Prior metabolite extraction fully preserves RNAseq quality and enables integrative multi-'omics analysis of the liver metabolic response to viral infection.

Here, we provide an in-depth analysis of the usefulness of single-sample metabolite/RNA extraction for multi-'omics readout. Using pulverized frozen livers of mice injected with lymphocytic choriomeningitis virus (LCMV) or vehicle (Veh), we isolated RNA prior (RNA) or following metabolite extraction (MetRNA). RNA sequencing (RNAseq) data were evaluated for differential expression analysis and dispersion, and differential metabolite abundance was determined. Both RNA and MetRNA clustered together by principal component analysis, indicating that inter-individual differences were the largest source of variance. Over 85% of LCMV versus Veh differentially expressed genes were shared between extraction methods, with the remaining 15% evenly and randomly divided between groups. Differentially expressed genes unique to the extraction method were attributed to randomness around the 0.05 FDR cut-off and stochastic changes in variance and mean expression. In addition, analysis using the mean absolute difference showed no difference in the dispersion of transcripts between extraction methods. Altogether, our data show that prior metabolite extraction preserves RNAseq data quality, which enables us to confidently perform integrated pathway enrichment analysis on metabolomics and RNAseq data from a single sample. This analysis revealed pyrimidine metabolism as the most LCMV-impacted pathway. Combined analysis of genes and metabolites in the pathway exposed a pattern in the degradation of pyrimidine nucleotides leading to uracil generation. In support of this, uracil was among the most differentially abundant metabolites in serum upon LCMV infection. Our data suggest that hepatic uracil export is a novel phenotypic feature of acute infection and highlight the usefulness of our integrated single-sample multi-'omics approach.

A high-throughput screening platform for discovering bacterial species and small molecules that modify animal physiology.

The gut microbiome has been proposed to influence many aspects of animal development and physiology. However, both the specific bacterial species and the molecular mechanisms by which bacteria exert these effects are unknown in most cases. Here, we established a high throughput screening platform using the model animal Caenorhabditis elegans for identifying bacterial species and mechanisms that influence animal development and physiology. From our initial screens we found that many Bacillus species can restore normal animal development to insulin signaling mutant animals that otherwise do not develop to adulthood. To determine how Bacilli influence animal development we screened a complete non-essential gene knockout library of Bacillus subtilis for mutants that no longer restored development to adulthood. We found the Bacillus gene speB is required for animal development. In the absence of speB, B. subtilis produces excess N1-aminopropylagmatine. This polyamine is taken up by animal intestinal cells via the polyamine transporter CATP-5. When this molecule is taken up in sufficient quantities it inhibits animal mitochondrial function and causes diverse species of animals to arrest their development. To our knowledge, these are the first observations that B. subtilis can produce N1-aminopropylagmatine and that polyamines produced by intestinal microbiome species can antagonize animal development and mitochondrial function. Given that Bacilli species are regularly isolated from animal intestinal microbiomes, including from humans, we propose that altered polyamine production from intestinal Bacilli is likely to also influence animal development and metabolism in other species and potentially even contribute developmental and metabolic pathologies in humans. In addition, our findings demonstrate that C. elegans can be used as a model animal to conduct high throughput screens for bacterial species and bioactive molecules that alter animal physiology.

A diverse proteome is present and enzymatically active in metabolite extracts.

Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.

Itaconate confers tolerance to late NLRP3 inflammasome activation.

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.

Fungal fermentation on anaerobic digestate for lipid-based biofuel production.

BACKGROUND: Anaerobic digestate is the effluent from anaerobic digestion of organic wastes. It contains a significant amount of nutrients and lignocellulosic materials, even though anaerobic digestion consumed a large portion of organic matters in the wastes. Utilizing the nutrients and lignocellulosic materials in the digestate is critical to significantly improve efficiency of anaerobic digestion technology and generate value-added chemical and fuel products from the organic wastes. Therefore, this study focused on developing an integrated process that uses biogas energy to power fungal fermentation and converts remaining carbon sources, nutrients, and water in the digestate into biofuel precursor-lipid. RESULTS: The process contains two unit operations of anaerobic digestion and digestate utilization. The digestate utilization includes alkali treatment of the mixture feed of solid and liquid digestates, enzymatic hydrolysis for mono-sugar release, overliming detoxification, and fungal fermentation for lipid accumulation. The experimental results conclude that 5 h and 30 °C were the preferred conditions for the overliming detoxification regarding lipid accumulation of the following fungal cultivation. The repeated-batch fungal fermentation enhanced lipid accumulation, which led to a final lipid concentration of 3.16 g/L on the digestate with 10% dry matter. The mass and energy balance analysis further indicates that the digestate had enough water for the process uses and the biogas energy was able to balance the needs of individual unit operations. CONCLUSIONS: A fresh-water-free and energy-positive process of lipid production from anaerobic digestate was achieved by integrating anaerobic digestion and fungal fermentation. The integration addresses the issues that both biofuel industry and waste management encounter-high water and energy demand of biofuel precursor production and few digestate utilization approaches of organic waste treatment.

Effects of inhibitory compounds in lignocellulosic hydrolysates on Mortierella isabellina growth and carbon utilization.

Oleaginous fungus Mortierella isabellina showed excellent lipid conversion on non-detoxified lignocellulosic hydrolysate. This study investigated the effects of inhibitory compounds (furfural, hydroxymethylfurfural, and ferulic and coumaric acids) in lignocellulosic hydrolysate on M. isabellina growth and lipid production. M. isabellina can tolerate furfural (∼1 g/L), hydroxymethylfurfural (∼2.5 g/L), ferulic (∼0.5 g/L) and coumaric acid (∼0.5 g/L) with normal growth rates. Synergistic effect of these inhibitors (2 g/L furfural, 0.4 g/L hydroxymethylfurfural, 0.02 g/L ferulic acid and 0.02 g/L coumaric acid) moderately reduces total fungal growth (by 28%), while the presence of these inhibitors has minor impact on cell lipid contents and lipid profiles. In the presence of inhibitory compounds, (13)C-tracing has revealed that M. isabellina can simultaneously utilize glucose and acetate, and acetate is mainly assimilated for synthesis of lipid and TCA cycle amino acids. The results also demonstrate that glucose has strong catabolite repression for xylose utilization for biomass and lipid production in the presence of inhibitors.