Extension of Yeast Chronological Lifespan by Methylamine

Background: Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast Hansenula polymorpha grown at conditions that require primary peroxisome metabolism for growth. Methodology/Principal Findings: The chronological lifespan of H. polymorpha is strongly enhanced when cells are grown on methanol or ethanol, metabolized by peroxisome enzymes, relative to growth on glucose that does not require peroxisomes. The short lifespan of H. polymorpha on glucose is mainly due to medium acidification, whereas most likely ROS do not play an important role. Growth of cells on methanol/methylamine instead of methanol/ammonium sulphate resulted in further lifespan enhancement. This was unrelated to medium acidification. We show that oxidation of methylamine by peroxisomal amine oxidase at carbon starvation conditions is responsible for lifespan extension. The methylamine oxidation product formaldehyde is further oxidized resulting in NADH generation, which contributes to increased ATP generation and reduction of ROS levels in the stationary phase. Conclusion/Significance: We conclude that primary peroxisome metabolism enhanced chronological lifespan of H. polymorpha. Moreover, the possibility to generate NADH at carbon starvation conditions by an organic nitrogen source supports further extension of the lifespan of the cell. Consequently, the interpretation of CLS analyses in yeast should include possible effects on the energy status of the cell.

Dietary Restriction Depends on Nutrient Composition to Extend Chronological Lifespan in Budding Yeast Saccharomyces cerevisiae

10.1371/journal.pone.0064448PLoS ONE85

A Microarray-Based Genetic Screen for Yeast Chronological Aging Factors

Model organisms have played an important role in the elucidation of multiple genes and cellular processes that regulate aging. In this study we utilized the budding yeast, Saccharomyces cerevisiae, in a large-scale screen for genes that function in the regulation of chronological lifespan, which is defined by the number of days that non-dividing cells remain viable. A pooled collection of viable haploid gene deletion mutants, each tagged with unique identifying DNA “bar-code” sequences was chronologically aged in liquid culture. Viable mutants in the aging population were selected at several time points and then detected using a microarray DNA hybridization technique that quantifies abundance of the barcode tags. Multiple short- and long-lived mutants were identified using this approach. Among the confirmed short-lived mutants were those defective for autophagy, indicating a key requirement for the recycling of cellular organelles in longevity. Defects in autophagy also prevented lifespan extension induced by limitation of amino acids in the growth media. Among the confirmed long-lived mutants were those defective in the highly conserved de novo purine biosynthesis pathway (the ADE genes), which ultimately produces IMP and AMP. Blocking this pathway extended lifespan to the same degree as calorie (glucose) restriction. A recently discovered cell-extrinsic mechanism of chronological aging involving acetic acid secretion and toxicity was suppressed in a long-lived ade4Δ mutant and exacerbated by a short-lived atg16Δ autophagy mutant. The identification of multiple novel effectors of yeast chronological lifespan will greatly aid in the elucidation of mechanisms that cells and organisms utilize in slowing down the aging process

Rule-Based Cell Systems Model of Aging using Feedback Loop Motifs Mediated by Stress Responses

Investigating the complex systems dynamics of the aging process requires integration of a broad range of cellular processes describing damage and functional decline co-existing with adaptive and protective regulatory mechanisms. We evolve an integrated generic cell network to represent the connectivity of key cellular mechanisms structured into positive and negative feedback loop motifs centrally important for aging. The conceptual network is casted into a fuzzy-logic, hybrid-intelligent framework based on interaction rules assembled from a priori knowledge. Based upon a classical homeostatic representation of cellular energy metabolism, we first demonstrate how positive-feedback loops accelerate damage and decline consistent with a vicious cycle. This model is iteratively extended towards an adaptive response model by incorporating protective negative-feedback loop circuits. Time-lapse simulations of the adaptive response model uncover how transcriptional and translational changes, mediated by stress sensors NF-κB and mTOR, counteract accumulating damage and dysfunction by modulating mitochondrial respiration, metabolic fluxes, biosynthesis, and autophagy, crucial for cellular survival. The model allows consideration of lifespan optimization scenarios with respect to fitness criteria using a sensitivity analysis. Our work establishes a novel extendable and scalable computational approach capable to connect tractable molecular mechanisms with cellular network dynamics underlying the emerging aging phenotype

Genome-Wide Screen in Saccharomyces cerevisiae Identifies Vacuolar Protein Sorting, Autophagy, Biosynthetic, and tRNA Methylation Genes Involved in Life Span Regulation

