Microbiome variation during culture growth of the European house dust mite, Dermatophagoides pteronyssinus.

In culture, the house dust mite, Dermatophagoides pteronyssinus, shows different growth patterns, but the composition and changes in the associated microbial community during mite culture growth are poorly known. In this study, we analyzed temporal changes in microbial communities including 'internal' communities (inside mites, ingested) and 'environmental' communities (from culture environment). Microbial community structure was correlated with guanine content (a nitrogenous waste product of mites) and mite population density. Both internal and environmental microbial communities were remarkably consistent between biological replicates from the same culture age group and were composed of relatively few dominant taxa-11 bacterial and 3 fungal operational taxonomic units (OTUs). Significant changes over time in microbial community structure in the bulk culture environment and in internal mite samples were observed. The yeast, Saccharomyces cerevisiae, a main component of the mite diet, gradually disappeared during mite culture growth and was replaced by fungi from the genera Aspergillus and Candida in both 'internal' and 'environmental' samples. In environmental samples, bacteria from the genus Lactobacillus and S. cerevisiae were negatively correlated, and Aspergillus and Candida positively correlated, with guanine content. The relative abundance of bacteria from the genus Kocuria increased with mite density but declined with increasing guanine content. The relative abundance of bacteria from the genus Virgibacillus was negatively correlated with mite density in 'internal' samples. Gram-positive bacteria dominated bacterial microbiomes at all time points in our experiments, indicating a more limited possibility for vaccine contamination by bacterial endotoxins (heat-stable lipopolysaccharides produced mostly by Gram-negative bacteria) in our experimental cultures.

A single honey proteome dataset for identifying adulteration by foreign amylases and mining various protein markers natural to honey.

Honey adulteration is a common practice that deceives consumers and devalues the unique curative and food properties of honey. For marketing, each honey must satisfy an internationally valid Codex standard. One of the quality parameters is diastase/amylase activity, which, if lowered, may be compensated for by the addition of foreign amylases. However, the estimation of enzyme activity does not enable identification of artificially added amylases. 45 honey samples were analyzed using label-free nanoLC-MS/MS proteomics. Four honeys were found to contain the foreign amylases from Aspergillus niger, Bacillus amyloliquefaciens and/or Bacillus licheniformis. This result was confirmed via proof of specificity at multiple levels. Furthermore, we identified a series of plant-related protein groups. Despite plant-related proteins constituting a significant portion of honey proteins, they were minor components compared to the major honey bee-derived proteins. Bioinformatic analysis also provided evidence for aphid and catalase proteins in honey, but the limited specificity of the MS/MS identified peptides must be considered. Overall, we demonstrate a proteomics approach employing LC-MS/MS that is useful for proving adulteration and assessing honey quality. As an resource useful for reference, we provide curated sequence databases. In addition, we provide many markers that are naturally found in honey for future studies. SIGNIFICANCE: Honey is unique natural product used since ancient times as a food and natural medicine. Humans strive to understand honey components because they can characterize different types of honey and be used for authentication and origin assessment. One of the important honey components are proteins. The proteins present in honey can naturally occur in honey, but some of them can be used to mask deficiencies in some honey quality properties. Diastases/amylases are such proteins, and their activity, a measure of honey freshness, can decrease in time or due to processing. To our knowledge, we for the first time specifically identify foreign amylases in honey. However, this study provided new information on other non-honey bee proteins in honey. Thus, this study is also of importance due to its identification of plant and aphid proteins and catalase-related proteins. This study provides a clue explaining the controversial presence of catalase in honey, since catalases can be identified and their origin determined via proteomics.

Dynamics of the microbial community during growth of the house dust mite Dermatophagoides farinae in culture.

The variation in house dust mite microbial communities is important because various microorganisms modulate the production of allergens by their mite hosts and/or contaminate immunotherapeutic extracts. Temporal changes in mite microbiomes and the mite culture environment occurring at different stages of mite culture development are particularly understudied in this system. Here, we analyzed the dynamics of microbial communities during the culture growth of Dermatophagoides farinae. Changes in microbiomes were related to three key variables: the mite population density, microbial microcosm respiration and concentration of guanine (the mite nitrogenous waste metabolite). Mite populations exhibited the following phases: exponential growth, plateau and exponential decline. The intracellular bacterium Cardinium and the yeast Saccharomyces cerevisiae prevailed in the internal mite microbiomes, and the bacterium Lactobacillus fermentum was prevalent in the mite diet. The reduction in the mite population size during the late phases of culture development was related to the changes in their microbial profiles: the intracellular bacterium Cardinium was replaced by Staphylococcus, Oceanobacillus and Virgibacillus, and S. cerevisiae was replaced by the antagonistic fungi Aspergillus penicillioides and Candida. Increases in the guanine content were positively correlated with increases in the Staphylococcus and A. penicillioides profiles in the culture environment. Our results show that the mite microbiome exhibits strong, dynamic alterations in its profiles across different mite culture growth stages.

The Unique Protein Composition of Honey Revealed by Comprehensive Proteomic Analysis: Allergens, Venom-like Proteins, Antibacterial Properties, Royal Jelly Proteins, Serine Proteases, and Their Inhibitors.

Honey is a unique natural product produced by European honeybees. Due to its high economic value, honey is considered to be well characterized chemically, and it is often discovered to be an adulterated commodity. However, this study shows that our knowledge of honey protein composition, which is of high medical and pharmaceutical importance, is incomplete. In this in-depth proteomic study of 13 honeys, we identified a number of proteins that are important for an understanding of honey properties and merit additional pharmaceutical research. Our major result is an expanded understanding of the proteins underlying honey's antimicrobial properties, such as hymenoptaecin and defensin-1, glucose dehydrogenase isoforms, venom allergens and other venom-like proteins, serine proteases and serine protease inhibitors, and a series of royal jelly proteins. In addition, we performed quantitative comparisons of all of the proteins previously known or newly identified. The honey proteins, determined using label-free nLC-MS/MS in which the same protein quantity was analyzed in one series, were found in relatively similar proportions, although eucalyptus honey differed most widely from the remaining honeys. Overall, the proteome analysis indicated that honeybees supply proteins to honey in a relatively stable ratio within each proteome, but total protein quantity can differ by approximately an order of magnitude in different honeys.

Monoaurated vs. diaurated intermediates: causality or independence?

Diaurated intermediates of gold-catalysed reactions have been a long-standing subject of debate. Although diaurated complexes were regarded as a drain of active monoaurated intermediates in catalytic cycles, they were also identified as the products of gold-gold cooperation in dual-activation reactions. This study shows investigation of intermediates in water addition to alkynes catalysed by [(IPr)Au(CH3CN)(BF4)]. Electrospray ionisation mass spectrometry (ESI-MS) allowed us to detect both monoaurated and diaurated complexes in this reaction. Infrared photodissociation spectra of the trapped complexes show that the structure of the intermediates corresponds to α-gold ketone intermediates protonated or aurated at the oxygen atom. Delayed reactant labelling experiments provided the half life of the intermediates in reaction of 1-phenylpropyne (∼7 min) and the kinetic isotope effects for hydrogen introduction to the carbon atom (KIE ∼ 4-6) and for the protodeauration (KIE ∼ 2). The results suggest that the ESI-MS detected monoaurated and diaurated complexes report on species with a very similar or the same kinetics in solution. Kinetic analysis of the overall reaction showed that the reaction rate is first-order dependent on the concentration of the gold catalyst. Finally, all results are consistent with the reaction mechanism proceeding via monoaurated neutral α-gold ketone intermediates only.