Delineating distinct heme-scavenging and -binding functions of domains in MF6p/helminth defense molecule (HDM) proteins from parasitic flatworms.

MF6p/FhHDM-1 is a small protein secreted by the parasitic flatworm (trematode) Fasciola hepatica that belongs to a broad family of heme-binding proteins (MF6p/helminth defense molecules (HDMs)). MF6p/HDMs are of interest for understanding heme homeostasis in trematodes and as potential targets for the development of new flukicides. Moreover, interest in these molecules has also increased because of their immunomodulatory properties. Here we have extended our previous findings on the mechanism of MF6p/HDM-heme interactions and mapped the protein regions required for heme binding and for other biological functions. Our data revealed that MF6p/FhHDM-1 forms high-molecular-weight complexes when associated with heme and that these complexes are reorganized by a stacking procedure to form fibril-like and granular nanostructures. Furthermore, we showed that MF6p/FhHDM-1 is a transitory heme-binding protein as protein·heme complexes can be disrupted by contact with an apoprotein (e.g. apomyoglobin) with higher affinity for heme. We also demonstrated that (i) the heme-binding region is located in the MF6p/FhHDM-1 C-terminal moiety, which also inhibits the peroxidase-like activity of heme, and (ii) MF6p/HDMs from other trematodes, such as Opisthorchis viverrini and Paragonimus westermani, also bind heme. Finally, we observed that the N-terminal, but not the C-terminal, moiety of MF6p/HDMs has a predicted structural analogy with cell-penetrating peptides and that both the entire protein and the peptide corresponding to the N-terminal moiety of MF6p/FhHDM-1 interact in vitro with cell membranes in hemin-preconditioned erythrocytes. Our findings suggest that MF6p/HDMs can transport heme in trematodes and thereby shield the parasite from the harmful effects of heme.

Comparison of recombinant cathepsins L1, L2, and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis.

Infections caused by Fasciola hepatica are of great importance in the veterinary field, as they cause important economic losses to livestock producers. Serodiagnostic methods, typically ELISA (with either native or recombinant antigens), are often used for early diagnosis. The use of native antigens, as in the MM3-SERO ELISA (commercialized as BIO K 211, BIO-X Diagnostics), continues to be beneficial in terms of sensitivity and specificity; however, there is interest in developing ELISA tests based on recombinant antigens to avoid the need to culture parasites. Of the antigens secreted by adult flukes, recombinant procathepsin L1 (rFhpCL1) is the most commonly tested in ELISA to date. However, although adult flukes produce three different clades of CLs (FhCL1, FhCL2, and FhCL5), to our knowledge, the diagnostic value of recombinant FhCL2 and FhCL5 has not yet been investigated. In the present study, we developed and tested three indirect ELISAs using rFhpCL1, rFhpCL2, and rFhpCL5 and evaluated their recognition by sera from sheep and cattle naturally infected with F. hepatica. Although the overall antibody response to these three rFhpCLs was similar, some animals displayed preferential recognition for particular rFhpCLs. Moreover, for cattle sera, the highest sensitivity was obtained using rFhpCL2 (97%), being equal for both rFhpCL1 and rFhpCL5 (87.9%), after adjusting cut-offs for maximum specificity. By contrast, for sheep sera, the sensitivity was 100% for the three rFhpCLs. Finally, the presence of truncated and/or partially unfolded molecules in antigen preparations is postulated as a possible source of cross-reactivity.

A study of the Immune Epitope Database for some fungi species using network topological indices.

In the last years, the encryption of system structure information with different network topological indices has been a very active field of research. In the present study, we assembled for the first time a complex network using data obtained from the Immune Epitope Database for fungi species, and we then considered the general topology, the node degree distribution, and the local structure of this network. We also calculated eight node centrality measures for the observed network and compared it with three theoretical models. In view of the results obtained, we may expect that the present approach can become a valuable tool to explore the complexity of this database, as well as for the storage, manipulation, comparison, and retrieval of information contained therein.

The MF6p/FhHDM-1 major antigen secreted by the trematode parasite Fasciola hepatica is a heme-binding protein.

