Comparative toxicological assessment of two bisphenols using a systems approach: Evaluation of the behavioral and transcriptomic responses of Danio rerio to Bisphenol A and Tetrabromobisphenol A.

The zebrafish (Danio rerio) is becoming a critical component of New Approach Methods (NAMs) in chemical risk assessment. As a whole organism in vitro NAM, the zebrafish model offers significant advantages over individual cell-line testing, including toxicokinetic and toxicodynamic competencies. A transcriptomic approach not only allows for insight into mechanism of action for both apical endpoints and unobservable adverse outcomes, but also changes in gene expression induced by lower, environmentally relevant concentrations. In this study, we used a larval zebrafish model to assess the behavioral and transcriptomic alterations caused by sub-phenotypic concentrations of two chemicals with the same structural backbone, the endocrine disrupting chemicals: Bisphenol A and Tetrabromobisphenol A. Following assessment of behavioral toxicity, we used a transcriptomic approach to identify molecular pathways associated with previously described phenotypes. We also determined the transcriptomic Point of Departure (POD) for each chemical by modelling gene expression changes as continuous systems which allows for the identification of a single concentration at which toxic effects can be predicted. This can then be investigated with confirmatory cell-based testing in an integrated approach to testing and assessment (IATA) to determine risk to human health and the environment with greater confidence. This paper demonstrates the impact of using a multi-faceted approach for evaluating the physiological and neurotoxic effects of exposure to structurally related chemicals. By comparing phenotypic effects with transcriptomic outcomes, we were able to differentiate, characterize and rank the toxicities of related bisphenols, which demonstrates methodological advantages unique to the larval zebrafish NAM.

Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy.

The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ T cells ex vivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ T cells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated T cells synergized in vivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8+ T cells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance T cell metabolism and anti-tumor activity.

A large-scale, targeted metabolomics method for the analysis and quantification of metabolites in human plasma via liquid chromatography-mass spectrometry.

Metabolomics is the study of small molecules, primarily metabolites, that are produced during metabolic processes. Analysis of the composition of an organism's metabolome can yield useful information about an individual's health status at any given time. In recent years, the development of large-scale, targeted metabolomic methods has allowed for the analysis of biological samples using analytical techniques such as LC-MS/MS. This paper presents a large-scale metabolomics method for analysis of biological samples, with a focus on quantification of metabolites found in blood plasma. The method comprises a 10-min chromatographic separation using HILIC and RP stationary phases combined with positive and negative electrospray ionization in order to maximize metabolome coverage. Complete analysis of a single sample can be achieved in as little as 40 min using the two columns and dual modes of ionization. With 540 metabolites and the inclusion of over 200 analytical standards, this method is comprehensive and quantitatively robust when compared to current targeted metabolomics methods. This study uses a large-scale evaluation of metabolite recovery from plasma that enables absolute quantification of metabolites by correcting for analyte loss throughout processes such as extraction, handling, or storage. In addition, the method was applied to plasma collected from adjuvant breast cancer patients to confirm the suitability of the method to clinical samples.

Analysis of the Circulating Metabolome of Patients with Cutaneous, Mucosal and Uveal Melanoma Reveals Distinct Metabolic Profiles with Implications for Response to Immunotherapy.

