Characterization of three 2-hydroxy-acid dehydrogenases in the context of a biotechnological approach to short-circuit photorespiration

Photorespiration results from the incorporation of oxygen into ribulose-1,5-bisphosphate due to the failure of RuBisCO to properly discriminate between oxygen and carbon dioxide. This process lowers photosynthetic efficiency in that CO2 and ammonia should be re-assimilated with the concomitant consumption of both ATP and reducing power. Two recent approaches, aimed at decreasing the detrimental effects of photorespiration by introducing novel metabolic pathways into plant chloroplasts, show great promise. The goal of this work was to identify and biochemically characterize a single-gene glycolate dehydrogenase for use in further improving the synthetic pathways. Forward and reverse genetics were used to identify three candidate genes in Arabidopsis thaliana; At5g06580, At4g36400 and At4g18360. The proteins encoded by these genes were expressed in Escherichia coli, purified and characterized. Moreover, in silico analysis and the analysis of loss-of-function mutants yielded insights into the significance of these novel enzymatic activities in plant metabolism. AtD-LDH, encoded by At5g06580, is a homodimeric FAD-binding flavoprotein that catalyzes the cytochrome c- dependent oxidation of substrates. The enzyme has high activity with D- and L-lactate, D-2-hydroxybutyrate and D-glycerate, but of these only D-lactate and D-2-hydroxybutyrate are bound with high affinity. Knock-out mutants show impaired growth on medium containing methylglyoxal and D-lactate. Together, the data indicates a role for AtD-LDH in the mitochondrial intermembrane space where it oxidizes D-lactate to pyruvate in the final step of methylglyoxal detoxification. AtD-2HGDH, encoded by At4g36400, is a homodimeric FAD-binding flavoprotein. The enzyme only has activity with D-2-hydroxyglutarate and uses a synthetic electron acceptor in vitro. Metabolic analysis of knock-out mutants reveals high accumulation of D-2-hydroxyglutarate in plants exposed to long periods of extended darkness, confirming that this is the in vivo substrate for the enzyme. Co-expression analysis reveals that AtD-2HGDH is co-expressed with enzymes and transporters participating in the breakdown of lipids, branched-chain amino acids and chlorophyll, all pathways that converge in the production of propionyl-CoA. Together, the data suggest a role for AtD-2HGDH in the mitochondrial matrix where it oxidizes D-2-hydroxyglutarate, most probably originating from propionyl-CoA metabolism, to 2-oxoglutarate, using an electron transfer flavoprotein as an electron acceptor. Finally, AtGOX3, encoded by At4g18360, is a peroxisomal (S)-2-hydroxy-acid oxidase with specificity towards glycolate, L-lactate and L-2-hydroxybutyrate. AtGOX3 is almost exclusively expressed in roots where it might participate in either the metabolism of L-lactate produced during hypoxia, or glycolate produced from glycolaldehyde. In this work, the identification and thorough characterization of three novel enzymatic activities in the model plant A. thaliana are described. Moreover, novel plant metabolic pathways in which these enzymes participate were discovered. The biochemical characterization of these enzymes indicated that they are not suited for use in pathways aimed at decreasing photorespiration and thus, the search for a single-gene glycolate dehydrogenase should continue