Blood-feeding parasites have developed biochemical mechanisms to control heme intake and detoxification. Here we show that a major antigen secreted by Fasciola hepatica, previously reported as MF6p, of unknown function (gb|CCA61804.1), and as FhHDM-1, considered to be a helminth defense molecule belonging to the family of cathelicidin-like proteins (gb|ADZ24001.1), is in fact a heme-binding protein. The heme-binding nature of the MF6p/FhHDM-1 protein was revealed in two independent experiments: (i) immunopurification of the secreted protein·heme complexes with mAb MF6 and subsequent analysis by C8 reversed-phase HPLC and MS/MS spectrometry and (ii) analysis of the binding ability of the synthetic protein to hemin in vitro. By immunohistochemistry analysis, we have observed that MF6p/FhHDM-1 is produced by parenchymal cells and transported to other tissues (e.g. vitellaria and testis). Interestingly, MF6p/FhHDM-1 is absent both in the intestinal cells and in the lumen of cecum, but it can be released through the tegumental surface to the external medium, where it binds to free heme molecules regurgitated by the parasite after hemoglobin digestion. Proteins that are close analogs of the Fasciola MF6p/FhHDM-1 are present in other trematodes, including Clonorchis, Opistorchis, Paragonimus, Schistosoma, and Dicrocoelium. Using UV-visible spectroscopy and immunoprecipitation techniques, we observed that synthetic MF6p/FhHDM-1 binds to hemin with 1:1 stoichiometry and an apparent Kd of 1.14 × 10(-6) M(-1). We also demonstrated that formation of synthetic MF6p/FhHDM-1·hemin complexes inhibited hemin degradation by hydrogen peroxide and hemin peroxidase-like activity in vitro. Our results suggest that MF6p/FhHDM-1 may be involved in heme homeostasis in trematodes.

Influence of life history traits on the population genetic structure of parasitic helminths: a minireview.

Parasite life history traits influence the rate of gene flow between populations and the effective population size, both of which determine the levels of genetic variability and the geographic distribution of such variability. In this short review targeted to parasitologists, we summarise how life history traits influence the population genetic structure of parasitic helminths. These organisms are characterised by a wide variety of life cycles and are ecologically different from microparasites, which have been studied in more detail. In order to provide the reader a concise review that illustrates key aspects of the subject matter, we have limited ourselves to studying examples selected for their clarity and relevance.

A QSPR-like model for multilocus genotype networks of Fasciola hepatica in Northwest Spain.

Fasciola hepatica is a parasitic trematode that infects wild and domesticated mammals, particularly cattle and sheep, and causes significant economic losses to global livestock production. In the present study, we used codominant genetic markers to define and build, for the first time, complex genotype networks for F. hepatica isolated from cattle and sheep in NW Spain. We generated three types of random networks with a number of nodes and edges as close as possible to the observed networks, and we then calculated 14 node centrality measures for both observed and random networks. Finally, using Linear Discriminant Analysis (LDA) and these measures as inputs, we constructed a quantitative structure-property relationship (QSPR)-like model able to predict the propensity of a specific genotype of F. hepatica to infect different infrapopulations, farms and/or host species. The accuracy, sensitivity and specificity of the model were >90% for both training and cross-validation series. We also assessed the applicability domain of the model. This type of QSPR model is a potentially powerful tool for epidemiological studies and could be used to manage and prevent the spread of fasciolosis.

Bayesian hierarchical modelling of the geospatial distribution of fasciolosis in dairy cattle and the impact on production: Application to the main milk-producing region (Galicia) in Spain.