Cutaneous melanoma (CM) patients respond better to immune checkpoint inhibitors (ICI) than mucosal and uveal melanoma patients (MM/UM). Aiming to explore these differences and understand the distinct response to ICI, we evaluated the serum metabolome of advanced CM, MM, and UM patients. Levels of 115 metabolites were analyzed in samples collected before ICI, using a targeted metabolomics platform. In our analysis, molecules involved in the tryptophan-kynurenine axis distinguished UM/MM from CM. UM/MM patients had higher levels of 3-hydroxykynurenine (3-HKyn), whilst patients with CM were found to have higher levels of kynurenic acid (KA). The KA/3-HKyn ratio was significantly higher in CM versus the other subtypes. UM, the most ICI-resistant subtype, was also associated with higher levels of sphingomyelin-d18:1/22:1 and the polyamine spermine (SPM). Overall survival was prolonged in a cohort of CM patients with lower SPM levels, suggesting there are also conserved metabolic factors promoting ICI resistance across melanoma subtypes. Our study revealed a distinct metabolomic profile between the most resistant melanoma subtypes, UM and MM, compared to CM. Alterations within the kynurenine pathway, polyamine metabolism, and sphingolipid metabolic pathway may contribute to the poor response to ICI. Understanding the different metabolomic profiles introduces opportunities for novel therapies with potential synergic activity to ICI, to improve responses of UM/MM.

Successful Dietary Therapy in Paediatric Crohn's Disease is Associated with Shifts in Bacterial Dysbiosis and Inflammatory Metabotype Towards Healthy Controls.

BACKGROUND AND AIMS: Nutritional therapy with the Crohn's Disease Exclusion Diet + Partial Enteral Nutrition [CDED+PEN] or Exclusive Enteral Nutrition [EEN] induces remission and reduces inflammation in mild-to-moderate paediatric Crohn's disease [CD]. We aimed to assess if reaching remission with nutritional therapy is mediated by correcting compositional or functional dysbiosis. METHODS: We assessed metagenome sequences, short chain fatty acids [SCFA] and bile acids [BA] in 54 paediatric CD patients reaching remission after nutritional therapy [with CDED + PEN or EEN] [NCT01728870], compared to 26 paediatric healthy controls. RESULTS: Successful dietary therapy decreased the relative abundance of Proteobacteria and increased Firmicutes towards healthy controls. CD patients possessed a mixture of two metabotypes [M1 and M2], whereas all healthy controls had metabotype M1. M1 was characterised by high Bacteroidetes and Firmicutes, low Proteobacteria, and higher SCFA synthesis pathways, and M2 was associated with high Proteobacteria and genes involved in SCFA degradation. M1 contribution increased during diet: 48%, 63%, up to 74% [Weeks 0, 6, 12, respectively.]. By Week 12, genera from Proteobacteria reached relative abundance levels of healthy controls with the exception of E. coli. Despite an increase in SCFA synthesis pathways, remission was not associated with increased SCFAs. Primary BA decreased with EEN but not with CDED+PEN, and secondary BA did not change during diet. CONCLUSION: Successful dietary therapy induced correction of both compositional and functional dysbiosis. However, 12 weeks of diet was not enough to achieve complete correction of dysbiosis. Our data suggests that composition and metabotype are important and change quickly during the early clinical response to dietary intervention. Correction of dysbiosis may therefore be an important future treatment goal for CD.

Metabolome Changes With Diet-Induced Remission in Pediatric Crohn's Disease.

BACKGROUND & AIMS: The Crohn's disease (CD) exclusion diet (CDED) plus partial enteral nutrition (PEN) and exclusive enteral nutrition (EEN) both induce remission in pediatric CD. CDED+PEN is better tolerated and able to sustain remission. We characterized the changes in fecal metabolites induced by CDED+PEN and EEN and their relationship with remission. METHODS: A total of 216 fecal metabolites were measured in 80 fecal samples at week (W) 0, W6, and W12, of children with mild to moderate CD in a prospective randomized trial comparing CDED+PEN vs EEN. The metabolites were measured using liquid chromatography coupled to mass spectrometry. Metagenome Kyoto Encyclopedia of Genes and Genomes Orthology analysis was performed to investigate the differential functional gene abundance involved in specific metabolic pathways. Data were analyzed according to clinical outcome of remission (W6_rem), no remission (W6_nr), sustained remission (W12_sr), and nonsustained (W12_nsr) remission. RESULTS: A decrease in kynurenine and succinate synthesis and an increase in N-α-acetyl-arginine characterized CDED+PEN W6_rem, whereas changes in lipid metabolism characterized EEN W6_rem, especially reflected by lower levels in ceramides. In contrast, fecal metabolites in EEN W6_nr were comparable to baseline/W0 samples. CDED+PEN W6_rem children maintained metabolome changes through W12. In contrast, W12_nsr children in the EEN group, who resumed a free diet after week 6, did not. The metabolome of CDED+PEN differed from EEN in the purine, pyrimidine, and sphingolipid pathways. A significant differential abundance in several genes involved in these pathways was detected. CONCLUSION: CDED+PEN- and EEN-induced remission are associated with significant changes in inflammatory bowel disease-associated metabolites such as kynurenine, ceramides, amino acids, and others. Sustained remission with CDED+PEN, but not EEN, was associated with persistent changes in metabolites. CLINICALTRIALS: gov, Number NCT01728870.

Coenzyme A fuels T cell anti-tumor immunity.

Metabolic programming is intricately linked to the anti-tumor properties of T cells. To study the metabolic pathways associated with increased anti-tumor T cell function, we utilized a metabolomics approach to characterize three different CD8+ T cell subsets with varying degrees of anti-tumor activity in murine models, of which IL-22-producing Tc22 cells displayed the most robust anti-tumor activity. Tc22s demonstrated upregulation of the pantothenate/coenzyme A (CoA) pathway and a requirement for oxidative phosphorylation (OXPHOS) for differentiation. Exogenous administration of CoA reprogrammed T cells to increase OXPHOS and adopt the CD8+ Tc22 phenotype independent of polarizing conditions via the transcription factors HIF-1α and the aryl hydrocarbon receptor (AhR). In murine tumor models, treatment of mice with the CoA precursor pantothenate enhanced the efficacy of anti-PDL1 antibody therapy. In patients with melanoma, pre-treatment plasma pantothenic acid levels were positively correlated with the response to anti-PD1 therapy. Collectively, our data demonstrate that pantothenate and its metabolite CoA drive T cell polarization, bioenergetics, and anti-tumor immunity.

Gut bacterial gene changes following pegaspargase treatment in pediatric patients with acute lymphoblastic leukemia.

Treatment of pediatric acute lymphoblastic leukemia (ALL) with pegaspargase exploits ALL cells dependency on asparagine. Pegaspargase depletes asparagine, consequentially affecting aspartate, glutamine and glutamate. The gut as a confounding source of these amino acids (AAs) and the role of gut microbiome metabolism of AAs has not been examined. We examined asparagine, aspartate, glutamine and glutamate in stool samples from patients over pegaspargase treatment. Microbial gene-products, which interact with these AAs were identified. Stool asparagine declined significantly, and 31 microbial genes changed over treatment. Changes were complex, and included genes involved in AA metabolism, nutrient sensing, and pathways increased in cancers. While we identified changes in a gene (iaaA) with limited asparaginase activity, it lacked significance after correction leaving open other mechanisms for asparagine decline, possibly including loss from gut to blood. Understanding pathways that change AA availability, including by microbes in the gut, could be useful in optimizing pegaspargase therapy.

Heat stress response in the closest algal relatives of land plants reveals conserved stress signaling circuits.

All land plants (embryophytes) share a common ancestor that likely evolved from a filamentous freshwater alga. Elucidating the transition from algae to embryophytes - and the eventual conquering of Earth's surface - is one of the most fundamental questions in plant evolutionary biology. Here, we investigated one of the organismal properties that might have enabled this transition: resistance to drastic temperature shifts. We explored the effect of heat stress in Mougeotia and Spirogyra, two representatives of Zygnematophyceae - the closest known algal sister lineage to land plants. Heat stress induced pronounced phenotypic alterations in their plastids, and high-performance liquid chromatography-tandem mass spectroscopy-based profiling of 565 transitions for the analysis of main central metabolites revealed significant shifts in 43 compounds. We also analyzed the global differential gene expression responses triggered by heat, generating 92.8 Gbp of sequence data and assembling a combined set of 8905 well-expressed genes. Each organism had its own distinct gene expression profile; less than one-half of their shared genes showed concordant gene expression trends. We nevertheless detected common signature responses to heat such as elevated transcript levels for molecular chaperones, thylakoid components, and - corroborating our metabolomic data - amino acid metabolism. We also uncovered the heat-stress responsiveness of genes for phosphorelay-based signal transduction that links environmental cues, calcium signatures and plastid biology. Our data allow us to infer the molecular heat stress response that the earliest land plants might have used when facing the rapidly shifting temperature conditions of the terrestrial habitat.

Molecular characterization of zebrafish embryogenesis via DNA microarrays and multiplatform time course metabolomics studies.

One of the greatest strengths of "-omics" technologies is their ability to capture a molecular snapshot of multiple cellular processes simultaneously. Transcriptomics, proteomics, and metabolomics have, individually, been used in wide-ranging studies involving cell lines, tissues, model organisms, and human subjects. Nonetheless, despite the fact that their power lies in the global acquisition of parallel data streams, these methods continue to be employed separately. We highlight work done to merge transcriptomics and metabolomics technologies to study zebrafish (Danio rerio) embryogenesis. We combine information from three bioanalytical platforms, that is, DNA microarrays, (1)H nuclear magnetic resonance ((1)H NMR), and mass spectrometry (MS)-based metabolomics, to identify and provide insights into the organism's developmental regulators. We apply a customized approach to the analysis of such time-ordered measurements to provide temporal profiles that depict the modulation of metabolites and gene transcription. Initially, the three data sets were analyzed individually but later they were fused to highlight the advantages gained through such an integrated approach. Unique challenges posed by fusion of such data are discussed given differences in the measurement error structures, the wide dynamic range for the molecular species, and the analytical platforms used to measure them (i.e., fluorescence ratios, NMR, and MS intensities). Our data analysis reveals that changes in transcript levels at specific developmental stages correlate with previously published data with over 90% accuracy. In addition, transcript profiles exhibited trends that were similar to the accumulation of metabolites over time. Profiles for metabolites such as choline-like compounds (Trimethylamine-N-oxide, phosphocholine, betaine), creatinine/creatine, and other metabolites involved in energy metabolism exhibited a steady increase from 15 hours post fertilization (hpf) to 48 hpf. Other metabolite and transcript profiles were transiently rising and then falling back to baseline. The "house keeping" metabolites such as branched chain amino acids exhibited a steady presence throughout embryogenesis. Although the transcript profiling corresponds to only 16 384 genes, a subset of the total number of genes in the zebrafish genome, we identified examples where gene transcript and metabolite profiles correlate with one another, reflective of a relationship between gene and metabolite regulation over the course of embryogenesis.

Identification of genes differentially expressed in Atlantic salmon (Salmo salar) in response to infection by Aeromonas salmonicida using cDNA microarray technology.

The response of Atlantic salmon, Salmo salar, to infection by the bacterial pathogen Aeromonas salmonicida (the causative agent of furunculosis), was investigated using a cohabitation model and a custom Atlantic salmon cDNA microarray consisting of over 4000 different amplicons. Pooled samples of each of three immune-relevant tissues (spleen, head kidney and liver) were obtained from fish exposed to infected salmon for 13 days. Reverse transcription-PCR assays were used to verify the differential expression of 12 candidate genes uncovered by microarray analysis. Among the differentially expressed genes were several previously revealed by suppression subtractive hybridization and EST surveys and that are recognized to encode humoral components of the innate immune system. Other genes identified in this study were not previously associated with infection. In addition, a number of genes with no known homologs were uncovered. Determination of their specific roles during infection may lead to a better understanding of innate immunity.

cpnDB: a chaperonin sequence database.

Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.