseline CD4 T cell count of 119 cells/mm(3) [standard deviation (SD) = 99] and a mean viral load of 5.09 log(10) copies/mL (SD = 0.49). The GWGR motif was not associated with a CD4 T cell response, but predicted R5 tropism by the geno2pheno([clinical20%]) algorithm correlated with higher CD4 T cell levels at all monitoring points (p < 0.05). Moreover, higher false-positive rates (FPR) values from this analysis revealed a strong correlation with CD4 T cell recovery (p < 0.0001). Transmitted drug resistance mutations, documented in 3/41 (7.3%) cases, were unrelated to the assigned antiretroviral regimen and had no impact on patient outcomes. In conclusion, na?ve HIV-1 R5 infected patients exhibited higher CD4 T cell counts at baseline; this difference was sustained throughout therapy. The geno2pheno([clinical]) option FPR positively correlated with CD4 T cell gain and may be useful in predicting CD4 T cell recovery.