Fasciolosis caused by Fasciola hepatica is a common parasitic infection among cattle in many countries. Although infected adult cows rarely show overt clinical signs, milk production may be impaired. Thus, significant production losses may occur in dairy herds with a high prevalence of fasciolosis. In this study, Bayesian hierarchical modelling was used to estimate the geospatial distribution of dairy cattle fasciolosis and its impact on milk production. The study was conducted in Galicia, the main milk producing region in Spain and a geographically heterogeneous area. The aims were: 1) to model the geospatial distribution of fasciolosis in dairy herds in the study area, 2) to identify clusters of herds with a high prevalence of fasciolosis, and 3) to assess the effect of fasciolosis on milk yield and quality. A large number of dairy cattle farms (n = 4907), of which 1660 provided production records, were surveyed. Fasciola infection status was determined by applying the MM3-SERO ELISA test to bulk tank milk samples. A high probability of infection was predicted in several zones, particularly in the centre, northeast and southeast of Galicia. Conversely, the predicted probability was very low in some parts of the northwest of the region. Infections with high within-herd prevalence (> 25% lactating cows infected) predominated. High within-herd prevalence was associated with loss of milk production (-1.387 kg/cow/ day, on average). No association between Fasciola infection and either milk fat or protein content was observed. This study has generated the first maps of the spatial distribution of the probability of Fasciola infection in dairy cattle herds in Galicia. The maps presented here can be used for reference purposes, enabling the design of better targeted fasciolosis control programmes in the region. Use of Bayesian hierarchical statistical analysis enabled us to ascertain the uncertainty of the predictions and to account for the spatial autocorrelation in the data. It also enabled us to generate maps showing the residual spatial variation in milk production, a topic that may deserve more detailed study.

Increased specificity of Fasciola hepatica excretory-secretory antigens combining negative selection on hydroxyapatite and salt precipitation.

A single and rapid method to obtain an antigenic fraction of excretory-secretory antigens (ESAs) from Fasciola hepatica suitable for serodiagnosis of fascioliasis is reported. The procedure consists in the negative selection of F. hepatica ESAs by hydroxyapatite (HA) chromatography (HAC; fraction HAC-NR) followed by antigen precipitation with 50% ammonium sulphate (AS) and subsequent recovery by means of a Millex-GV or equivalent filter (Fi-SOLE fraction). Tested in indirect ELISA, the Fi-SOLE antigens detected natural infections by F. hepatica with 100% sensitivity and 98.9% specificity in sheep, and 97.7% sensitivity and 97.7% specificity in cattle, as determined by ROC analysis. The SDS-PAGE and proteomic nano-UHPLC-Tims-QTOF MS/MS analysis of fractions showed that the relative abundance of L-cathepsins and fragments thereof was 57% in fraction HAC-NR and 93.8% in fraction Fi-SOLE. The second most abundant proteins in fraction HAC-NR were fatty-acid binding proteins (11.9%). In contrast, free heme, and heme:MF6p/FhHDM-1 complexes remained strongly bond to the HA particles during HAC. Interestingly, phosphorylcholine (PC)-bearing antigens, which are a frequent source of cross-reactivity, were detected with an anti-PC mAb (BH8) in ESAs and fraction HAC-NR but were almost absent in fraction Fi-SOLE.

Different fish-eating habits and cytokine production in chronic urticaria with and without sensitization against the fish-parasite Anisakis simplex.

BACKGROUND: Anisakis simplex sensitization has been associated with acute, but also with chronic urticaria. The objective of this study is to characterize chronic urticaria with (CU+) and without sensitization (CU-) against the ubiquitous fish parasite A. simplex in a transversal and longitudinal evaluation. METHODS: 16 CU+ and 22 CU- patients were included and assessed for Urticaria activity score (UAS), fish-eating habits by standardized questionnaire and cytokine production (assessed by flow cytometric bead-based array) of peripheral blood mononuclear cells after stimulation with A. simplex extract or Concanavalin A (Con A). Patients were randomly put on a fish-free diet for three months and UAS, as well as cytokine production were again assessed. A difference of ≥1 in UAS was defined as improvement. RESULTS: There was no difference in UAS in both groups. Anisakis induced IL-2, IL-4 and IFN-γ production was higher in CU+. Con A induced IL-6 and IL-10 production was higher in CU+. CU+ was associated with higher total fish intake, whereas CU- was associated with oily fish intake. The correlation of UAS was positive with oily fish, but negative with total fish intake. There was a better UAS-based prognosis in CU+ without diet. Improvement was associated with higher Con A induced IL-10/IFN-γ as well as IL-10/IL-6 ratios. Further, previous higher oily fish intake was associated with improvement. CONCLUSIONS: Our data confirm the different clinical and immunological phenotype of CU+. Our results show a complex relationship between fish-eating habits, cytokine production and prognosis, which could have important consequences in dietary advice in patients with CU. When encountering A. simplex sensitization, patients should not be automatically put on a diet without fish in order to reduce contact with A. simplex products.