Cell lineage tracing in the human pancreas

Background and aims: The human pancreas is normally proliferatively quiescent; however loss of cells gives a rapid regenerative response to restore a functionally healthy tissue mass. The mechanism for this is unclear and to date research has failed to identify a definitive pancreatic stem cell. Cell lineage analyses are possible in animals by labelling cells with radioactive tags or genetic markers, but this is not feasible in humans. Current human studies rely on stem cell markers, thus there is a need to develop a better technique to track, isolate and identify cells of related origin. Mitochondrial DNA mutations amass naturally within our cells as we age. Therefore, cells formed by division of a stem cell carrying mutated mitochondrial DNA should, given a qualitative assay, permit the progeny of this cell to be traced. Methods and results: Mitochondrial DNA mutations often result in a defect in the mitochondrial DNA-encoded protein cytochrome c oxidase, and can be identified using a histochemical stain. Individual mutant patches were studied by staining serial sections of normal pancreas. This revealed the presence of clonal units of cells, strong evidence that pancreatic cells within each patch are derived from a common progenitor. We tried to show that these cells are normal in synthetic and proliferative function, although this showed that the mitochondrial DNA mutated cells didn't have normal synthetic function. However this did not negate our evidence of clonal proliferation. Conclusion: This novel means of tracing patterns of cell division and migration in human tissues is suitable for finding how cells proliferate and move in the human pancreas, and with continued work it might be useful as a marker for other changes e.g. pre-cancerous lesions.Cellerna i bukspottkörteln hos människa är normalt inte proliferativa, om dessa celler skadas är regenerationen snabb. Den bakomliggande mekanismen är inte känd men det innebär att pankreas funktion bibehålls. Forskning har ännu inte kunnat identifiera en definitiv stamcell i pankreas. I djurförsök är det möjligt att använda radioaktiva eller genetiska markörer för att följa cellinjer, vilket inte är möjligt i humanforskning utan där används istället stamcellsmarkörer. Det finns därför ett behov att utveckla bättre teknik för att kunna följa, isolera och identifiera celler av samma ursprung (stamceller). Kvalitativ teknik skulle kunna möjliggöra kartläggning av ursprunget till celler, vilka härstammar från stamceller med mutationer, eftersom mutationer av mitokondriell-DNA ackumuleras natruligt i våra celler allt eftersom vi åldras. Eftersom proteinet cytokrom c oxidas kodas från mitokondriellt-DNA uppstår ofta defekter i cytokrom c oxidas om DNA:t är muterat. I den redovisade studien undersöktes muterade områden i normal pankreas där klonala grupper med celler kunde påvisas med hjälp av immunohistokemi avseende förekomsten av cytokrom c oxidas. Dessa grupper av celler har troligtvis samma ursprung. I studien försökte vi visa att dessa celler även uppvisade normal funktion, även vad gäller proliferationskapacitet. Undersökningen visade dock att celler med muterat mitokondriellt-DNA inte har normal funktion. Tekniken att spåra cellers delning och rörelse i human vävnad är användbar för att följa hur celler prolifererar och förflyttar sig i pankreas. Fortsatta studier inom området skulle kunna identifiera markörer för att påvisa förändringar i pankreasceller, som exempelvis förstadier till cancer

Mechanical behaviour of glassy polymers: experiments and modelling

This thesis presents experimental investigation and modelling of the mechanical response of glassy polycarbonate (PC) during deformation. The mechanical response is studied experimentally over a wide range of length-scales using X-ray scattering techniques and optical full-field deformation measurement by Digital Image Correlation (DIC). Results from the experimental work have been used to develop an elasto-viscoplastic model for glassy polymers. The thesis includes an introductory section on glassy polymers, aspects of the experimental procedures and a summary of the key aspects of the constitutive modelling, and four papers.An experimental method combining X-ray scattering, full-field DIC and tensile loading has been developed and used within this thesis. Details about the experimental method are presented in Paper A. By combining the, individually well established, experimental techniques, the deformation of a material can be studied simultaneously over a wide range of length-scales, from the macroscopic response down to the behaviour of the molecular structure. Results from experiments performed using the developed method are also presented in Paper B. Novel observations of the deformation and reorientation of the microstructure of glassy PC are presented and related to relevant local macroscopic measures of deformation.The experimental results presented in Paper B have been used to develop a constitutive model for glassy polymers in Paper C. A separate microstructural deformation gradient is introduced to model the deformation of the polymer network. Moreover, the reorientation of the microstructure, shown in Paper B, is introduced by an evolution of the directions of the network chains. By incorporating the evolving reorientation and the deformation of the microstructure shown by the experiments, the model is able to capture the deformation at the macroscopic, the mesoscopic and the microscopic levels. In Paper D, the mechanical behaviour of glassy PC is studied using biaxial tension loading and DIC. The experiments performed in Paper D show a significant influence of the multi-axial loading on the localisation behaviour. It is also found that the commonly used quadratic form of the elastic free energy results in a too stiff initial response during biaxial loading. To this end, a new format for the volumetric part of the elastic free energy is proposed which results in a softer response with increasing volumetric deformation. The proposed format also improves the ability to capture the non-linear, pre-peak behaviour exhibited by PC

