Methionine Sources Differently Affect Production of Reactive Oxygen Species, Mitochondrial Bioenergetics, and Growth of Murine and Quail Myoblasts In Vitro.

An optimal supply of L-methionine (L-Met) improves muscle growth, whereas over-supplementation exerts adverse effects. To understand the underlying mechanisms, this study aims at exploring effects on the growth, viability, ROS production, and mitochondrial bioenergetics of C2C12 (mouse) and QM7 (quail) myoblasts additionally supplemented (100 or 1000 µM) with L-Met, DL-methionine (DL-Met), or DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA). In both cell lines, all the supplements stimulated cell growth. However, in contrast to DL-Met, 1000 µM of L-Met (C2C12 cells only) or DL-HMTBA started to retard growth. This negative effect was stronger with DL-HMTBA and was accompanied by significantly elevated levels of extracellular H2O2, an indicator for OS, in both cell types. In addition, oversupplementation with DL-HMTBA (1000 µM) induced adaptive responses in mitochondrial bioenergetics, including reductions in basal (C2C12 and QM7) and ATP-synthase-linked (C2C12) oxygen consumption, maximal respiration rate, and reserve capacity (QM7). Only QM7 cells switched to nonmitochondrial aerobic glycolysis to reduce ROS production. In conclusion, we found a general negative effect of methionine oversupplementation on cell proliferation. However, only DL-HMTBA-induced growth retardation was associated with OS and adaptive, species-specific alterations in mitochondrial functionality. OS could be better compensated by quail cells, highlighting the role of species differences in the ability to cope with methionine oversupplementation.

Effects of supplementation of Bacillus amyloliquefaciens on performance, systemic immunity, and intestinal microbiota of weaned pigs experimentally infected with a pathogenic enterotoxigenic E. coli F18.

The objective of this study was to investigate the effects of dietary supplementation of Bacillus (B.) amyloliquefaciens on growth performance, diarrhea, systemic immunity, and intestinal microbiota of weaned pigs experimentally infected with F18 enterotoxigenic Escherichia coli (ETEC). A total of 50 weaned pigs (7.41 ± 1.35 kg BW) were individually housed and randomly allotted to one of the following five treatments: sham control (CON-), sham B. amyloliquefaciens (BAM-), challenged control (CON+), challenged B. amyloliquefaciens (BAM+), and challenged carbadox (AGP+). The experiment lasted 28 days, with 7 days of adaptation and 21 days after the first ETEC inoculation. ETEC challenge reduced (P < 0.05) average daily gain (ADG) of pigs. Compared with CON+, AGP+ enhanced (P < 0.05) ADG, while B. amyloliquefaciens supplementation tended (P < 0.10) to increase ADG in pigs from days 0 to 21 post-inoculation (PI). The ETEC challenge increased (P < 0.05) white blood cell (WBC) count on days 7 and 21 PI, while BAM+ pigs tended (P < 0.10) to have low WBC on day 7 PI and had lower (P < 0.05) WBC on day 21 PI compared with CON+. In comparison to AGP+ fecal microbiota, BAM+ had a lower (P < 0.05) relative abundance of Lachnospiraceae on day 0 and Clostridiaceae on day 21 PI, but a higher (P < 0.05) relative abundance of Enterobacyeriaceae on day 0. In ileal digesta, the Shannon index was higher (P < 0.05) in BAM+ than in AGP+. Bray-Curtis PCoA displayed a difference in bacterial community composition in ileal digesta collected from sham pigs vs. ETEC-infected pigs on day 21 PI. Pigs in BAM+ had a greater (P < 0.05) relative abundance of Firmicutes, but a lower (P < 0.05) relative abundance of Actinomycetota and Bacteroidota in ileal digesta than pigs in AGP+. Ileal digesta from AGP+ had a greater (P < 0.05) abundance of Clostridium sensu stricto 1 but lower (P < 0.05) Bifidobacterium than pigs in BAM+. In conclusion, supplementation of B. amyloliquefaciens tended to increase ADG and had limited effects on the diarrhea of ETEC-infected pigs. However, pigs fed with B. amyloliquefaciens exhibit milder systemic inflammation than controls. B. amyloliquefaciens differently modified the intestinal microbiota of weaned pigs compared with carbadox.

Dietary methionine source alters the lipidome in the small intestinal epithelium of pigs.

Methionine (Met) as an essential amino acid has key importance in a variety of metabolic pathways. This study investigated the influence of three dietary Met supplements (0.21% L-Met, 0.21% DL-Met and 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA)) on the metabolome and inflammatory status in the small intestine of pigs. Epithelia from duodenum, proximal jejunum, middle jejunum and ileum were subjected to metabolomics analysis and qRT-PCR of caspase 1, NLR family pyrin domain containing 3 (NLRP3), interleukins IL1ß, IL8, IL18, and transforming growth factor TGFß. Principal component analysis of the intraepithelial metabolome revealed strong clustering of samples by intestinal segment but not by dietary treatment. However, pathway enrichment analysis revealed that after L-Met supplementation polyunsaturated fatty acids (PUFA) and tocopherol metabolites were lower across small intestinal segments, whereas monohydroxy fatty acids were increased in distal small intestine. Pigs supplemented with DL-HMTBA showed a pronounced shift of secondary bile acids (BA) and sphingosine metabolites from middle jejunum to ileum. In the amino acid super pathway, only histidine metabolism tended to be altered in DL-Met-supplemented pigs. Diet did not affect the expression of inflammation-related genes. These findings suggest that dietary supplementation of young pigs with different Met sources selectively alters lipid metabolism without consequences for inflammatory status.