Targeted proteomics and specific immunoassays reveal the presence of shared allergens between the zoonotic nematodes Anisakis simplex and Pseudoterranova decipiens.

The family Anisakidae, mainly represented by Anisakis simplex s.l. and Pseudoterranova decipiens, encompasses zoonotic nematodes infecting many marine fish. Both are responsible for gastrointestinal disease in humans after ingestion of a live larva by consumption of undercooked fish, and, in the case of A. simplex, an allergic reaction may occur after consuming or even handling infected fish. Due to its phylogenetic relatedness with A. simplex, few studies investigated the allergenic potential of P. decipiens, yet none of them focused on its excretory/secretory (E/S) proteins that easily get missed when working solely on extracts from crushed nematodes. Moreover, these E/S allergens remain behind even when the larva has been removed during fish quality processing. Therefore, the aim was to investigate if Anisakis-like allergens could also be detected in both crushed and E/S P. decipiens protein extract using targeted mass spectrometry analysis and immunological methods. The results confirmed that at least five A. simplex allergens have homologous proteins in P. decipiens; a result that emphasizes the importance of also including E/S protein extracts in proteomic studies. Not only A. simplex, but also P. decipiens should therefore be considered a potential source of allergens that could lead to hypersensitivity reactions in humans.

Field evaluation of the enhanced MM3-COPRO ELISA test for the diagnosis of Fasciola hepatica infection in sheep.

Fasciolosis is a severe zoonosis responsible for major economic losses in livestock. The enhanced MM3-COPRO test (eMM3-COPRO) and the commercial version BIO K 201 (Bio-X Diagnostics, Rochefort, Belgium) are widely used as immunodiagnostic tools for the specific detection of coproantigens released by Fasciola during the late prepatent and patent stages of infection. However, performance of the eMM3-COPRO has never been evaluated under field conditions. To address this gap, a large number of ovine faecal samples, collected in a region where fasciolosis is endemic (Galicia, NW Spain), were analyzed. Two groups of sheep flocks were selected according to the Fasciola infection status: 'Fasciola-free' and 'Fasciola-infected' flocks. 'Fasciola-free' flocks were seronegative flocks with no history of fasciolosis detected by either coproscopy or necropsy in the last 5 years. Faecal samples from these sheep were used to calculate a cut-off value for infection (OD = 0.021). The cut-off was calculated using a bootstrap resampling method that enables estimation of the sampling distribution of the statistical parameters without making assumptions about the underlying data distribution. 'Fasciola-infected' flocks were characterized by high seroprevalence, a history of fasciolosis and periodical treatment with flukicides. Samples from these flocks were used to estimate the diagnostic accuracy of the eMM3-COPRO relative to coproscopy, which although limited by poor sensitivity is the only reference test available for diagnosing fasciolosis in vivo. To overcome this limitation, all animals classified positive by eMM3-COPRO were treated with triclabendazole and then retested. The eMM3-COPRO displayed higher sensitivity than coproscopy, as it detected coproantigens in all samples with positive coproscopy and in 12% of samples with negative coproscopy. The test also proved highly specific as coproantigens disappeared after the treatment. The eMM3-COPRO was less time consuming than coproscopy, particularly when the procedure involved numerous samples, and showed promise as a tool for monitoring flukicide efficacy.

MIND-BEST: Web server for drugs and target discovery; design, synthesis, and assay of MAO-B inhibitors and theoretical-experimental study of G3PDH protein from Trichomonas gallinae.

