Education- and income-related differences in processed meat consumption across Europe: The role of food-related attitudes.

PURPOSE: Dietary behaviors differ between socio-economic groups and are one key determinant of health inequalities. Psychological factors such as attitudes are assumed to underlie the relation between inequality and dietary behaviors, but this assumption has rarely been tested empirically. We focus on a specific food group shown as detrimental to health: processed meat. METHODS: In two representative international surveys (Survey 1: N = 10,226 participants from nine European countries - Austria, France, Germany, Italy, Netherlands, Poland, Russia, Spain, UK; Survey 2: N = 9149 participants from the same countries, except not including Austria and the Netherlands), participants reported inequality indicators (education, income), processed meat consumption as well as their attitudes toward nutrition and food. PRINCIPAL RESULTS: There were diverging relationships between indicators of inequality and processed meat consumption: the higher the educational attainment, the lower the consumption of processed meat (rSurvey1 = -0.062, p < .001; rSurvey2 = -0.071, p < .001). At the same time, higher income was related to higher processed meat consumption (rSurvey1 = 0.088, p < .001; rSurvey2 = 0.152, p < .001). A path model showed that four of seven attitude factors mediated the relation between education and processed meat consumption (i.e., indifference toward nutrition and food, preference for regional and fresh food, processed food consumption, health efforts); none of the attitude factors mediated the relation between income and overall processed meat consumption. CONCLUSIONS: Processed meats are consumed very frequently across European countries. The relation between inequality and processed meat consumption is heterogeneous and partially mediated by attitudes. More research is needed to better understand how psychological factors explain social inequality in nutrition behaviors and health in general.

Serous cystadenoma of the tunica testis: A case of malignancy mimicry.

Cystadenoma of the tunica of the adult male testis has rarely been reported in the literature. We report a case of an adult serous cystadenoma along with the radiological and pathological findings. The patient was a 40-year-old male with a slowly growing mass on the left testis. An ultrasound scan of left testis showed features highly suspicious for malignancy. Lactate dehydrogenase (LDH) was mildly elevated. Alpha feta-protein (AFP) and beta-human chorionic gonadotropin (beta hCG) were negative. We performed left radical orchiectomy. The pathological findings showed serous cystadenoma of the tunica of the testis. The patient remains asymptomatic.

Autoantibody Formation and Mapping of Immunogenic Epitopes against Cold-Shock-Protein YB-1 in Cancer Patients and Healthy Controls.

Cold shock Y-box binding protein-1 participates in cancer cell transformation and mediates invasive cell growth. It is unknown whether an autoimmune response against cancerous human YB-1 with posttranslational protein modifications or processing develops. We performed a systematic analysis for autoantibody formation directed against conformational and linear epitopes within the protein. Full-length and truncated recombinant proteins from prokaryotic and eukaryotic cells were generated. Characterization revealed a pattern of spontaneous protein cleavage, predominantly with the prokaryotic protein. Autoantibodies against prokaryotic, but not eukaryotic full-length and cleaved human YB-1 protein fragments were detected in both, healthy volunteers and cancer patients. A mapping of immunogenic epitopes performed with truncated E. coli-derived GST-hYB-1 proteins yielded distinct residues in the protein N- and C-terminus. A peptide array with consecutive overlapping 15mers revealed six distinct antigenic regions in cancer patients, however to a lesser extent in healthy controls. Finally, a protein cleavage assay was set up with recombinant pro- and eukaryotic-derived tagged hYB-1 proteins. A distinct cleavage pattern developed, that is retarded by sera from cancer patients. Taken together, a specific autoimmune response against hYB-1 protein develops in cancer patients with autoantibodies targeting linear epitopes.

Characterization of the IS200/IS605 Insertion Sequence Family in Halanaerobium Hydrogeniformans.

Mobile DNA elements play a significant evolutionary role by promoting genome plasticity. Insertion sequences are the smallest prokaryotic transposable elements. They are highly diverse elements, and the ability to accurately identify, annotate, and infer the full genomic impact of insertion sequences is lacking. Halanaerobium hydrogeniformans is a haloalkaliphilic bacterium with an abnormally high number of insertion sequences. One family, IS200/IS605, showed several interesting features distinct from other elements in this genome. Twenty-three loci harbor elements of this family in varying stages of decay, from nearly intact to an ends-only sequence. The loci were characterized with respect to two divergent open reading frames (ORF), tnpA and tnpB, and left and right ends of the elements. The tnpB ORF contains two nearly identical insert sequences that suggest recombination between tnpB ORF is occurring. From these results, insertion sequence activity can be inferred, including transposition capability and element interaction.