Are the Main Methionine Sources Equivalent? A Focus on DL-Methionine and DL-Methionine Hydroxy Analog Reveals Differences on Rainbow Trout Hepatic Cell Lines Functions.

The replacement of fishmeal by plant proteins in aquafeeds imposes the use of synthetic methionine (MET) sources to balance the amino acid composition of alternative diets and so to meet the metabolic needs of fish of agronomic interest such as rainbow trout (RT-Oncorhynchus mykiss). Nonetheless, debates still exist to determine if one MET source is more efficiently used than another by fish. To address this question, the use of fish cell lines appeared a convenient strategy, since it allowed to perfectly control cell growing conditions notably by fully depleting MET from the media and studying which MET source is capable to restore cell growth/proliferation and metabolism when supplemented back. Thus, results of cell proliferation assays, Western blots, RT-qPCR and liquid chromatography analyses from two RT liver-derived cell lines revealed a better absorption and metabolization of DL-MET than DL-Methionine Hydroxy Analog (MHA) with the activation of the mechanistic Target Of Rapamycin (mTOR) pathway for DL-MET and the activation of integrated stress response (ISR) pathway for MHA. Altogether, the results clearly allow to conclude that both synthetic MET sources are not biologically equivalent, suggesting similar in vivo effects in RT liver and, therefore, questioning the MHA efficiencies in other RT tissues.

Bioaccumulation of therapeutic drugs by human gut bacteria.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

SPEED2: inferring upstream pathway activity from differential gene expression.

Extracting signalling pathway activities from transcriptome data is important to infer mechanistic origins of transcriptomic dysregulation, for example in disease. A popular method to do so is by enrichment analysis of signature genes in e.g. differentially regulated genes. Previously, we derived signatures for signalling pathways by integrating public perturbation transcriptome data and generated a signature database called SPEED (Signalling Pathway Enrichment using Experimental Datasets), for which we here present a substantial upgrade as SPEED2. This web server hosts consensus signatures for 16 signalling pathways that are derived from a large number of transcriptomic signalling perturbation experiments. When providing a gene list of e.g. differentially expressed genes, the web server allows to infer signalling pathways that likely caused these genes to be deregulated. In addition to signature lists, we derive 'continuous' gene signatures, in a transparent and automated fashion without any fine-tuning, and describe a new algorithm to score these signatures.

Nutritional preferences of human gut bacteria reveal their metabolic idiosyncrasies.

Bacterial metabolism plays a fundamental role in gut microbiota ecology and host-microbiome interactions. Yet the metabolic capabilities of most gut bacteria have remained unknown. Here we report growth characteristics of 96 phylogenetically diverse gut bacterial strains across 4 rich and 15 defined media. The vast majority of strains (76) grow in at least one defined medium, enabling accurate assessment of their biosynthetic capabilities. These do not necessarily match phylogenetic similarity, thus indicating a complex evolution of nutritional preferences. We identify mucin utilizers and species inhibited by amino acids and short-chain fatty acids. Our analysis also uncovers media for in vitro studies wherein growth capacity correlates well with in vivo abundance. Further value of the underlying resource is demonstrated by correcting pathway gaps in available genome-scale metabolic models of gut microorganisms. Together, the media resource and the extracted knowledge on growth abilities widen experimental and computational access to the gut microbiota.

Perturbation-response genes reveal signaling footprints in cancer gene expression.

Aberrant cell signaling can cause cancer and other diseases and is a focal point of drug research. A common approach is to infer signaling activity of pathways from gene expression. However, mapping gene expression to pathway components disregards the effect of post-translational modifications, and downstream signatures represent very specific experimental conditions. Here we present PROGENy, a method that overcomes both limitations by leveraging a large compendium of publicly available perturbation experiments to yield a common core of Pathway RespOnsive GENes. Unlike pathway mapping methods, PROGENy can (i) recover the effect of known driver mutations, (ii) provide or improve strong markers for drug indications, and (iii) distinguish between oncogenic and tumor suppressor pathways for patient survival. Collectively, these results show that PROGENy accurately infers pathway activity from gene expression in a wide range of conditions.

Computational tools for modeling xenometabolism of the human gut microbiota.

The gut microbiota is increasingly being recognized as a key site of metabolism for drugs and other xenobiotic compounds that are relevant to human health. The molecular complexity of the gut microbiota revealed by recent metagenomics studies has highlighted the need as well as the challenges for system-level modeling of xenobiotic metabolism in the gut. Here, we outline the possible pathways through which the gut microbiota can modify xenobiotics and review the available computational tools towards modeling complex xenometabolic processes. We put these diverse computational tools and relevant experimental findings into a unified perspective towards building holistic models of xenobiotic metabolism in the gut.