Many drugs with very different affinity to a large number of receptors are described. Thus, in this work, we selected drug-target pairs (DTPs/nDTPs) of drugs with high affinity/nonaffinity for different targets. Quantitative structure-activity relationship (QSAR) models become a very useful tool in this context because they substantially reduce time and resource-consuming experiments. Unfortunately, most QSAR models predict activity against only one protein target and/or they have not been implemented on a public Web server yet, freely available online to the scientific community. To solve this problem, we developed a multitarget QSAR (mt-QSAR) classifier combining the MARCH-INSIDE software for the calculation of the structural parameters of drug and target with the linear discriminant analysis (LDA) method in order to seek the best model. The accuracy of the best LDA model was 94.4% (3,859/4,086 cases) for training and 94.9% (1,909/2,012 cases) for the external validation series. In addition, we implemented the model into the Web portal Bio-AIMS as an online server entitled MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (MIND-BEST), located at http://miaja.tic.udc.es/Bio-AIMS/MIND-BEST.php . This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally, we illustrated two practical uses of this server with two different experiments. In experiment 1, we report for the first time a MIND-BEST prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of eight rasagiline derivatives, promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF and -TOF/TOF MS, MASCOT search, 3D structure modeling with LOMETS, and MIND-BEST prediction for different peptides as new protein of the found in the proteome of the bird parasite Trichomonas gallinae, which is promising for antiparasite drug targets discovery.

NL MIND-BEST: a web server for ligands and proteins discovery--theoretic-experimental study of proteins of Giardia lamblia and new compounds active against Plasmodium falciparum.

There are many protein ligands and/or drugs described with very different affinity to a large number of target proteins or receptors. In this work, we selected Ligands or Drug-target pairs (DTPs/nDTPs) of drugs with high affinity/non-affinity for different targets. Quantitative Structure-Activity Relationships (QSAR) models become a very useful tool in this context to substantially reduce time and resources consuming experiments. Unfortunately most QSAR models predict activity against only one protein target and/or have not been implemented in the form of public web server freely accessible online to the scientific community. To solve this problem, we developed here a multi-target QSAR (mt-QSAR) classifier using the MARCH-INSIDE technique to calculate structural parameters of drug and target plus one Artificial Neuronal Network (ANN) to seek the model. The best ANN model found is a Multi-Layer Perceptron (MLP) with profile MLP 20:20-15-1:1. This MLP classifies correctly 611 out of 678 DTPs (sensitivity=90.12%) and 3083 out of 3408 nDTPs (specificity=90.46%), corresponding to training accuracy=90.41%. The validation of the model was carried out by means of external predicting series. The model classifies correctly 310 out of 338 DTPs (sensitivity=91.72%) and 1527 out of 1674 nDTP (specificity=91.22%) in validation series, corresponding to total accuracy=91.30% for validation series (predictability). This model favorably compares with other ANN models developed in this work and Machine Learning classifiers published before to address the same problem in different aspects. We implemented the present model at web portal Bio-AIMS in the form of an online server called: Non-Linear MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (NL MIND-BEST), which is located at URL: http://miaja.tic.udc.es/Bio-AIMS/NL-MIND-BEST.php. This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally we illustrated two practical uses of this server with two different experiments. In experiment 1, we report by first time Quantum QSAR study, synthesis, characterization, and experimental assay of antiplasmodial and cytotoxic activities of oxoisoaporphine alkaloids derivatives as well as NL MIND-BEST prediction of potential target proteins. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF, and -TOF/TOF MS, MASCOT search, MM/MD 3D structure modeling, and NL MIND-BEST prediction for different peptides a new protein of the found in the proteome of the human parasite Giardia lamblia, which is promising for anti-parasite drug-targets discovery.

Current Challenges for Fasciolicide Treatment in Ruminant Livestock.

