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The DNMT3A DNA methyltransferase is an important epigenetic enzyme that is frequently mutated in cancers, particularly in AML. The heterozygous R736H mutation in the FF-interface of the tetrameric enzyme is the second most frequently observed DNMT3A cancer mutation, but its pathogenic mechanism is unclear. We show here that R736H leads to a moderate reduction in catalytic activity of 20-40% depending on the substrate, but no changes in CpG specificity, flanking sequence preferences and subnuclear localization. Strikingly, R736H showed a very strong stimulation by DNMT3L and the R736H/DNMT3L complex was 3-fold more active than WT/DNMT3L. Similarly, formation of mixed R736H/DNMT3A WT FF-interfaces led to an increased activity. R736H/DNMT3L and mixed R736H/DNMT3A WT FF-interfaces were less stable than interfaces not involving R736H, suggesting that an increased flexibility of the mixed interfaces stimulates catalytic activity. Our data suggest that aberrant activity of DNMT3A R736H may lead to DNA hypermethylation in cancer cells which could cause changes in gene expression., Competing Interests: Declaration of competing interest None declared., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

TET dioxygenases convert 5-methylcytosine (5mC) preferentially in a CpG context into 5-hydroxymethylcytosine (5hmC) and higher oxidized forms, thereby initiating DNA demethylation, but details regarding the effects of the DNA sequences flanking the target 5mC site on TET activity are unknown. We investigated oxidation of libraries of DNA substrates containing one 5mC or 5hmC residue in randomized sequence context using single molecule readout of oxidation activity and sequence and show pronounced 20 and 70-fold flanking sequence effects on the catalytic activities of TET1 and TET2, respectively. Flanking sequence preferences were similar for TET1 and TET2 and also for 5mC and 5hmC substrates. Enhanced flanking sequence preferences were observed at non-CpG sites together with profound effects of flanking sequences on the specificity of TET2. TET flanking sequence preferences are reflected in genome-wide and local patterns of 5hmC and DNA demethylation in human and mouse cells indicating that they influence genomic DNA modification patterns in combination with locus specific targeting of TET enzymes., (© 2022. The Author(s).)

Scope: Chickpea (Cicer arietinum) allergy has frequently been reported particularly in Spain and India. Nevertheless, chickpea allergens are poorly characterized. The authors aim to identify and characterize potential allergens from chickpea., Methods and Results: Candidate proteins are selected by an in silico approach or immunoglobuline E (IgE)-testing. Potential allergens are prepared as recombinant or natural proteins and characterized for structural integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD)-spectroscopy, and mass spectrometry (MS) analysis. IgE-sensitization pattern of Spanish chickpea allergic and German peanut and birch pollen sensitized patients are investigated using chickpea extracts and purified proteins. Chickpea allergic patients show individual and heterogeneous IgE-sensitization profiles with extracts from raw and boiled chickpeas. Chickpea proteins pathogenesis related protein family 10 (PR-10), a late embryogenesis abundant protein (LEA/DC-8), and a vicilin-containing fraction, but not 2S albumin, shows IgE reactivity with sera from chickpea, birch pollen, and peanut sensitized patients. Remarkably, allergenic vicilin, DC-8, and PR-10 are detected in the extract of boiled chickpeas., Conclusion: Several IgE-reactive chickpea allergens are identified. For the first time a yet not classified DC-8 protein is characterized as minor allergen (Cic a 1). Finally, the data suggest a potential risk for peanut allergic patients by IgE cross-reactivity with homologous chickpea proteins., (© 2020 Wiley-VCH GmbH.)

Allergic reactions to food are among the major food safety concerns in industrialized countries, and it is estimated that approximately 5% of the population suffers from immunoglobulin-E-mediated food allergy. High-resolution mass spectrometry has become one of the most important techniques for the molecular characterization of allergens, including structural modification, degradation in the gastrointestinal environment, or identification of suitable marker peptides for the development of novel analytical approaches, in the past decade. This perspective aims to briefly summarize the current situation and discuss future developments.

Scope: Allergy to hazelnut seeds ranks among the most prevalent food allergies in Europe. The aim of this study was to elucidate the gastrointestinal digestion of hazelnut allergens on molecular level., Methods and Results: Hazelnut flour was digested in vitro following the Infogest consensus model. For six allergenic proteins, the time-dependent course of digestion was monitored by SDS-PAGE and HPLC-MS/MS, and degradation products were characterized by a bottom-up proteomics approach. Depending on the molecular structure, a specific biochemical fate was observed for each allergen, and degradation kinetics were traced back to the peptide level. 1183 peptides were characterized, including 130 peptides that carry known IgE-binding epitopes and may represent sensitizers for hazelnut allergy. The kinetics of peptide formation and degradation were determined by label-free quantification and follow a complex multi-stage mechanism., Conclusion: We present a comprehensive survey on the gastrointestinal digestion of a relevant allergenic food on level of the peptidome, including the first systematic characterization and quantification of degradation products. This provides information on the differential resistance of plant food allergens and their structural elements undergoing digestion and forms the basis for a deeper understanding of the molecular principles responsible for sensitization to food allergy., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An enhanced enrichment using the Portable Microbe Enrichment Unit (PMEU) with the microaerobic bubbling of broths was applied for the detection of thermotolerant Campylobacter species from water. This PMEU enrichment was compared with the conventional static enrichment of the international standard ISO 17995:2005. In addition, Campylobacter detection after enrichment using a real-time PCR detection was compared with colony counts. The tests with stressed Campylobacter jejuni cells in drinking water indicated that the PMEU enrichment yielded a significantly higher number of Campylobacter cells in the Bolton broth compared with the conventional static incubation. Application of the real-time PCR technique shortened the Campylobacter detection time. This combination of method modifications can be used for Campylobacter detection from water and adds methodological repertoire for the rapid survey and management of waterborne outbreaks.