Serological Analysis of Herpes B Virus at Individual Epitope Resolution: From Two-Dimensional Peptide Arrays to Multiplex Bead Flow Assays.

Macacine herpesvirus or B Virus (BV) is a zoonotic agent that leads to high mortality rates in humans if transmitted and untreated. Here, BV is used as a test case to establish a two-step procedure for developing high throughput serological assays based on synthetic peptides. In step 1, peptide microarray analysis of 42 monkey sera (30 of them tested BV positive by ELISA) revealed 1148 responses against 369 different peptides. The latter could be grouped into 142 different antibody target regions (ATRs) in six different glycoproteins (gB, gC, gD, gG, gH, and gL) of BV. The high number of newly detected ATRs was made possible inter alia by a new preanalytical protocol that reduced unspecific binding of serum components to the cellulose-based matrix of the microarray. In step 2, soluble peptides corresponding to eight ATRs of particularly high antigenicity were synthesized and coupled to fluorescently labeled beads, which were subsequently employed in immunochemical bead flow assays. Their outcome mirrored the ELISA results used as reference. Hence, convenient, fast, and economical screening of arbitrarily large macaque colonies for BV infection is now possible. The study demonstrates that a technology platform switch from two-dimensional high-resolution peptide arrays used for epitope discovery to a readily available bead array platform for serology applications is feasible.

Whole-Genome Identification and Characterization of Bacterial Insertion Sequences Using Bioinformatic Tools.

Insertion sequences are small mobile regions of DNA (transposable elements) found primarily in prokaryotes. The identification of insertion sequences in bacteria is a growing field of study because of their applications in evolution, genetics, and medicine. One of the first steps in characterizing the insertion sequences found in an organism is to perform a genome-wide survey to identify all insertion sequences using in silico methods. This includes a thorough scan of the genome to locate all copies of different families of insertion sequences and the identification of the key characteristics of each element. The results provide an extensive catalog of the insertion sequences which can be used to further other analyses or manipulation of the genome.

Von Willebrand Factor Mediates Pneumococcal Aggregation and Adhesion in Blood Flow.

Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture infection analyses with primary endothelial cells characterized VWF as bridging molecule that mediates bacterial adherence to endothelial cells in a heparin-sensitive manner. Due to the mechanoresponsive changes of the VWF protein conformation and multimerization status, which occur in the blood stream, we used a microfluidic pump system to generate shear flow-induced multimeric VWF strings on endothelial cell surfaces and analyzed attachment of RFP-expressing pneumococci in flow. By applying immunofluorescence visualization and additional electron microscopy, we detected a frequent and enduring bacterial attachment to the VWF strings. Bacterial attachment to the endothelium was confirmed in vivo using a zebrafish infection model, which is described in many reports and acknowledged as suitable model to study hemostasis mechanisms and protein interactions of coagulation factors. Notably, we visualized the recruitment of zebrafish-derived VWF to the surface of pneumococci circulating in the blood stream and detected a VWF-dependent formation of bacterial aggregates within the vasculature of infected zebrafish larvae. Furthermore, we identified the surface-exposed bacterial enolase as pneumococcal VWF binding protein, which interacts with the VWF domain A1 and determined the binding kinetics by surface plasmon resonance. Subsequent epitope mapping using an enolase peptide array indicates that the peptide 181YGAEIFHALKKILKS195 might serve as a possible core sequence of the VWF interaction site. In conclusion, we describe a VWF-mediated mechanism for pneumococcal anchoring within the bloodstream via surface-displayed enolase, which promotes intravascular bacterial aggregation.

Diagnosing Zika virus infection against a background of other flaviviruses: Studies in high resolution serological analysis.