Pharmacological treatment remains essential to control fasciolosis in areas where infection is endemic. However, there are major constraints to treating food-producing animals. Of particular concern is the lack of flukicides for treating early Fasciola infections in ruminant livestock in some countries. In addition, the information provided in package leaflets, particularly regarding withdrawal periods, is often incomplete, confusing, and/or contradictory. International regulatory bodies should harmonize the use of flukicides in livestock in favor of fairer, safer international trade. In addition, monitoring the efficacy of fasciolicides on farms is also essential to minimize the spread of drug-resistant populations of Fasciola. The current situation regarding flukicide formulations in the European Union and other, non-European countries is analyzed in this review paper.

Evidence of population structuring following population genetic analyses of Fasciola hepatica from Argentina.

Fasciola hepatica, the liver fluke, is a trematode parasite that causes disease of economic importance in livestock. As a zoonosis this parasite also poses a risk to human health in areas where it is endemic. Population genetic studies can reveal the mechanisms responsible for genetic structuring (non-panmixia) within parasite populations and provide valuable insights into population dynamics, which in turn enables theoretical predictions of evolutionary dynamics such as the evolution of drug resistance. Here we genotyped 320 F. hepatica collected from 14 definitive hosts from four provinces in Argentina. STRUCTURE analysis indicated three population clusters, and principal coordinate analysis confirmed this, showing population clustering across provinces. Similarly, pairwise FST values amongst all four provinces were significant, with standardised pairwise FST (F'ST) ranging from 0.0754 to 0.6327. Therefore, population genetic structure was evident across these four provinces in Argentina. However, there was no evidence of deviation from Hardy-Weinberg equilibrium, so it appears that within these sub-populations there is largely random mating. We identified 263 unique genotypes, which gave a clonal diversity of 82%. Parasites with identical genotypes, clones, accounted for 26.6% of the parasites studied and were found in 12 of the 14 hosts studied, suggesting some clonemate transmission.

Unified QSAR & network-based computational chemistry approach to antimicrobials. II. Multiple distance and triadic census analysis of antiparasitic drugs complex networks.

In the previous work, we reported a multitarget Quantitative Structure-Activity Relationship (mt-QSAR) model to predict drug activity against different fungal species. This mt-QSAR allowed us to construct a drug-drug multispecies Complex Network (msCN) to investigate drug-drug similarity (González-Díaz and Prado-Prado, J Comput Chem 2008, 29, 656). However, important methodological points remained unclear, such as follows: (1) the accuracy of the methods when applied to other problems; (2) the effect of the distance type used to construct the msCN; (3) how to perform the inverse procedure to study species-species similarity with multidrug resistance CNs (mdrCN); and (4) the implications and necessary steps to perform a substructural Triadic Census Analysis (TCA) of the msCN. To continue the present series with other important problem, we developed here a mt-QSAR model for more than 700 drugs tested in the literature against different parasites (predicting antiparasitic drugs). The data were processed by Linear Discriminate Analysis (LDA) and the model classifies correctly 93.62% (1160 out of 1239 cases) in training. The model validation was carried out by means of external predicting series; the model classified 573 out of 607, that is, 94.4% of cases. Next, we carried out the first comparative study of the topology of six different drug-drug msCNs based on six different distances such as Euclidean, Chebychev, Manhattan, etc. Furthermore, we compared the selected drug-drug msCN and species-species mdsCN with random networks. We also introduced here the inverse methodology to construct species-species msCN based on a mt-QSAR model. Last, we reported the first substructural analysis of drug-drug msCN using Triadic Census Analysis (TCA) algorithm.

QSAR for RNases and theoretic-experimental study of molecular diversity on peptide mass fingerprints of a new Leishmania infantum protein.

The toxicity and low success of current treatments for Leishmaniosis determines the search of new peptide drugs and/or molecular targets in Leishmania pathogen species (L. infantum and L. major). For example, Ribonucleases (RNases) are enzymes relevant to several biologic processes; then, theoretical and experimental study of the molecular diversity of Peptide Mass Fingerprints (PMFs) of RNases is useful for drug design. This study introduces a methodology that combines QSAR models, 2D-Electrophoresis (2D-E), MALDI-TOF Mass Spectroscopy (MS), BLAST alignment, and Molecular Dynamics (MD) to explore PMFs of RNases. We illustrate this approach by investigating for the first time the PMFs of a new protein of L. infantum. Here we report and compare new versus old predictive models for RNases based on Topological Indices (TIs) of Markov Pseudo-Folding Lattices. These group of indices called Pseudo-folding Lattice 2D-TIs include: Spectral moments pi ( k )(x,y), Mean Electrostatic potentials xi ( k )(x,y), and Entropy measures theta ( k )(x,y). The accuracy of the models (training/cross-validation) was as follows: xi ( k )(x,y)-model (96.0%/91.7%)>pi ( k )(x,y)-model (84.7/83.3) > theta ( k )(x,y)-model (66.0/66.7). We also carried out a 2D-E analysis of biological samples of L. infantum promastigotes focusing on a 2D-E gel spot of one unknown protein with M<20, 100 and pI <7. MASCOT search identified 20 proteins with Mowse score >30, but not one >52 (threshold value), the higher value of 42 was for a probable DNA-directed RNA polymerase. However, we determined experimentally the sequence of more than 140 peptides. We used QSAR models to predict RNase scores for these peptides and BLAST alignment to confirm some results. We also calculated 3D-folding TIs based on MD experiments and compared 2D versus 3D-TIs on molecular phylogenetic analysis of the molecular diversity of these peptides. This combined strategy may be of interest in drug development or target identification.

Analysis of Ani s 7 and Ani s 1 allergens as biomarkers of sensitization and allergy severity in human anisakiasis.

The high frequency of infection by Anisakis simplex (A. simplex) has led to an increase in IgE sensitization, turning allergy to this parasite a relevant contemporary health problem. Improving the lack of conventional diagnosis test specificity is crucial to better understand these clinical scenarios. Specific IgE (sIgE) to A. simplex extract by ImmunoCAP (Anisakis-sIgE) was determined in sera from 403 blood donors (BD) from Cantabria (North of Spain) of which 51 subjects resulted sensitized. Among these latter, 47 were asymptomatic (sABD). The values of total IgE, prick-test, Anisakis-sIgE, and sIgE to Ani s 1 (anti-rAni s 1) and Ani s 7 (anti-rAni s 7) were compared between 46 sABD and 49 A. simplex allergic patients. The IgE seroprevalence by ImmunoCAP among BD was 12.65%. Allergic patients and sABD showed significant differences in all serum biomarkers evaluated. The area under the curve was assessed for Anisakis-sIgE (0.892), sIgE-rAni s 1 (0.672) and sIgE-rAni s 7 (0.668). After a severe reaction, significantly higher levels of Anisakis-sIgE and sIgE anti-rAni s 1 were detected. Determinations of sIgE by ImmunoCAP, Ani s 1 and Ani s 7 presented different sensitization patterns between allergic and asymptomatic individuals. The Ani s 1 allergen arises as a possible biomarker to detect patients at risk of suffering severe allergic reactions.

Author Correction: Analysis of Ani s 7 and Ani s 1 allergens as biomarkers of sensitization and allergy severity in human anisakiasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic diversity of Fasciola hepatica in Spain and Peru.

In the present study, molecular characterization of Fasciola flukes from Spain was performed to reveal the relation with the previously reported Peruvian F. hepatica population. The nuclear DNA markers, phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), were used for species identification of Fasciola flukes. A total of 196 Fasciola flukes were identified as F. hepatica by pepck and pold, and 26 haplotypes were detected in mitochondrial NADH dehydrogenase subunit 1 (nad1). Only one of them was previously found in Spanish samples; which indicates the existence of high genetic diversity and population structure in F. hepatica from Spain. Three haplotypes were identical to those from Peruvian F. hepatica. The pairwise fixation index value confirmed a relatively close relationship between the Spanish and Peruvian F. hepatica samples. The Spanish samples showed clearly higher genetic variability than the Peruvian population. These results are discussed in relation with the hypothesis of the introduction of the parasite in America from Europe and recent evidence of pre-Hispanic F. hepatica from Argentina revealed by ancient DNA.