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved

A morphogenetic EphB/EphrinB code controls hepatopancreatic duct formation

© 2019 The Authors. Published by Springer. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1038/s41467-019-13149-7The hepatopancreatic ductal (HPD) system connects the intrahepatic and intrapancreatic ducts to the intestine and ensures the afferent transport of the bile and pancreatic enzymes. Yet the molecular and cellular mechanisms controlling their differentiation and morphogenesis into a functional ductal system are poorly understood. Here, we characterize HPD system morphogenesis by high-resolution microscopy in zebrafish. The HPD system differentiates from a rod of unpolarized cells into mature ducts by de novo lumen formation in a dynamic multi-step process. The remodeling step from multiple nascent lumina into a single lumen requires active cell intercalation and myosin contractility. We identify key functions for EphB/EphrinB signaling in this dynamic remodeling step. Two EphrinB ligands, EphrinB1 and EphrinB2a, and two EphB receptors, EphB3b and EphB4a, control HPD morphogenesis by remodeling individual ductal compartments, and thereby coordinate the morphogenesis of this multi-compartment ductal system.This work was funded by the Novo Nordisk Foundation (NNF17CC0027852) and Danish National Research Foundation (DNRF116). J.C. and D.G.W. were supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001217), the UK Medical Research Council (FC001217), and the Wellcome Trust (FC001217). S.C. was supported by an SNSF Early Postdoc Mobility fellowship (P2ZHP3_164840) and a Long Term EMBO Postdoc fellowship (ALTF 511-2016), and L.S. and J.B.A. by the Independent Research Fund Denmark (DFF; Sapere Aude2 4183-00118B).Published versio

Single continuous lumen formation in the zebrafish gut is mediated by smoothened-dependent tissue remodeling.

The formation of a single lumen during tubulogenesis is crucial for the development and function of many organs. Although 3D cell culture models have identified molecular mechanisms controlling lumen formation in vitro, their function during vertebrate organogenesis is poorly understood. Using light sheet microscopy and genetic approaches we have investigated single lumen formation in the zebrafish gut. Here we show that during gut development multiple lumens open and enlarge to generate a distinct intermediate, which consists of two adjacent unfused lumens separated by basolateral contacts. We observed that these lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate into a single, continuous lumen requires the remodeling of contacts between adjacent lumens and subsequent lumen fusion. We show that lumen resolution, but not lumen opening, is impaired in smoothened (smo) mutants, indicating that fluid-driven lumen enlargement and resolution are two distinct processes. Furthermore, we show that smo mutants exhibit perturbations in the Rab11 trafficking pathway and demonstrate that Rab11-mediated trafficking is necessary for single lumen formation. Thus, lumen resolution is a distinct genetically controlled process crucial for single, continuous lumen formation in the zebrafish gut

An Ontology to represent Knowledge on Animal Testing Alternatives

EU Directive 86/609/EEC for the protection of laboratory animals obliges scientists toconsider whether a planned animal experiment can be replaced, reduced or refined (3Rsprinciple). To meet this regulatory obligation, scientists must consult the relevant scientificliterature prior to any experimental study using laboratory animals. More than 50 millionpotentially 3Rs relevant documents are spread over the World Wide Web, biomedicalliterature and patent databases. In April 2008, the beta version of Go3R (www.Go3R.org),the first knowledge-based semantic search engine for alternative methods to animalexperiments, was released. Go3R is free of charge and enables scientists and regulatoryauthorities involved in the planning, authorisation and performance of animal experiments todetermine the availability of alternative methods in a fast and comprehensive manner.The technical basis of this search engine is specific 3Rs expert knowledge captured withinthe Go3R Ontology containing 87,218 labels and synonyms. A total of 16,620 concepts werestructured in 28 branches, where 1,227 concepts were newly defined to specifically describedirectly 3Rs relevant knowledge. Additionally relevant headings from MeSH wherereferenced to reflect the topics associated with the definition of Animal Testing Alternatives.Therefore it is distinguished between thematic-defining and directly 3Rs relevant branches.In addition to the assignment of direct parent-child relationships, further relationship typeswere introduced to allow to model 3Rs relevant domain knowledge. Examples for suchknowledge are e.g. (1) the characteristics of cell culture tests methods, which usually utilize"specific cell types" or "cell lines" and are associated with a specific "endpoint" and "endpointdetection method" or (2) named test methods like "PREDISAFE TM", which replaces ananimal test namely the "eye irritation test" in rabbits and uses specific cells namely "SIRCCells" or (3) the "Haemagglutinin-Neuraminidase Protein Assay", which detects a protein ofthe "Newcastle disease virus".Thereby, an article in which e.g. a specific 3Rs method is not explicitly mentioned could stillbe recognized as relevant for the specific topic searched for in an indirect manner, forexample if it mentions specific cells, endpoints or endpoint detection methods, which arerelevant for the respective application.The search engine Go3R with its novel ontology is already well recognized by the3Rs community and will be further maintained and developed.Methods: A platinum nanoparticle aerosol was generated using a spark discharge generatorin nitrogen as the carrier gas. The Pt NP aerosol was diluted by the factor of 10 withsynthetic air directly after the generation process. The aerosol was directed to the KarlsruheExposure system (Paur et al., 2008; Diabaté et al., 2008) to analyze the toxicologicalpotential of the freshly generated Pt NP aerosol.For the bioassay we employed the human alveolar epithelial cells A549 and the bronchialepithelial cells BEAS-2B, which was co-cultured with differentiated THP-1 macrophages,growing on Transwell inserts. The responses of the cells were analyzed by measuringviability (AlamarBlue assay), release of lactate dehydrogenase (LDH) as an indicator ofmembrane integrity, induction of heme oxygenase-1 (HO-1) as an indicator of an antioxidativeresponse and release of Interleukin-8 (IL-8) as an indicator of a pro-inflammatoryresponse. Additionally, Pt NPs collected on polycarbonate filters (pore diameter 0.4 ìm) wereuse