Zika virus (ZIKV) is a mosquito-borne flavivirus. Homologous proteins of different flaviviruses display high degrees of sequence identity, especially within subgroups. This leads to extensive immunological cross-reactivity and corresponding problems for developing a ZIKV-specific serological assay. In this study, peptide microarrays were employed to identify individual ZIKV antibody targets with promise in differential diagnosis. A total of 1643 overlapping oligopeptides were synthesized and printed onto glass slides. Together, they encompass the full amino acid sequences of ZIKV proteomes of African, Brazilian, USA, and French Polynesian origins. The resulting ZIKV scanning microarray chips were used to screen three pools of sera from recent Zika outbreaks in Senegal and Cape Verde, in Brazil, and from overseas travelers returning to the EU. Together with a mixed pool of well characterized, archived sera of patients suffering from infections by dengue, yellow fever, tick-borne encephalitis, and West Nile viruses, a total of 42 sera went into the study. Sixty-eight antibody target regions were identified. Most of which were hitherto unknown. Alignments and sequence comparisons revealed 13 of which could be classified as bona fide ZIKV-specific. These identified antibody target regions constitute a founding set of analytical tools for serological discrimination of ZIKV from other flaviviruses.

Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.

Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs.

UNLABELLED: High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein. IMPORTANCE: In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.

Higher body mass index, less exercise, but healthier eating in married adults: Nine representative surveys across Europe.

Numerous studies show that married individuals enjoy better health than those who were never married. This representative survey examines whether they also have a healthier body mass index (BMI) and weight-related behaviors, and tests four independent explanations. Face-to-face interviews were conducted with representative samples (N = 4555) from nine European countries (Austria, France, Germany, Italy, the Netherlands, Poland, Russia, Spain, UK). On average, never married respondents had a lower BMI than married respondents (p = .048). Married individuals reported stronger preferences for organic/fair trade food and regional/unprocessed food, and paying less attention to dietary convenience or dietary fat and body weight. Importantly, married men also exercised less (all ps < .05). Despite these behavioral differences, only attention to dietary fat and body weight (p = .001) predicted BMI differently for married versus never married men. There were few country differences in the relationship between marital status and BMI. All analyses were controlled for age and socio-economic status. In conclusion, despite more favorable eating-related cognitions and behaviors, married respondents had a higher BMI than never married respondents, but differences were small. The link between marital status and BMI cannot be fully described by one single explanation. Obesity interventions may benefit from considering specific weight-related behaviors in married versus never married individuals.

Characterization of the interaction between heterodimeric αvß6 integrin and urokinase plasminogen activator receptor (uPAR) using functional proteomics.

Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvß6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvß6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvß6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo ß6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvß6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, ß1, ß3, and ß6 alone). Our data suggest that interaction with uPAR requires expression of the complete αß heterodimer (e.g., αvß6), not individual subunits (i.e., αv, ß1, ß3, or ß6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvß6 and uPAR are located in uPAR domains II and III.

EU-OPENSCREEN-chemical tools for the study of plant biology and resistance mechanisms.

EU-OPENSCREEN is an academic research infrastructure initiative in Europe for enabling researchers in all life sciences to take advantage of chemical biology approaches to their projects. In a collaborative effort of national networks in 16 European countries, EU-OPENSCREEN will develop novel chemical compounds with external users to address questions in, among other fields, systems and network biology (directed and selective perturbation of signalling pathways), structural biology (compound-target interactions at atomic resolution), pharmacology (early drug discovery and toxicology) and plant biology (response of wild or crop plants to environmental and agricultural substances). EU-OPENSCREEN supports all stages of a tool development project, including assay adaptation, high-throughput screening and chemical optimisation of the 'hit' compounds. All tool compounds and data will be made available to the scientific community. EU-OPENSCREEN integrates high-capacity screening platforms throughout Europe, which share a rationally selected compound collection comprising up to 300,000 (commercial and proprietary compounds collected from European chemists). By testing systematically this chemical collection in hundreds of assays originating from very different biological themes, the screening process generates enormous amounts of information about the biological activities of the substances and thereby steadily enriches our understanding of how and where they act.

Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B.

BACKGROUND: Tuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic. RESULTS: In this work, the first human single chain fragment variable (scFv) antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection. CONCLUSIONS: Recombinant antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important.

Design of a general-purpose European compound screening library for EU-OPENSCREEN.

