High-throughput Saccharomyces cerevisiae cultivation method for credentialing-based untargeted metabolomics.

peer reviewedIdentifying metabolites in model organisms is critical for many areas of biology, including unravelling disease aetiology or elucidating functions of putative enzymes. Even now, hundreds of predicted metabolic genes in Saccharomyces cerevisiae remain uncharacterized, indicating that our understanding of metabolism is far from complete even in well-characterized organisms. While untargeted high-resolution mass spectrometry (HRMS) enables the detection of thousands of features per analysis, many of these have a non-biological origin. Stable isotope labelling (SIL) approaches can serve as credentialing strategies to distinguish biologically relevant features from background signals, but implementing these experiments at large scale remains challenging. Here, we developed a SIL-based approach for high-throughput untargeted metabolomics in S. cerevisiae, including deep-48 well format-based cultivation and metabolite extraction, building on the peak annotation and verification engine (PAVE) tool. Aqueous and nonpolar extracts were analysed using HILIC and RP liquid chromatography, respectively, coupled to Orbitrap Q Exactive HF mass spectrometry. Of the approximately 37,000 total detected features, only 3-7% of the features were credentialed and used for data analysis with open-source software such as MS-DIAL, MetFrag, Shinyscreen, SIRIUS CSI:FingerID, and MetaboAnalyst, leading to the successful annotation of 198 metabolites using MS2 database matching. Comparable metabolic profiles were observed for wild-type and sdh1Δ yeast strains grown in deep-48 well plates versus the classical shake flask format, including the expected increase in intracellular succinate concentration in the sdh1Δ strain. The described approach enables high-throughput yeast cultivation and credentialing-based untargeted metabolomics, providing a means to efficiently perform molecular phenotypic screens and help complete metabolic networks

BSA4Yeast: Web-based quantitative trait locus linkage analysis and bulk segregant analysis of yeast sequencing data

Quantitative Trait Loci (QTL) mapping using bulk segregants is an effective approach for identifying genetic variants associated with phenotypes of interest in model organisms. By exploiting next-generation sequencing technology, the QTL mapping accuracy can be improved significantly, providing a valuable means to annotate new genetic variants. However, setting up a comprehensive analysis framework for this purpose is a time-consuming and error prone task, posing many challenges for scientists with limited experience in this domain. Findings: Here, we present BSA4Yeast, a comprehensive web-application for QTL mapping via bulk segregant analysis of yeast sequencing data. The software provides an automated and efficiency-optimized data processing, up-to-date functional annotations, and an interactive web-interface to explore identified QTLs. Conclusion: BSA4Yeast enables researchers to identify plausible candidate genes in QTL regions efficiently in order to validate their genetic variations experimentally as causative for a phenotype of interest. BSA4Yeast is freely available at https://bsa4yeast.lcsb.uni.lu

Millifluidic culture improves human midbrain organoid vitality and differentiation

Human midbrain-specific organoids (hMOs) serve as an experimental in vitro model for studying the pathogenesis of Parkinson's disease (PD). In hMOs, neuroepithelial stem cells (NESCs) give rise to functional midbrain dopaminergic (mDA) neurons that are selectively degenerating during PD. A limitation of the hMO model is an under-supply of oxygen and nutrients to the densely packed core region, which leads eventually to a "dead core". To reduce this phenomenon, we applied a millifluidic culture system that ensures media supply by continuous laminar flow. We developed a computational model of oxygen transport and consumption in order to predict oxygen levels within the hMOs. The modelling predicts higher oxygen levels in the hMO core region under millifluidic conditions. In agreement with the computational model, a significantly smaller "dead core" was observed in hMOs cultured in a bioreactor system compared to those ones kept under conventional shaking conditions. Comparing the necrotic core regions in the organoids with those obtained from the model allowed an estimation of the critical oxygen concentration necessary for ensuring cell vitality. Besides the reduced "dead core" size, the differentiation efficiency from NESCs to mDA neurons was elevated in hMOs exposed to medium flow. Increased differentiation involved a metabolic maturation process that was further developed in the millifluidic culture. Overall, bioreactor conditions that improve hMO quality are worth considering in the context of advanced PD modelling

Natural variation of chronological aging in the Saccharomyces cerevisiae species reveals diet-dependent mechanisms of life span control

Aging is a complex trait of broad scientific interest, especially because of its intrinsic link with common human diseases. Pioneering work on aging-related mechanisms has been made in Saccharomyces cerevisiae, mainly through the use of deletion collections isogenic to the S288c reference strain. In this study, using a recently published high-throughput approach, we quantified chronological life span (CLS) within a collection of 58 natural strains across seven different conditions. We observed a broad aging variability suggesting the implication of diverse genetic and environmental factors in chronological aging control. Two major Quantitative Trait Loci (QTLs) were identified within a biparental population obtained by crossing two natural isolates with contrasting aging behavior. Detection of these QTLs was dependent upon the nature and concentration of the carbon sources available for growth. In the first QTL, the RIM15 gene was identified as major regulator of aging under low glucose condition, lending further support to the importance of nutrient-sensing pathways in longevity control under calorie restriction. In the second QTL, we could show that the SER1 gene, encoding a conserved aminotransferase of the serine synthesis pathway not previously linked to aging, is causally associated with CLS regulation, especially under high glucose condition. These findings hint toward a new mechanism of life span control involving a trade-off between serine synthesis and aging, most likely through modulation of acetate and trehalose metabolism. More generally it shows that genetic linkage studies across natural strains represent a promising strategy to further unravel the molecular basis of aging