Långsiktig hållbarhet i det urbana trädbeståndet vid klimatförändringar

Sommaren 2018 drabbades Sverige av den längsta värmebölja som uppmätts sedan 1941. Klimatförändringarna höjer den globala medeltemperaturen och värmeböljorna förväntas bli fler och längre för varje år. De urbana miljöerna växer och därav blir de hårdgjorda ytorna fler. Hårdgjorda ytor lagrar värme vilket gör att temperaturen höjs lokalt och värmeöar skapas. De förhöjda temperaturerna bidrar till att de inhemska trädarterna kan få försämrad vitalitet samt minskad eller ingen leverans av ekosystemtjänster då de är vana vid en sval och fuktig naturmiljö. För att skapa långsiktig hållbarhet behövs välmående träd som är anpassade till ståndorten i den urbana miljön, detta för att skapa resilienta trädbestånd som kan leverera ekosystemtjänster samt bevara den biologiska mångfalden. Arbetets syfte är att undersöka risker och möjligheter med icke inhemska trädarter i den svenska urbana miljön kopplat till de pågående klimatförändringarna. För att svara på syftet har en enkätstudie samt en intervjustudie genomförts. Enkätstudien omfattar 18 svenska kommuner och intervjustudien sex personer med kunskap inom trädfrågor. Informanterna i studierna värnar om att använda inhemska trädarter på ståndorter där de kan trivas i den urbana miljön, framför allt för att gynna den biologiska mångfalden. De belyser även vikten av att använda icke inhemska trädarter i den urbana miljön på de ståndorter som är utmanande för de inhemska träden. Det finns dock en viss rädsla kopplat till import av icke inhemska träd, som införsel av sjukdomar och skadedjur samt eventuell invasivitet. För att träd ska kunna leverera maximalt med ekosystemtjänster behöver de en hög vitalitet samt många år att växa till sig, vilket är en process som kräver långsiktig planering, kunskap och ekonomiska resurser. Av resultatet går att utläsa att informanterna framhåller vikten av samarbeten mellan olika aktörer och yrkesgrupper för att få en bred kunskap inom ämnet.In the summer of 2018, Sweden was affected by the longest heat wave measured since 1941. Climate change raises the global average temperature and the heat waves are expected to be more common every year and last for a long time. The urban environments are growing and as a result, the areas with hardened surfaces are increasing. Hardened surfaces store heat which results in locally increased temperatures that create heat islands. The elevated temperatures contribute to reduced vitality in native tree species as well as reduced or no delivery of ecosystem services as they are used to cool and humid natural environments. To create long-term sustainability, healthy trees that are adapted to the location in the urban environment are needed. This creates resilient tree stands that can deliver ecosystem services and preserve biodiversity. The purpose of this work is to investigate risks and opportunities with the use of non-native tree species in the Swedish urban environment; taking into consideration actual climate change. To answer the purpose, a survey study and an interview study were conducted. The survey study includes 32 Swedish municipalities and the interview study includes six informants who have knowledge in tree issues. The informants in the studies emphasize the importance of using native tree species in sites where they can thrive in the urban environment and benefit biological diversity. They also highlight the importance of using non-native tree species in the urban environment in the sites that are challenging for the native trees. There is a certain fear associated with non-native trees such as the introduction of diseases, pests and possible invasiveness. In order for trees to be able to deliver maximum ecosystem services, they need high vitality and many years to grow which is a process that requires long-term planning, knowledge and financial resources. The results show that several informants emphasize the importance of collaboration between different actors and professional groups in order to gain broad knowledge on the subject

Highlighting the Need for Systems-Level Experimental Characterization of Plant Metabolic Enzymes

The biology of living organisms is determined by the action and interaction of a large number of individual gene products, each with specific functions. Discovering and annotating the function of gene products is key to our understanding of these organisms. Controlled experiments and bioinformatic predictions both contribute to functional gene annotation. For most species it is difficult to gain an overview of what portion of gene annotations are based on experiments and what portion represent predictions. Here, I survey the current state of experimental knowledge of enzymes and metabolism in Arabidopsis thaliana as well as eleven economically important crops and forestry trees – with a particular focus on reactions involving organic acids in central metabolism. I illustrate the limited availability of experimental data for functional annotation of enzymes in most of these species. Many enzymes involved in metabolism of citrate, malate, fumarate, lactate, and glycolate in crops and forestry trees have not been characterized. Furthermore, enzymes involved in key biosynthetic pathways which shape important traits in crops and forestry trees have not been characterized. I argue for the development of novel high-throughput platforms with which limited functional characterization of gene products can be performed quickly and relatively cheaply. I refer to this approach as systems-level experimental characterization. The data collected from such platforms would form a layer intermediate between bioinformatic gene function predictions and in-depth experimental studies of these functions. Such a data layer would greatly aid in the pursuit of understanding a multiplicity of biological processes in living organisms

Inaccurate mimicry and predator ecology

Reinhold K, Engqvist L. Inaccurate mimicry and predator ecology. Journal of Theoretical Biology. 2004;229(4):433-434

Experimental and computational investigation of enzyme functional annotations uncovers misannotation in the EC 1.1.3.15 enzyme class

Only a small fraction of genes deposited to databases have been experimentally characterised. The majority of proteins have their function assigned automatically, which can result in erroneous annotations. The reliability of current annotations in public databases is largely unknown; experimental attempts to validate the accuracy within individual enzyme classes are lacking. In this study we performed an overview of functional annotations to the BRENDA enzyme database. We first applied a high-throughput experimental platform to verify functional annotations to an enzyme class of S-2-hydroxyacid oxidases (EC 1.1.3.15). We chose 122 representative sequences of the class and screened them for their predicted function. Based on the experimental results, predicted domain architecture and similarity to previously characterised S-2-hydroxyacid oxidases, we inferred that at least 78% of sequences in the enzyme class are misannotated. We experimentally confirmed four alternative activities among the misannotated sequences and showed that misannotation in the enzyme class increased over time. Finally, we performed a computational analysis of annotations to all enzyme classes in the BRENDA database, and showed that nearly 18% of all sequences are annotated to an enzyme class while sharing no similarity or domain architecture to experimentally characterised representatives. We showed that even well-studied enzyme classes of industrial relevance are affected by the problem of functional misannotation. Copyright

Adaptation of a Microfluidic qPCR System for Enzyme Kinetic Studies

Microfluidic platforms offer a drastic increase in throughput while minimizing sample usage and hands-on time, which make them important tools for large-scale biological studies. A range of such systems have been developed for enzyme activity studies, although their complexity largely hinders their application to the wider scientific community. Here, we present adaptation of an easy-to-use commercial microfluidic qPCR system for performing enzyme kinetic studies. We demonstrate the functionality of the Fluidigm Biomark HD system (the Fluidigm system) by determining the kinetic properties of three oxidases in a resorufin-based fluorescence assay. The results obtained in the microfluidic system proved reproducible and comparable to the ones obtained in a standard microplate-based assay. With a wide range of easy-to-use, off-the-shelf components, the microfluidic system presents itself as a simple and customizable platform for high-throughput enzyme activity studies