41 research outputs found

    Millifluidic culture improves human midbrain organoid vitality and differentiation

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    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

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    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

    A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level

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    Grünberger A, Paczia N, Probst C, et al. A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level. Lab on a Chip. 2012;12(11):2060-2068

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

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    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

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    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.

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    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

    Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione

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    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

    Metabolomanalyse von Mikroorganismen : Methodenetablierung und Konzepte zur Automatisierung

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    Paczia N. Metabolomanalyse von Mikroorganismen : Methodenetablierung und Konzepte zur Automatisierung. Bielefeld: Universität Bielefeld; 2012.Die vorliegende Arbeit behandelt die Untersuchung und Optimierung der Probengenerierung zur quantitativen Metabolomanalyse von Mikroorganismen. Durch eine detaillierte Betrachtung aller Arbeitsschritte wie Biomassebestimmung, Probenahme, Quenching, Zellseparation, Analytik und Datenprozessierung konnten alle Einflussgrößen des Gesamtverfahrens charakterisiert werden. Insbesondere konnte gezeigt werden, dass die wesentlichen Grundlagen einer belastbaren Metabolomanalyse die Berücksichtigung der Heterogenität von bakteriellen Populationen und die differenzierte Erfassung des Biovolumens als Referenzgröße sind. Zudem belegt die vorliegende Arbeit, dass im Verlauf von Batch-Kultivierungen teilweise hohe extrazelluläre Metabolitkonzentrationen auftreten und damit ein simultanes Quenching nur in ausgewählten Fällen anwendbar ist. Darüber hinaus wurde nachgewiesen, dass die Messmatrix bei der Durchführung von LC-ESI-MS/MS-Messungen auch unter Verwendung interner Standards einen erheblichen Einfluss besitzt. Auf Basis eines umfangreichen Methodenvergleichs für die Durchführung jedes Teilschrittes und der daraus resultierenden Optimierung ausgewählter Einzelmethoden konnte ein Standardprozess zur Metabolomanalyse etabliert werden. Die Funktionalität des Standardprozesses wurde anhand einer vergleichenden Metabolomanalyse von vier Corynebacterium glutamicum-Stämmen erfolgreich nachgewiesen. Abschließend konnte ein Konzept zur automatisierten Anwendung des optimierten Protokolls erstellt werden. Für die Arbeitsschritte Quenching und Zellseparation wurden dazu eigene technische Lösungen entwickelt und umgesetzt

    Error Propagation Analysis for Quantitative Intracellular Metabolomics

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    Model-based analyses have become an integral part of modern metabolic engineering and systems biology in order to gain knowledge about complex and not directly observable cellular processes. For quantitative analyses, not only experimental data, but also measurement errors, play a crucial role. The total measurement error of any analytical protocol is the result of an accumulation of single errors introduced by several processing steps. Here, we present a framework for the quantification of intracellular metabolites, including error propagation during metabolome sample processing. Focusing on one specific protocol, we comprehensively investigate all currently known and accessible factors that ultimately impact the accuracy of intracellular metabolite concentration data. All intermediate steps are modeled, and their uncertainty with respect to the final concentration data is rigorously quantified. Finally, on the basis of a comprehensive metabolome dataset of Corynebacterium glutamicum, an integrated error propagation analysis for all parts of the model is conducted, and the most critical steps for intracellular metabolite quantification are detected
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