This work describes a collaborative effort to define and apply a protocol for the rational selection of a general-purpose screening library, to be used by the screening platforms affiliated with the EU-OPENSCREEN initiative. It is designed as a standard source of compounds for primary screening against novel biological targets, at the request of research partners. Given the general nature of the potential applications of this compound collection, the focus of the selection strategy lies on ensuring chemical stability, absence of reactive compounds, screening-compliant physicochemical properties, loose compliance to drug-likeness criteria (as drug design is a major, but not exclusive application), and maximal diversity/coverage of chemical space, aimed at providing hits for a wide spectrum of drugable targets. Finally, practical availability/cost issues cannot be avoided. The main goal of this publication is to inform potential future users of this library about its conception, sources, and characteristics. The outline of the selection procedure, notably of the filtering rules designed by a large committee of European medicinal chemists and chemoinformaticians, may be of general methodological interest for the screening/medicinal chemistry community. The selection task of 200K molecules out of a pre-filtered set of 1.4M candidates was shared by five independent European research groups, each picking a subset of 40K compounds according to their own in-house methodology and expertise. An in-depth analysis of chemical space coverage of the library serves not only to characterize the collection, but also to compare the various chemoinformatics-driven selection procedures of maximal diversity sets. Compound selections contributed by various participating groups were mapped onto general-purpose self-organizing maps (SOMs) built on the basis of marketed drugs and bioactive reference molecules. In this way, the occupancy of chemical space by the EU-OPENSCREEN library could be directly compared with distributions of known bioactives of various classes. This mapping highlights the relevance of the selection and shows how the consensus reached by merging the five different 40K selections contributes to achieve this relevance. The approach also allows one to readily identify subsets of target- or target-class-oriented compounds from the EU-OPENSCREEN library to suit the needs of the diverse range of potential users. The final EU-OPENSCREEN library, assembled by merging five independent selections of 40K compounds from various expert groups, represents an excellent example of a Europe-wide collaborative effort toward the common objective of building best-in-class European open screening platforms.

Multiple antibody targets on herpes B glycoproteins B and D identified by screening sera of infected rhesus macaques with peptide microarrays.

Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1) is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any) in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV). 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs) were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test.

The myxobacterial metabolite ratjadone A inhibits HIV infection by blocking the Rev/CRM1-mediated nuclear export pathway.

BACKGROUND: The nuclear export of unspliced and partially spliced HIV-1 mRNA is mediated by the recognition of a leucine-rich nuclear export signal (NES) in the HIV Rev protein by the host protein CRM1/Exportin1. This makes the CRM1-Rev complex an attractive target for the development of new antiviral drugs. Here we tested the anti-HIV efficacy of ratjadone A, a CRM1 inhibitor derived from myxobacteria. RESULTS: Ratjadone A inhibits HIV infection in vitro in a dose-dependent manner with EC50 values at the nanomolar range. The inhibitory effect of ratjadone A occurs around 12 hours post-infection and is specific for the Rev/CRM1-mediated nuclear export pathway. By using a drug affinity responsive target stability (DARTS) assay we could demonstrate that ratjadone A interferes with the formation of the CRM1-Rev-NES complex by binding to CRM1 but not to Rev. CONCLUSION: Ratjadone A exhibits strong anti-HIV activity but low selectivity due to toxic effects. Although this limits its potential use as a therapeutic drug, further studies with derivatives of ratjadones might help to overcome these difficulties in the future.

Symptom recognition of heart attack and stroke in nine European countries: a representative survey.

BACKGROUND: Cardiovascular diseases are the number one cause of death and a source of chronic disability. OBJECTIVES: To assess recognition of and reaction to symptoms of heart attack and stroke, and how recognition is related to the frequency of consulting physicians and other information sources. DESIGN: Face-to-face computer-assisted personal interviews. PARTICIPANTS: Representative sample of 10,228 persons in Austria, France, Germany, Italy, the Netherlands, Poland, Russia, Spain and UK, aged 14-98. MAIN OUTCOME VARIABLES: Recognition of heart attack and stroke symptoms and proper reaction to symptoms. RESULTS: Chest pain was the only heart attack symptom recognized by more than 50% of participants. Eight percent knew no symptoms. Of 14 stroke symptoms, none was recognized by more than 50% of participants; 19% could not identify any symptom. For both heart attack and stroke, Germans and Austrians recognized the largest number of symptoms. Persons in Italy, Poland, Russia and Spain knew only about half as many symptoms as in Germany or Austria. Only 51% of Europeans would call an ambulance when someone suffers a stroke, the fewest (33 and 34%) in Germany and Austria. In most countries, people who consulted their physician more frequently had no better recognition of heart attack or stroke symptoms. CONCLUSIONS: The majority of persons in nine European countries recognize few heart attack and stroke symptoms; many do not know how to react. This low level of knowledge constitutes a major health risk and likely leads to delay in treatment, contributing to the high mortality and morbidity from these diseases.