Connecting environmental exposure and neurodegeneration using cheminformatics and high resolution mass spectrometry: potential and challenges

Connecting chemical exposures over a lifetime to complex chronic diseases with multifactorial causes such as neurodegenerative diseases is an immense challenge requiring a long-term, interdisciplinary approach. Rapid developments in analytical and data technologies, such as non-target high resolution mass spectrometry (NT-HR-MS), have opened up new possibilities to accomplish this, inconceivable 20 years ago. While NT-HR-MS is being applied to increasingly complex research questions, there are still many unidentified chemicals and uncertainties in linking exposures to human health outcomes and environmental impacts. In this perspective, we explore the possibilities and challenges involved in using cheminformatics and NT-HR-MS to answer complex questions that cross many scientific disciplines, taking the identification of potential (small molecule) neurotoxicants in environmental or biological matrices as a case study. We explore capturing literature knowledge and patient exposure information in a form amenable to high-throughput data mining, and the related cheminformatic challenges. We then briefly cover which sample matrices are available, which method(s) could potentially be used to detect these chemicals in various matrices and what remains beyond the reach of NT-HR-MS. We touch on the potential for biological validation systems to contribute to mechanistic understanding of observations and explore which sampling and data archiving strategies may be required to form an accurate, sustained picture of small molecule signatures on extensive cohorts of patients with chronic neurodegenerative disorders. Finally, we reflect on how NT-HR-MS can support unravelling the contribution of the environment to complex diseases

Examining the growth and stable isotopes of phytoplankton and periphyton communities exposed to oil sands reclamation strategies

The impacts of oil sands processed materials (OSPM) on phytoplankton and periphyton community growth and stable carbon and nitrogen isotopes were examined. Estimates of plankton and periphyton community growth, measured as chl a and dry weight, were low and similar in reference and OSPM reclamation wetlands. The use of stable isotope analyses revealed higher δ15N of plankton and periphyton in OSPM wetlands than reference wetlands, possibly due to increased TN concentrations in some OSPM wetlands. In the laboratory, water-soluble fractions (WSF) of two types of OSPM (mature fine tailings, MFT and consolidated tailings, CT) and an amendment material (peat-mineral mixture), potential fill materials in wetland or end pit lake reclamation, were examined for phytoplankton community growth and stable carbon and nitrogen isotopes. All WSF treatments had higher chl a compared to reference water and maximum growth was observed at a 50:50 ratio of peat:CT or peat:MFT. In general, WSFs of peat had the highest concentration of total nitrogen (TN) whereas WSFs of MFT had the highest total phosphorus (TP; 3x higher). The results suggested that the addition of peat as an amendment to OSPM (particularly for MFT), contributing additional TN, could improve phytoplankton community growth in oil sands reclamation. At higher percentages of MFT WSF, there was increased turbidity due to fine clay particles that likely contributed to reduced phytoplankton growth. Turbidity could be an important factor limiting phytoplankton growth and thus reducing dietary resources and biological detritus (via sedimentation) in the initial development of an end pit lake. The WSFs also promoted the unfavourable growth of filamentous algae, highest at intermediate concentrations of peat and CT WSFs and inhibited in MFT WSFs due to light limitation. Stable N isotopes of plankton and filamentous algae suggests that 15N enrichment of algae could be a useful indicator of nutrient inputs, including OSPM seepage into natural aquatic systems, for oil sands regional monitoring programs

2-Hydroxyglutarate modulates histone methylation at specific loci and alters gene expression via Rph1 inhibition.

peer reviewed2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target

Regulation of N6-Methyladenosine after Myocardial Infarction

Development of heart failure (HF) after myocardial infarction (MI) is responsible for premature death. Complex cellular and molecular mechanisms are involved in this process. A number of studies have linked the epitranscriptomic RNA modification N6-methyladenosine (m6A) with HF, but it remains unknown how m6A affects the risk of developing HF after MI. We addressed the regulation of m6A and its demethylase fat mass and obesity-associated (FTO) after MI and their association with HF. Using liquid chromatography coupled to mass spectrometry, we observed an increase of m6A content in the infarcted area of rat hearts subjected to coronary ligation and a decrease in blood. FTO expression measured by quantitative PCR was downregulated in the infarcted hearts. In whole blood samples collected at the time of reperfusion in MI patients, m6A content was lower in patients who developed HF as attested by a 4-month ejection fraction (EF) of ≤40 as compared to patients who did not develop HF (EF \textgreater 50\%). M6A content was higher in females. These results show that m6A measured in blood is associated with HF development after MI and motivate further investigation of the potential role of m6A as a novel epitranscriptomics biomarker and therapeutic target of HF

Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione

The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable “repair enzyme” to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria