Systems Biology

Xenobiotics, including environmental pollutants such as bisphenols, phthalates, and parabens, are widely present in food, cosmetics, packaging, and water. These compounds can reach the gastrointestinal tract and interact with the gut microbiota (GM), a complex microbial community that plays a key role in host immunity, metabolism, and barrier function. The GM engages in bidirectional communication with the host via the production of bioactive metabolites, including short-chain fatty acids, neurotransmitter precursors, and bile acid derivatives. Dysbiosis induced by xenobiotics can disrupt microbial metabolite production, impair gut barrier integrity, and contribute to the development of systemic disorders affecting distant organs such as the liver or brain. On the other hand, the GM can biotransform xenobiotics into metabolites with altered bioactivity or toxicity. In vitro models of the human GM offer a valuable tool to complement population-based and in vivo studies, enabling controlled investigation of causative effects and underlying mechanisms. Here, we present an optimized protocol for the collection, cryopreservation, and cultivation of human GM under strictly anaerobic conditions for toxicomicrobiomics applications. The method allows the assessment of xenobiotic–GM interactions in a cost-effective and ethically sustainable way. It is compatible with a wide range of downstream applications, including 16S rDNA sequencing, metabolomics, and endocrine activity assays. The protocol has been optimized to minimize oxygen exposure to less than 2 min, ensuring the viability of obligate anaerobes that dominate the gut ecosystem. This approach facilitates reproducible, mechanistic studies on the impact of environmental xenobiotics on human GM.

Quantitative proteomic analysis plays a crucial role in understanding microbial co-culture systems. Traditional techniques, such as label-free quantification (LFQ) and label-based proteomics, provide valuable insights into the interactions and metabolic exchanges of microbial species. However, the complexity of microbial co-culture systems often leads to challenges in data normalization, especially when dealing with comparative LFQ data where ratios of different organisms can vary across experiments. This protocol describes the application of LFQRatio normalization, a novel normalization method designed to improve the reliability and accuracy of quantitative proteomics data obtained from microbial co-cultures. The method was developed following the analysis of factors that affect both the identification of proteins and the quantitative accuracy of co-culture proteomics. These include peptide physicochemical characteristics such as isoelectric point (pI), molecular weight (MW), hydrophobicity, dynamic range, and proteome size, as well as shared peptides between species. We then created a normalization method based on LFQ intensity values named LFQRatio normalization. This approach was demonstrated by analysis of a synthetic co-culture of two bacteria, Synechococcus elongatus cscB/SPS and Azotobacter vinelandii ΔnifL. Results showed enhanced accuracy of differentially expressed proteins, allowing for more reliable biological interpretation. This protocol provides a reliable and effective tool with wider application to analyze other co-culture systems to study microbial interactions.

Populations of some bumble bee species are in decline, prompting the need to better understand bumble bee biology and for assessing the effects of environmental stressors on these important pollinators. Microcolonies have been successfully used for investigating a range of endpoints, including behavior, gut microbiome, nutrition, development, pathogens, and the effects of pesticide exposure on bumble bee health. Here, we present a step-by-step protocol for initiating, maintaining, and monitoring microcolonies with Bombus impatiens. This protocol has been successfully used in two pesticide exposure-effects studies and can be easily expanded to investigate other aspects of bumble bee biology.