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Evidence exists that humans are exposed to plastic microparticles via diet. Data on intestinal particle uptake and health-related effects resulting from microplastic exposure are scarce. Aim of the study was to analyze the uptake and effects of microplastic particles in human in vitro systems and in rodents in vivo. The gastrointestinal uptake of microplastics was studied in vitro using the human intestinal epithelial cell line Caco-2 and thereof-derived co-cultures mimicking intestinal M-cells and goblet cells. Different sizes of spherical fluorescent polystyrene (PS) particles (1, 4 and 10 µm) were used to study particle uptake and transport. A 28-days in vivo feeding study was conducted to analyze transport at the intestinal epithelium and oxidative stress response as a potential consequence of microplastic exposure. Male reporter gene mice were treated three times per week by oral gavage with a mixture of 1 µm (4.55 × 107 particles), 4 µm (4.55 × 107 particles) and 10 µm (1.49 × 106 particles) microplastics at a volume of 10 mL/kg/bw. Effects of particles on macrophage polarization were investigated using the human cell line THP-1 to detect a possible impact on intestinal immune cells. Altogether, the results of the study demonstrate the cellular uptake of a minor fraction of particles. In vivo data show the absence of histologically detectable lesions and inflammatory responses. The particles did not interfere with the differentiation and activation of the human macrophage model. The present results suggest that oral exposure to PS microplastic particles under the chosen experimental conditions does not pose relevant acute health risks to mammals.

Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptide were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37) and in the eukaryotic system (BvTri a 37) as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR) and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN). Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of sequential IgE epitopes indicates that sensitization to alpha-purothionin occurs via the gut. Both allergens can be used for in-vitro diagnosis of wheat food allergy. The induction of blocking IgG antibodies suggests the usefulness for immunotherapy.

Background Worldwide, an increase in antimicrobial resistance (AMR) of Neisseria gonorrhoeae has been observed. Until now, no protocol for an external quality assessment (EQA) has been available for Germany. The German gonococcal resistance network (GORENET) performed an EQA of primary laboratories in Germany in order to assess quality of antibiotic susceptibility testing, to gain information about laboratory procedures and to assess the impact of these procedures on test results. Methods Laboratories assessed drug susceptibility to cefixime, ceftriaxone, azithromycin, penicillin and ciprofloxacin for five N. gonorrhoeae strains, using their standard laboratory protocols. Minimal inhibitory concentrations (MICs) were compared to World Health Organisation (WHO) consensus results (or, if not available, reference laboratory results), while deviation by +/− one doubling dilution was accepted. Data on laboratory procedures were collected via a standardised questionnaire. Generalized linear models and conditional inference trees (CTREE) were used to assess relationships between laboratory procedures and testing outcomes. Results Twenty-one primary laboratories participated in the EQA in June 2018. 96% of ciprofloxacin MICs were reported within accepted deviations, as well as 88% for cefixime, 85% for ceftriaxone, 79% for penicillin and 70% for azithromycin. The use of interpretation standards and general laboratory procedures like agar base, incubation settings or the use of control strains strongly differed between laboratories. In statistical analysis, incubation time of cultures < 24 h was associated with correct measurements. Additionally, a 5% CO 2 concentration was associated with correct results regarding azithromycin compared to 3%. CTREE analysis showed that incubation time, humidity and CO 2 concentration had the greatest influence on the average deviation from consensus results. Conclusions In conclusion, we report the development of a protocol for N. gonorrhoeae antimicrobial susceptibility testing in Germany. While testing results were in accordance with the expected consensus results in 70–96%, depending on the antibiotic agent, laboratory methodology was heterogeneous and may significantly affect the testing quality. We therefore recommend the development of a standard operating procedure (SOP) for N. gonorrhoeae susceptibility testing in Germany.

Alkaliphilic haloarchaea, a distinct physiological group from the closely related neutrophilic haloarchaea, represent an underutilized resource for basic research and industrial applications. In contrast to the neutrophilic haloarchaea, no reports on genomic manipulations in haloalkaliphiles have been published until now. Genomic manipulations via homologous recombination are useful for basic research. In this study, we demonstrate the possibility for this strategy in alkaliphilic haloarchaea for the first time. In a previous study, we developed a PEG-mediated transformation technique for alkaliphilic haloarchaea that was deployed in this study to deliver a gene disruption cassette into the model organism Natrialba magadii. The gene encoding for the well-studied Natrialba extracellular protease was successfully disrupted by a recombination marker gene, demonstrating a proof of principle for the usability of homologous recombination for genomic manipulations in alkaliphilic haloarchaea. Since halo(alkali)philic Archaea are polyploid, a selection process was applied in order to obtain a mutant strain containing exclusively disrupted genes. The resulting strain exhibited no proteolytic activity measurable by an azo-casein assay. Complementation was able to restore proteolytic activity. The expression pattern of the Natrialba extracellular protease was different in the complemented strain. The genomic manipulation of the Natrialba extracellular protease demonstrated for the first time a successful introduction of a specific mutation into the genome of Natrialba magadii.

The respiratory epithelium is a barrier against pathogens and allergens and a target for therapy in respiratory allergy, asthma and chronic obstructive pulmonary disease (COPD). We investigated barrier-damaging factors and protective factors by real-time measurement of respiratory cell barrier integrity. Barrier integrity to cigarette smoke extract (CSE), house dust mite (HDM) extract, interferon-γ (IFN-γ) or human rhinovirus (HRV) infection alone or in combination was assessed. Corticosteroids, lipopolysaccharide (LPS), and nasal mucus proteins were tested for their ability to prevent loss of barrier integrity. Real-time impedance-based measurement revealed different patterns of CSE-, HDM-, IFN-γ- and HRV-induced damage. When per se non-damaging concentrations of harmful factors were combined, a synergetic effect was observed only for CSE and HDM. Betamethasone prevented the damaging effect of HRV and CSE, but not damage caused by HDM or IFN-γ. Real-time impedance-based measurement of respiratory epithelial barrier function is useful to study factors, which are harmful or protective. The identification of a synergetic damaging effect of CSE and HDM as well as the finding that Betamethasone protects against HRV- and CSE-induced damage may be important for asthma and COPD.

This document provides supplementary guidance on specific topics for the allergenicity risk assessment of genetically modified plants. In particular, it supplements general recommendations outlined in previous EFSA GMO Panel guidelines and Implementing Regulation (EU) No 503/2013. The topics addressed are non-IgE-mediated adverse immune reactions to foods, in vitro protein digestibility tests and endogenous allergenicity. New scientific and regulatory developments regarding these three topics are described in this document. Considerations on the practical implementation of those developments in the risk assessment of genetically modified plants are discussed and recommended, where appropriate.