570 Biowissenschaften; Biologie
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- Korrelationsanalyse (2)
- Körperfett (2)
- Körperzusammensetzung (2)
- LC–MS/MS (2)
- LEA protein (2)
- LEM-domain protein (2)
- LMA (2)
- LPS (2)
- Lake (2)
- Lamin (2)
- Land use (2)
- Land-use history (2)
- Landscape (2)
- Landscape of fear (2)
- Landschaft der Angst (2)
- Large fragment deletion (2)
- Late Quaternary vegetation (2)
- Leaf senescence (2)
- Leopards (2)
- Limnology (2)
- Lipases (2)
- Lipidomics (2)
- Lysophosphatidylcholine (2)
- Lythrum salicaria (2)
- MADS-domain transcription factor (2)
- MAPK (2)
- MED16 (2)
- MHC (2)
- MSAP (2)
- Manganese (2)
- Markov cluster algorithm (2)
- Martini force-field (2)
- Mate choice (2)
- Maus (2)
- Mechanotransduction (2)
- Medicago truncatula (2)
- Meiosis (2)
- Melampyrum pratense (2)
- Membranproteine (2)
- Mesenchymal stem cells (2)
- Metabolismus (2)
- Metabolit (2)
- Metabolite (2)
- Metaboliten (2)
- Metabolites (2)
- Metabolom (2)
- Metabolomik (2)
- Metal Metabolism (2)
- Metschnikowia (2)
- Microarrays (2)
- Microbial ecology (2)
- Microbiology (2)
- Microcystin (2)
- Microcystis aeruginosa (2)
- Microsatellite analysis (2)
- Mikroorganismen (2)
- Mikrosatelliten (2)
- Mindfulness (2)
- Mitochondrial DNA (2)
- Mitochondrien (2)
- Mitogenome (2)
- Mitogenomes (2)
- Mitose (2)
- Mitosis (2)
- Mixed mating (2)
- Mixed methods (2)
- Modell (2)
- Molekularbiologie (2)
- Molybdänkofaktor (2)
- Monoclonal antibodies (2)
- Monoclonal antibody (2)
- Monte Carlo method (2)
- Morphology (2)
- Multiplex (2)
- Multiplex mutagenesis (2)
- Muntjac (2)
- Mutation rate (2)
- Mutator locus (2)
- Mykotoxine (2)
- NAB (2)
- NE Germany (2)
- NMR spectroscopy (2)
- NMR structure (2)
- NRPS (2)
- NZO (2)
- Nahrungsnetz (2)
- Namibia (2)
- Nanostruktur (2)
- Network clustering (2)
- Netzwerke (2)
- Neurotoxicity (2)
- Nighttime illumination (2)
- Nitrat (2)
- Nitrogen deposition (2)
- Northern Asia (2)
- Norway (2)
- Nostoc punctiforme (2)
- Nucleus (2)
- NutriAct Family Study (2)
- Nutrition (2)
- O-serotyping (2)
- ODBA (2)
- OMICs tools (2)
- Offspring weight (2)
- Ohrid-Prespa region (2)
- Older adults (2)
- Optogenetik (2)
- Organogenesis (2)
- Orthologous Matrix (OMA) Project (2)
- Outcrossing (2)
- Outcrossing rate (2)
- Outdoor enclosure (2)
- PBPK (2)
- PBS1 (2)
- PC-3 cells (2)
- PEI coating (2)
- PKS (2)
- PUFA (2)
- PXR (2)
- Palaeogenetics (2)
- Paläoökologie (2)
- Parasit (2)
- Parasitoid wasp (2)
- Partial Little Square (2)
- Partnership (2)
- Paternity analysis (2)
- Pathogen (2)
- Pathway design (2)
- Peptid (2)
- Periphyton (2)
- Periplaneta americana (2)
- Personality (2)
- Pex1 (2)
- Pex6 (2)
- Pflanze (2)
- Pflanzengemeinschaften (2)
- Pflanzenwachstum (2)
- PhIP (2)
- Phase variation (2)
- Phenole (2)
- Phosphat (2)
- Photosynthesis (2)
- Phylogenetics (2)
- Phylogenie (2)
- Phylogenomics (2)
- Pinus sylvestris (2)
- Pipistrellus nathusii (2)
- Plankton (2)
- Plant community model (2)
- Plant development (2)
- Plant-soil feedback (2)
- Plants (2)
- Plasma membrane (2)
- Plastid (2)
- Poecilia formosa (2)
- Poecilia latipinna (2)
- Polyadenylierung (2)
- Polyelektrolyt-Multischichten (2)
- Polyethylenglykol (2)
- Polysaccharide (2)
- Population dynamics (2)
- Predator-prey interactions (2)
- Pregnancy (2)
- Promiscuous enzymes (2)
- Promotor (2)
- Prostaglandin E2 (2)
- Proteinaggregation (2)
- Proteins (2)
- Pseudomonas aeruginosa (2)
- Puumala virus seroprevalence (2)
- QDs (2)
- R Shiny (2)
- RNA (2)
- RNA interference (2)
- RNA virus (2)
- RNA-guided Cas9 (2)
- RNAseq (2)
- RT-PCR (2)
- RUNX2 (2)
- Randomized-controlled trial (2)
- Raphidiopsis (2)
- Recombination (2)
- Redox marker (2)
- Redoxreaktion (2)
- Redoxregulation (2)
- Regulation (2)
- Rezeptor (2)
- Rice (2)
- Riesenvesikel (2)
- Rodent (2)
- Roridula gorgonias (2)
- Rotifera (2)
- RpoS (2)
- RubisCO (2)
- S-XRF (2)
- SELEX (2)
- SERS enhancement factor (2)
- SFON (2)
- SIMS techniques (2)
- SPB (2)
- SULT1A1 (2)
- SWL (2)
- Sailfin molly (2)
- Salmonella Typhimurium (2)
- Saprolegnia (2)
- Saprolegniaceae (2)
- Savannas (2)
- Schleudertrauma (2)
- Schmalwand <Arabidopsis> (2)
- Schwefel (2)
- Scopoletin (2)
- Seasonality (2)
- Sediment (2)
- Selenium (2)
- Senescence (2)
- Sequenzierung (2)
- Sequenzierung der nächsten Generation (2)
- Serine cycle (2)
- Sexual selection (2)
- Siberian tree line (2)
- Simulation (2)
- Simulationsmodell (2)
- Smpd1 (2)
- Soil (2)
- Soil function (2)
- Solanaceae (2)
- Space use (2)
- Specific wood density (2)
- Stickstoff (2)
- Strategic growth adjustment (2)
- Sulforaphan (2)
- Sulfotransferase (2)
- Sundaland (2)
- Sus scrofa (2)
- Säugetiere (2)
- TAS2R (2)
- TEM (2)
- TMS (2)
- TTR (2)
- Tailspike protein (2)
- Tanzania (2)
- Tas2r (2)
- Tas2rs (2)
- Temperate forest (2)
- Tenebrio molitor larvae (2)
- Tocopherol (2)
- Toll and Imd pathways (2)
- Tomate (2)
- Tomato (2)
- Transcription factors (2)
- Transcriptome assembly (2)
- Transporters (2)
- Tree allometry (2)
- Turkey (2)
- TusA (2)
- Type 2 Diabetes (2)
- UV radiation (2)
- Ulcerative colitis (2)
- Ursus arctos (2)
- V-ATPase (2)
- Venom proteins (2)
- Verhalten (2)
- Verkhoyansk mountains (2)
- Virus (2)
- Wachstum (2)
- Walker A motif (2)
- Weakly electric fish (2)
- Weizen (2)
- Weißstorch (2)
- Wildschwein (2)
- Wind (2)
- Wood specific gravity (2)
- Woody aboveground biomass (2)
- X-ray structure (2)
- XMRV (2)
- Xpr1 (2)
- Yap1/Wwtr1 (Taz) (2)
- Zebrafish (2)
- Zelladhäsion (2)
- Zellkern (2)
- Zellweger (2)
- Zellweger syndrome spectrum disorder (ZSSD) (2)
- Zinypr-1 (2)
- Zweizustandsmodell (2)
- Zytoskelett (2)
- aberrations (2)
- accelerometer (2)
- acclimation (2)
- acid mine drainage (2)
- acoustic communication (2)
- actin (2)
- actin polymerization (2)
- activated carbon (2)
- activator–inhibitor models (2)
- activity (2)
- adaptive introgression (2)
- adaptive processes (2)
- adaptive radiation (2)
- adhesion (2)
- adiponectin (2)
- age (2)
- ageing (2)
- agricultural landscape (2)
- agrin (2)
- agro-infiltration (2)
- aldehyde oxidoreductase (2)
- alignment (2)
- alignment sensitivity / specificity (2)
- alkylierte polyzyklische aromatische Kohlenwasserstoffe (2)
- alkylphospholipids (2)
- all-cause mortality (2)
- allocation (2)
- alphaherpesvirus (2)
- amino acid (2)
- animal cognition (2)
- animal migration (2)
- anthropogenic environment (2)
- anthropogenic food subsidies (2)
- anthropometric measures (2)
- anthropometry (2)
- anti-oxidative response (2)
- antibody producing cell selection (2)
- antioxidant capacity (2)
- anxiety-like behavior (2)
- apis mellifera (2)
- apple (2)
- aquatic (2)
- aquatic ecosystems (2)
- aqueous dispersion (2)
- aqueous-solution (2)
- arable land (2)
- arable weeds (2)
- arbuscular mycorrhizal symbiosis (2)
- area-based conservation (2)
- arsenic (2)
- arthropod (2)
- artificial light at night (ALAN) (2)
- ascorbate peroxidase (2)
- assembly (2)
- assembly processes (2)
- assembly rules (2)
- autocorrelation (2)
- automated radio telemetry (2)
- autophagy (2)
- axillary bud (2)
- baltic sea (2)
- basal body (2)
- base excision repair (incision activity) (2)
- bat fatalities (2)
- behavior (2)
- behavioral plasticity (2)
- behavioral type (2)
- behavioural syndrome (2)
- benzylic alcohol (2)
- benzylischer Alkohol (2)
- beta diversity (2)
- bias (2)
- bifurcation (2)
- bifurcation theory (2)
- biodiversity conservation (2)
- biodiversity decline (2)
- biodiversity exploratories (2)
- biodiversity facets (2)
- biofortification (2)
- biogene Amine (2)
- biological age (2)
- biological control (2)
- biological soil crusts (2)
- biomarker detection (2)
- biomimetic sensors (2)
- biosignatures (2)
- biostimulant (2)
- biosynthesis (2)
- biotic filtering (2)
- biotin sulfoxide reductase (2)
- bisphosphonates (2)
- bitter (2)
- bitter taste receptors (2)
- body composition (2)
- body proportions (2)
- bone mineral density (2)
- bone pathologies (2)
- brackish waters (2)
- brain insulin signaling (2)
- branched chain amino acids (2)
- branching (2)
- breeding (2)
- buffer zones (2)
- bush encroachment (2)
- cAMP (2)
- cTBS (2)
- cadmium-free (2)
- caffeine (2)
- calcination (2)
- calcite (2)
- calcium (2)
- calcium carbonate inclusions (2)
- calcium imaging (2)
- calcium influx (2)
- canalization (2)
- cancer epidemiology (2)
- capture enrichment (2)
- carbohydrates (2)
- carbon dots (2)
- carbon isotopes (2)
- carbon labeling (2)
- cardiac development (2)
- cardiomyocyte (2)
- cardiomyogenic differentiation (2)
- carotenoid biosynthesis (2)
- carrion ecology (2)
- cascading effects (2)
- cave fish (2)
- cell adhesion (2)
- cell division (2)
- cell shape (2)
- cell signaling (2)
- cells (2)
- cellular bioenergetics (2)
- cellular bioimaging (2)
- cellulose polymeric organic matter (2)
- cerami-des (2)
- ceramides (2)
- cereal leaf beetle (2)
- cereal meals (2)
- chemostat experiments (2)
- child growth (2)
- chimeric transcription factors (2)
- chlamydomonas (2)
- chlorophyll a (2)
- chloroplast (2)
- chloroplasts (2)
- chronic diseases (2)
- chytridiomycota (2)
- cis-regulatory evolution (2)
- click chemistry (2)
- climate adaptation (2)
- climate dynamics (2)
- climate extremes (2)
- climate warming (2)
- co-expression (2)
- co-limitation (2)
- coefficient (2)
- coffee by-products (2)
- cold stress (2)
- collagen (2)
- colon carcinogenesis (2)
- colony viability (2)
- common‐garden experiment (2)
- community model (2)
- community structure (2)
- community theory (2)
- competition–defense trade‐off (2)
- composition (2)
- comprehensive analysis (2)
- concepts (2)
- confocal microscopy (2)
- conformational change (2)
- conformational rearrangement (2)
- congeneric species (2)
- conscripts (2)
- constitutive activity (2)
- constraint-based modeling (2)
- converting factor (2)
- copper complex (2)
- copper(II) (2)
- copper-related disorders (2)
- cord blood (2)
- core shell UCNP (2)
- cori cycle (2)
- cortisol (2)
- counting (2)
- coviability analysis (2)
- crop (2)
- crop diversity (2)
- cropping system (2)
- cross-species capture (2)
- cryolithology (2)
- cryptomycota (2)
- cyanobacterial bloom (2)
- cyanobacterial sucrose-phosphatase (2)
- cytochrome c (2)
- cytoplasmic polyadenylation (2)
- cytosine methylation (2)
- cytosolic tRNA thiolation (2)
- cytotoxicity (2)
- dark virus (2)
- data integration (2)
- ddRAD (2)
- de novo genome assembly (2)
- dead Cas9 (2)
- decline (2)
- deep-sea (2)
- defense against predation (2)
- defensive symbiosis (2)
- degraded DNA (2)
- dehydration (2)
- demographic noise (2)
- depressive-like behavior (2)
- desiccation (2)
- design of experiment (2)
- developing brain (2)
- developmental canalization (2)
- developmental dyslexia (2)
- developmental plasticity (2)
- diacylglycerol (2)
- dietary patterns (2)
- dietary restriction (2)
- differential expression analysis (2)
- digestive enzymes quantification (2)
- dimerization of 4-nitrothiophenol (2)
- dispersal filtering (2)
- diversity profiles (2)
- dominance effect (2)
- droughts (2)
- drug delivery (2)
- drug metabolism (2)
- drug release (2)
- dynamic equilibrium (2)
- eastern continental Asia (2)
- eavesdropping (2)
- echolocation (2)
- ecophysiology (2)
- ecosystem services provisioning (2)
- ecosystems (2)
- education (2)
- egg ratio (2)
- eicosapentaenoic acid (2)
- electric fish (2)
- electroencephalography (EEG) (2)
- electron microscopy (2)
- electron paramagnetic resonance (2)
- electronic tool integration (2)
- emotional imagery (2)
- emotions (2)
- endocardium (2)
- endothelial cell (2)
- endotoxin (2)
- energy expenditure (2)
- enrichment experiments (2)
- entropy (2)
- environmental DNA (2)
- environmental genomics (2)
- environmental noise (2)
- enzymology (2)
- epidemiology (2)
- epigenetic variation (2)
- epiphytes (2)
- epithionitrile (2)
- epitope prediction (2)
- epizoochory (2)
- equalizing and stabilizing mechanisms (2)
- error reduction (2)
- establishment (2)
- event coincidence analysis (2)
- evolutionary ecology (2)
- evolutionary rescue (2)
- expansion microscopy (2)
- exploitation (2)
- expression profile (2)
- extinction (2)
- extinction drivers (2)
- extra-cytoplasmic pockets (2)
- extracellular DNA (2)
- extracellular enzymes (2)
- extracellular matrix (2)
- extracellular signaling (2)
- extremophiles (2)
- eye-tracking (2)
- facilitation (2)
- fatty acid changes (2)
- feeding (2)
- feeding behaviour (2)
- fence interaction (2)
- fertilization (2)
- fetal origins hypothesis (2)
- fire (2)
- fisheries (2)
- fitness gradient (2)
- fitness response (2)
- floating mat (2)
- flooded grasslands (2)
- floral scent (2)
- flow cytometry (2)
- fluorescent probe (2)
- food frequency questionnaire (2)
- food web dynamics (2)
- food webs (2)
- forage availability (2)
- forage gaps (2)
- foraging (2)
- foraging behaviour (2)
- forebrain (2)
- forest (2)
- forest management (2)
- forestREplot (2)
- formaldehyde assimilation (2)
- formate dehydrogenase (2)
- fractionation (2)
- fractionation factors (2)
- fragmentation (2)
- free zinc (2)
- freshwater algae (2)
- freshwater heterotrophic bacteria (2)
- functional complementation (2)
- functional inhibitors of acid sphin-gomyelinase (2)
- functional response (2)
- functional richness (2)
- fungal diversity (2)
- fungal pathogens (2)
- funktionelle Diversität (2)
- galactolipids (2)
- gamma diversity (2)
- gas-production (2)
- gene delivery (2)
- generalized dissimilarity modelling (2)
- genetic accommodation (2)
- genetic adaptation (2)
- genetic engineering (2)
- genetic rescue (2)
- genetic screen (2)
- genetischer Screen (2)
- genome (2)
- genome scan (2)
- genomic prediction (2)
- geo-bio interaction (2)
- geographic distribution (2)
- glacial maximum (2)
- global and regional change (2)
- glucocorticoid receptor (2)
- glucose (2)
- glucosinolate hydrolysis (2)
- glutathione (2)
- glycine cleavage system (2)
- governance (2)
- grazing (2)
- gross primary production (2)
- groundwater (2)
- groundwater recharge (2)
- growth behavior (2)
- growth restriction (2)
- guard cell (2)
- gut microbiota (2)
- gwas (2)
- habitat (2)
- habitat selection (2)
- habitat use (2)
- habituation (2)
- hantavirus (2)
- heart regeneration (2)
- heat (2)
- heat shock protein (2)
- heliozoa (2)
- hepcidin-25 (2)
- heterocyclic aromatic amine (2)
- heterotrophic bacteria (2)
- hilly loes plateau (2)
- histone modification (2)
- holding capability (2)
- holding isometric muscle action (HIMA) (2)
- holocene (2)
- honey bee (2)
- honey bees (2)
- hoverflies (2)
- human aldehyde oxidase (2)
- human endotoxemia (2)
- human excised skin (2)
- human introduction (2)
- human-wildlife conflict (2)
- hydrogels (2)
- hyena (2)
- hyperoxia (2)
- hypertension (2)
- hyperthermia (2)
- immunoassay (2)
- immunogenicity (2)
- importin (2)
- in silico (2)
- in vitro intestinal model (2)
- in-vitro-synthesis (2)
- inbreeding depression (2)
- indirect facilitation (2)
- individual based modeling (2)
- individual-based modeling (2)
- infancy (2)
- infiltration (2)
- inhibition (2)
- inner-mongolia (2)
- integrated assessments (2)
- inter-brain synchronization (2)
- inter-muscle-brain synchronization (2)
- interpersonal muscle action (2)
- interspecific interactions (2)
- intestinal mucins (2)
- intestinal zinc resorption (2)
- intra-organ-communication (2)
- intraguild predation (2)
- intraspecific variation (2)
- invasibility (2)
- invasion success (2)
- invasive species (2)
- ion channel (2)
- ion mobility spectrometry (2)
- ion transport (2)
- ion-exchange chromatography (2)
- ionic liquid (2)
- ionic strength (2)
- ionogel (2)
- iron-sulfur clusters (2)
- island biogeography (2)
- island disharmony (2)
- island syndromes (2)
- islands (2)
- isomerization (2)
- jasmonate (2)
- kelp (2)
- l-cysteine desulfurase (2)
- lake monitoring (2)
- lake periphyton (2)
- land sharing vs. land sparing (2)
- land use change (2)
- land-use change (2)
- landscape diversity (2)
- landscape generator (2)
- landscape homogenization (2)
- larch (2)
- late pleistocene (2)
- later health (2)
- leaf (2)
- lichens (2)
- life cycle (2)
- life‐history traits (2)
- ligand exchange (2)
- light adaptation (2)
- light variability (2)
- limits (2)
- lipid classes (2)
- lipid limitation thresholds (2)
- lipid membranes (2)
- lipidomics (2)
- lipid–lipid interactions (2)
- lipoplexes (2)
- loci (2)
- lokale Anpassung (2)
- longitudinal (2)
- low-energy electrons (2)
- luminescence (2)
- lysosomal hydrolases (2)
- lysosomal storage disorders (2)
- lysosome (2)
- mRNA degradation (2)
- magnitude estimation (2)
- maintenance (2)
- maintenance of functional diversity (2)
- maintenance of genomic integrity (2)
- major histocompatibility complex (2)
- male Daphnia (2)
- maltooligosaccharides (2)
- mammalian-cells (2)
- manganese (2)
- manual muscle test (2)
- many-to-one genotype–phenotype map (2)
- mapping (2)
- marine (2)
- mass conservation (2)
- mass index (2)
- mate-pairs (2)
- mathematical modelling (2)
- mathematical precursor (2)
- maturation (2)
- maximal isometric Adaptive Force (2)
- meal timing (2)
- mechanical (2)
- mechanical strength (2)
- mechanisms (2)
- mechanomyography (MMG) (2)
- mediated delivery (2)
- membrane biophysics (2)
- membrane fusion (2)
- membrane microdomains (2)
- membrane protein (2)
- membrane stabilization (2)
- memory (2)
- mercaptocarboxylic acids (2)
- mesenchymal stem cells (2)
- mesophyll cell (2)
- messenger-rna polyadenylation (2)
- meta-analysis (2)
- metabolic syndrome (2)
- metabolic-profiling (2)
- metabolische Netzwerke (2)
- metabolite (2)
- metagenome (2)
- metagenomic analysis (2)
- metagenomics 2.0 (2)
- metal complex (2)
- metal peptide (2)
- metallopeptide (2)
- metalloprotein (2)
- methane oxidation (2)
- methylotrophy (2)
- miRNAs (2)
- microarrays (2)
- microbial activity (2)
- microbial communities (2)
- microbiota (2)
- microclimate (2)
- microcomputed tomography (2)
- microeukaryotes (2)
- microfluidics (2)
- microstructure (2)
- microtubule-organization (2)
- mitochondrial genome (mtDNA) (2)
- mitogenomes (2)
- mixed cultures (2)
- model (2)
- model integration (2)
- model limitations (2)
- modeling (2)
- models (2)
- modern coexistence theory (2)
- mojave desert (2)
- molecular biology (2)
- molecular dynamics simulations (2)
- molecular evolution (2)
- molecular species identification (2)
- molybdoenzyme (2)
- molybdopterin synthase (2)
- morphology (2)
- motor control (2)
- movement speed (2)
- mucus layer (2)
- multiple sclerosis (2)
- multishell (2)
- multivalence (2)
- multi‐ year flooding cycle (2)
- muscle (2)
- musicality (2)
- mutation (2)
- mutual information (2)
- mycotoxins (2)
- myocardial infarction (2)
- myocardium (2)
- myoglobin (2)
- n-oxide reductase (2)
- nachhaltige Landnutzung (2)
- nanoelectrodes (2)
- nanogels (2)
- nanostructure (2)
- native American ancestry (2)
- necrobiome (2)
- neophilia (2)
- neophobia (2)
- net primary productivity (2)
- network analysis (2)
- network reconstruction (2)
- networks (2)
- neurodegeneration (2)
- neurodegenerative diseases (2)
- neuroendocrine (2)
- neuromuscular adaptation (2)
- niche and fitness differences (2)
- niche width (2)
- nickel (2)
- nitrile (2)
- nitrogen (2)
- nitrogen limitation (2)
- nitrous-oxide (2)
- noise color (2)
- non-alcoholic fatty liver disease (2)
- non-linear dynamics (2)
- nonlinear time series analysis (2)
- nonlinear waves (2)
- nuclear pore complex (2)
- nucleolus (2)
- nucleoporins (2)
- nucleus-associated body (2)
- number knowledge (2)
- nutrient (2)
- nutrient availability (2)
- nutrient spike (2)
- nutrient transport (2)
- odd chain fatty acids (2)
- oil palm (2)
- oligomerization (2)
- omics (2)
- ontogeny (2)
- ordinary differential equation (2)
- organ size (2)
- organic compounds adsorption (2)
- organic matter (2)
- organic–inorganic hybrid (2)
- organisches Material (2)
- osmotic-pressure (2)
- osteoporosis (2)
- other-race effect (2)
- outbreeding depression (2)
- overhunting (2)
- overweight (2)
- oxidase (2)
- oxidative stress tolerance (2)
- pH (2)
- pace-of-life syndrome (2)
- paleoenvironmental records (2)
- parallel beta-helix (2)
- parameter estimation (2)
- parameters (2)
- paraquat (2)
- parasitoid (2)
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Hepcidin-25 was identified as themain iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II) binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandemmass spectrometry (MS/MS), high-resolutionmass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or referencematerial comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
Hepcidin-25 was identified as themain iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II) binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandemmass spectrometry (MS/MS), high-resolutionmass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or referencematerial comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
Say it with double flowers
(2020)
Every year, lovers world-wide rely on mutants to show their feelings on Valentine's Day. This is because many of the most popular ornamental flowering plants have been selected to form extra petals at the expense of reproductive organs to enhance their attractiveness and aesthetic value to humans. This so-called 'double flower' (DF) phenotype, first described more than 2000 years ago (Meyerowitz et al., 1989) is present, for example, in many modern roses, carnations, peonies, and camellias. Gattolin et al. (2020) now identify a unifying explanation for the molecular basis of many of these DF cultivars.
Subsurface microbial communities undertake many terminal electron-accepting processes, often simultaneously. Using a tritium-based assay, we measured the potential hydrogen oxidation catalyzed by hydrogenase enzymes in several subsurface sedimentary environments (Lake Van, Barents Sea, Equatorial Pacific, and Gulf of Mexico) with different predominant electron-acceptors. Hydrogenases constitute a diverse family of enzymes expressed by microorganisms that utilize molecular hydrogen as a metabolic substrate, product, or intermediate. The assay reveals the potential for utilizing molecular hydrogen and allows qualitative detection of microbial activity irrespective of the predominant electron-accepting process. Because the method only requires samples frozen immediately after recovery, the assay can be used for identifying microbial activity in subsurface ecosystems without the need to preserve live material. We measured potential hydrogen oxidation rates in all samples from multiple depths at several sites that collectively span a wide range of environmental conditions and biogeochemical zones. Potential activity normalized to total cell abundance ranges over five orders of magnitude and varies, dependent upon the predominant terminal electron acceptor. Lowest per-cell potential rates characterize the zone of nitrate reduction and highest per-cell potential rates occur in the methanogenic zone. Possible reasons for this relationship to predominant electron acceptor include (i) increasing importance of fermentation in successively deeper biogeochemical zones and (ii) adaptation of H(2)ases to successively higher concentrations of H-2 in successively deeper zones.
Pancreatic steatosis associates with beta-cell failure and may participate in the development of type-2-diabetes. Our previous studies have shown that diabetes-susceptible mice accumulate more adipocytes in the pancreas than diabetes-resistant mice. In addition, we have demonstrated that the co-culture of pancreatic islets and adipocytes affect insulin secretion. The aim of this current study was to elucidate if and to what extent pancreas-resident mesenchymal stromal cells (MSCs) with adipogenic progenitor potential differ from the corresponding stromal-type cells of the inguinal white adipose tissue (iWAT). miRNA (miRNome) and mRNA expression (transcriptome) analyses of MSCs isolated by flow cytometry of both tissues revealed 121 differentially expressed miRNAs and 1227 differentially expressed genes (DEGs). Target prediction analysis estimated 510 DEGs to be regulated by 58 differentially expressed miRNAs. Pathway analyses of DEGs and miRNA target genes showed unique transcriptional and miRNA signatures in pancreas (pMSCs) and iWAT MSCs (iwatMSCs), for instance fibrogenic and adipogenic differentiation, respectively. Accordingly, iwatMSCs revealed a higher adipogenic lineage commitment, whereas pMSCs showed an elevated fibrogenesis. As a low degree of adipogenesis was also observed in pMSCs of diabetes-susceptible mice, we conclude that the development of pancreatic steatosis has to be induced by other factors not related to cell-autonomous transcriptomic changes and miRNA-based signals.
Functional characterization of ROS-responsive genes, ANAC085 and ATR7, in Arabidopsis thaliana
(2023)
Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set
(2022)
A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material.
Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set
(2022)
A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material.
Development and application of novel genetic transformation technologies in maize (Zea mays L.)
(2007)
Plant genetic engineering approaches are of pivotal importance to both basic and applied research. However, rapid commercialization of genetically engineered crops, especially maize, raises several ecological and environmental concerns largely related to transgene flow via pollination. In most crops, the plastid genome is inherited uniparentally in a maternal manner. Consequently, a trait introduced into the plastid genome would not be transferred to the sexually compatible relatives of the crops via pollination. Thus, beside its several other advantages, plastid transformation provides transgene containment, and therefore, is an environmentally friendly approach for genetic engineering of crop plants. Reliable in vitro regeneration systems allowing repeated rounds of regeneration are of utmost importance to development of plastid transformation technologies in higher plants. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. In one part of this work, a novel tissue culture and plant regeneration system was developed that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. Also, protocols were established for (i) the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants. Furthermore, several selection methods were tested for developing a plastid transformation system in maize. However, stable plastid transformed maize plants could not be yet recovered. Possible explanations as well as suggestions for future attempts towards developing plastid transformation in maize are discussed. Nevertheless, these results represent a first essential step towards developing chloroplast transformation technology for maize, a method that requires multiple rounds of plant regeneration and selection to obtain genetically stable transgenic plants. In order to apply the newly developed transformation system towards metabolic engineering of carotenoid biosynthesis, the daffodil phytoene synthase (PSY) gene was integrated into the maize genome. The results illustrate that expression of a recombinant PSY significantly increases carotenoid levels in leaves. The beta-carotene (pro-vitamin A) amounts in leaves of transgenic plants were increased by ~21% in comparison to the wild-type. These results represent evidence for maize to have significant potential to accumulate higher amounts of carotenoids, especially beta-carotene, through transgenic expression of phytoene synthases. Finally, progresses were made towards developing transformation technologies in Peperomia (Piperaceae) by establishing an efficient leaf-based regeneration system. Also, factors determining plastid size and number in Peperomia, whose species display great interspecific variation in chloroplast size and number per cell, were investigated. The results suggest that organelle size and number are regulated in a tissue-specific manner rather than in dependency on the plastid type. Investigating plastid morphology in Peperomia species with giant chloroplasts, plasmatic connections between chloroplasts (stromules) were observed under the light microscope and in the absence of tissue fixation or GFP overexpression demonstrating the relevance of these structures in vivo. Furthermore, bacteria-like microorganisms were discovered within Peperomia cells, suggesting that this genus provides an interesting model not only for studying plastid biology but also for investigating plant-microbe interactions.
Um das Immunsystem der Pflanze zu manipulieren translozieren gram-negative pathogene Bakterien Typ-III Effektorproteine (T3E) über ein Typ-III Sekretionssystem (T3SS) in die pflanzliche Wirtszelle. Dort lokalisieren T3Es in verschiedenen subzellulären Kompartimenten, wo sie Zielproteine modifizieren und so die Infektion begünstigen. HopZ1a, ein T3E des Pflanzenpathogens Pseudomonas syringae pv. syringae, ist eine Acetyltransferase und lokalisiert über ein Myristolierungsmotiv an der Plasmamembran der Wirtszelle. Obwohl gezeigt wurde, dass HopZ1a die frühe Signalweiterleitung an der Plasmamembran stört, wurde bisher kein mit der Plasmamembran assoziiertes Zielprotein für diesen T3E identifiziert. Um bisher unbekannte HopZ1a-Zieleproteine zu identifizieren wurde im Vorfeld dieser Arbeit eine Hefe-Zwei-Hybrid-Durchmusterung mit einer cDNA-Bibliothek aus Tabak durchgeführt, wobei ein nicht näher charakterisiertes Remorin als Interaktor gefunden wurde.
Bei dem Remorin handelt es sich um einen Vertreter der Gruppe 4 der Remorin-Familie, weshalb es in NbREM4 umbenannt wurde. Durch den Einsatz verschiedener Interaktionsstudien konnte demonstriert werden, dass HopZ1a mit NbREM4 in Hefe, in vitro und in planta wechselwirkt. Es wurde ferner deutlich, dass HopZ1a auf spezifische Weise mit dem konservierten C-Terminus von NbREM4 interagiert, das Remorin jedoch in vitro nicht acetyliert. Analysen mittels BiFC haben zudem ergeben, dass NbREM4 in Homodimeren an der Plasmamembran lokalisiert, wo auch die Interaktion mit HopZ1a stattfindet.
Eine funktionelle Charakterisierung von NbREM4 ergab, dass das Remorin eine spezifische Rolle im Immunsystem der Pflanze einnimmt. Die transiente Expression in N. benthamiana induziert die Expression von Abwehrgenen sowie einen veränderten Blattphänotyp. In A. thaliana wird HopZ1a über das Decoy ZED1 und das R-Protein ZAR1 erkannt, was zur Auslösung einer starken Hypersensitiven Antwort (HR von hypersensitive response) führt. Es konnte im Rahmen dieser Arbeit gezeigt werden, dass ZAR1 in N. benthamiana konserviert ist, NbREM4 jedoch nicht in der ETI als Decoy fungiert. Mit Hilfe einer Hefe-Zwei-Hybrid-Durchmusterung mit NbZAR1 als Köder konnten zwei Proteine, die Catalase CAT1 und der Protonenpumpeninteraktor PPI1, als Interaktoren von NbZAR1 identifiziert werden, welche möglicherweise in der Regulation der HR eine Rolle spielen.
Aus Voruntersuchungen war bekannt, dass NbREM4 mit weiteren, nicht näher charakterisierten Proteinen aus Tabak interagieren könnte. Eine phylogenetische Einordnung hat gezeigt, dass es sich um die bekannte Immun-Kinase PBS1 sowie zwei E3-Ubiquitin-Ligasen, NbSINA1 und NbSINAL3, handelt. PBS1 interagiert mit NbREM4 an der Plasmamembran und phosphoryliert das Remorin innerhalb des intrinsisch ungeordneten N-Terminus. Mittels Massenspektrometrie konnten die Serine an Position 64 und 65 innerhalb der Aminosäuresequenz von NbREM4 als PBS1-abhängige Phosphorylierungsstellen identifiziert wurden.
NbSINA1 und NbSINAL3 besitzen in vitro Ubiquitinierungsaktivität, bilden Homo- und Heterodimere und interagieren ebenfalls mit dem N-terminalen Teil von NbREM4, wobei sie das Remorin in vitro nicht ubiquitinieren.
Aus den in dieser Arbeit gewonnenen Ergebnissen lässt sich ableiten, dass der bakterielle T3E HopZ1a gezielt mit dem Tabak-Remorin NbREM4 an der Plasmamembran interagiert und über einen noch unbekannten Mechanismus mit dem Immunsystem der Pflanze interferiert, wobei NbREM4 möglicherweise eine Rolle als Adapter- oder Ankerprotein zukommt, über welches HopZ1a mit weiteren Immunkomponenten interagiert. NbREM4 ist Teil eines größeren Immunnetzwerkes, zu welchem die bekannte Immun-Kinase PBS1 und zwei E3-Ubiquitin-Ligasen gehören. Mit NbREM4 konnte damit erstmalig ein membranständiges Protein mit einer Funktion im Immunsystem der Pflanze als Zielprotein von HopZ1a identifiziert werden.
The plant pathogen Pseudomonas syringae is a gram-negative bacterium which infects a wide range of plant species including important crops plants. To suppress plant immunity and cause disease P.syringae injects type-III effector proteins (T3Es) into the plant cell cytosol. In this study, we identified a novel target of the well characterized bacterial T3E HopZ1a. HopZ1a is an acetyltransferase that was shown to disrupt vesicle transport during innate immunity by acetylating tubulin. Using a yeast-two-hybrid screen approach, we identified a REMORIN (REM) protein from tobacco as a novel HopZ1a target. HopZ1a interacts with REM at the plasma membrane (PM) as shown by split-YFP experiments. Interestingly, we found that PBS1, a well-known kinase involved in plant immunity also interacts with REM in pull-down assays, and at the PM as shown by BiFC. Furthermore, we confirmed that REM is phosphorylated by PBS1 in vitro. Overexpression of REM provokes the upregulation of defense genes and leads to disease-like phenotypes pointing to a role of REM in plant immune signaling. Further protein-protein interaction studies reveal novel REM binding partners with a possible role in plant immune signaling. Thus, REM might act as an assembly hub for an immune signaling complex targeted by HopZ1a. Taken together, this is the first report describing that a REM protein is targeted by a bacterial effector. How HopZ1a might mechanistically manipulate the plant immune system through interfering with REM function will be discussed.
The plasma membrane (PM) is at the interface of plant-pathogen interactions and, thus, many bacterial type-III effector (T3E) proteins target membrane-associated processes to interfere with immunity. The Pseudomonas syringae T3E HopZ1a is a host cell PM-localized effector protein that has several immunity-associated host targets but also activates effector-triggered immunity in resistant backgrounds. Although HopZ1a has been shown to interfere with early defense signaling at the PM, no dedicated PM-associated HopZ1a target protein has been identified until now. Here, we show that HopZ1a interacts with the PM-associated remorin protein NbREM4 from Nicotiana benthamiana in several independent assays. NbREM4 relocalizes to membrane nanodomains after treatment with the bacterial elicitor flg22 and transient overexpression of NbREM4 in N. benthamiana induces the expression of a subset of defense-related genes. We can further show that NbREM4 interacts with the immune-related receptor-like cytoplasmic kinase avrPphB-susceptible 1 (PBS1) and is phosphorylated by PBS1 on several residues in vitro. Thus, we conclude that NbREM4 is associated with early defense signaling at the PM. The possible relevance of the HopZ1a-NbREM4 interaction for HopZ1a virulence and avirulence functions is discussed.
The prevalence of contaminant microbial DNA in ancient bone samples represents the principal limiting factor for palaeogenomic studies, as it may comprise more than 99% of DNA molecules obtained. Efforts to exclude or reduce this contaminant fraction have been numerous but also variable in their success. Here, we present a simple but highly effective method to increase the relative proportion of endogenous molecules obtained from ancient bones. Using computed tomography (CT) scanning, we identify the densest region of a bone as optimal for sampling. This approach accurately identifies the densest internal regions of petrous bones, which are known to be a source of high-purity ancient DNA. For ancient long bones, CT scans reveal a high-density outermost layer, which has been routinely removed and discarded prior to DNA extraction. For almost all long bones investigated, we find that targeted sampling of this outermost layer provides an increase in endogenous DNA content over that obtained from softer, trabecular bone. This targeted sampling can produce as much as 50-fold increase in the proportion of endogenous DNA, providing a directly proportional reduction in sequencing costs for shotgun sequencing experiments. The observed increases in endogenous DNA proportion are not associated with any reduction in absolute endogenous molecule recovery. Although sampling the outermost layer can result in higher levels of human contamination, some bones were found to have more contamination associated with the internal bone structures. Our method is highly consistent, reproducible and applicable across a wide range of bone types, ages and species. We predict that this discovery will greatly extend the potential to study ancient populations and species in the genomics era.
Characterization of altered inflorescence architecture in Arabidopsis thaliana BG-5 x Kro-0 hybrid
(2018)
A reciprocal cross between two A. thaliana accessions, Kro-0 (Krotzenburg, Germany) and BG-5 (Seattle, USA), displays purple rosette leaves and dwarf bushy phenotype in F1 hybrids when grown at 17 °C and a parental-like phenotype when grown at 21 °C. This F1 temperature-dependent-dwarf-bushy phenotype is characterized by reduced growth of the primary stem together with an increased number of branches. The reduced stem growth was the strongest at the first internode. In addition, we found that a temperature switch from 21 °C to 17 °C induced the phenotype only before the formation of the first internode of the stem. Similarly, the F1 dwarf-bushy phenotype could not be reversed when plants were shifted from 17 °C to 21 °C after the first internode was formed. Metabolic analysis showed that the F1 phenotype was associated with a significant upregulation of anthocyanin(s), kaempferol(s), salicylic acid, jasmonic acid and abscisic acid. As it has been previously shown that the dwarf-bushy phenotype is linked to two loci, one on chromosome 2 from Kro-0 and one on chromosome 3 from BG-5, an artificial micro-RNA approach was used to investigate the necessary genes on these intervals. From the results obtained, it was found that two genes, AT2G14120 that encodes for a DYNAMIN RELATED PROTEIN3B and AT2G14100 that encodes a member of the Cytochrome P450 family protein CYP705A13, were necessary for the appearance of the F1 phenotype on chromosome 2. It was also discovered that AT3G61035 that encodes for another cytochrome P450 family protein CYP705A13 and AT3G60840 that encodes for a MICROTUBULE-ASSOCIATED PROTEIN65-4 on chromosome 3 were both necessary for the induction of the F1 phenotype. To prove the causality of these genes, genomic constructs of the Kro-0 candidate genes on chromosome 2 were transferred to BG-5 and genomic constructs of the chromosome 3 candidate genes from BG-5 were transferred to Kro-0. The T1 lines showed that these genes are not sufficient alone to induce the phenotype. In addition to the F1 phenotype, more severe phenotypes were observed in the F2 generations that were grouped into five different phenotypic classes. Whilst seed yield was comparable between F1 hybrids and parental lines, three phenotypic classes in the F2 generation exhibited hybrid breakdown in the form of reproductive failure. This F2 hybrid breakdown was less sensitive to temperature and showed a dose-dependent effect of the loci involved in F1 phenotype. The severest class of hybrid breakdown phenotypes was observed only in the population of backcross with the parent Kro-0, which indicates a stronger contribution of the BG-5 allele when compared to the Kro-0 allele on the hybrid breakdown phenotypes. Overall, the findings of my thesis provide a further understanding of the genetic and metabolic factors underlying altered shoot architecture in hybrid dysfunction.
Zinc is an essential trace element, making it crucial to have a reliable biomarker for evaluating an individual’s zinc status. The total serum zinc concentration, which is presently the most commonly used biomarker, is not ideal for this purpose, but a superior alternative is still missing. The free zinc concentration, which describes the fraction of zinc that is only loosely bound and easily exchangeable, has been proposed for this purpose, as it reflects the highly bioavailable part of serum zinc. This report presents a fluorescence-based method for determining the free zinc concentration in human serum samples, using the fluorescent probe Zinpyr-1. The assay has been applied on 154 commercially obtained human serum samples. Measured free zinc concentrations ranged from 0.09 to 0.42 nM with a mean of 0.22 ± 0.05 nM. It did not correlate with age or the total serum concentrations of zinc, manganese, iron or selenium. A negative correlation between the concentration of free zinc and total copper has been seen for sera from females. In addition, the free zinc concentration in sera from females (0.21 ± 0.05 nM) was significantly lower than in males (0.23 ± 0.06 nM). The assay uses a sample volume of less than 10 µL, is rapid and cost-effective and allows us to address questions regarding factors influencing the free serum zinc concentration, its connection with the body’s zinc status, and its suitability as a future biomarker for an individual’s zinc status.
Zinc is an essential trace element, making it crucial to have a reliable biomarker for evaluating an individual’s zinc status. The total serum zinc concentration, which is presently the most commonly used biomarker, is not ideal for this purpose, but a superior alternative is still missing. The free zinc concentration, which describes the fraction of zinc that is only loosely bound and easily exchangeable, has been proposed for this purpose, as it reflects the highly bioavailable part of serum zinc. This report presents a fluorescence-based method for determining the free zinc concentration in human serum samples, using the fluorescent probe Zinpyr-1. The assay has been applied on 154 commercially obtained human serum samples. Measured free zinc concentrations ranged from 0.09 to 0.42 nM with a mean of 0.22 ± 0.05 nM. It did not correlate with age or the total serum concentrations of zinc, manganese, iron or selenium. A negative correlation between the concentration of free zinc and total copper has been seen for sera from females. In addition, the free zinc concentration in sera from females (0.21 ± 0.05 nM) was significantly lower than in males (0.23 ± 0.06 nM). The assay uses a sample volume of less than 10 µL, is rapid and cost-effective and allows us to address questions regarding factors influencing the free serum zinc concentration, its connection with the body’s zinc status, and its suitability as a future biomarker for an individual’s zinc status.
Climate impacts on transocean dispersal and habitat in gray whales from the Pleistocene to 2100
(2015)
Arctic animals face dramatic habitat alteration due to ongoing climate change. Understanding how such species have responded to past glacial cycles can help us forecast their response to today's changing climate. Gray whales are among those marine species likely to be strongly affected by Arctic climate change, but a thorough analysis of past climate impacts on this species has been complicated by lack of information about an extinct population in the Atlantic. While little is known about the history of Atlantic gray whales or their relationship to the extant Pacific population, the extirpation of the Atlantic population during historical times has been attributed to whaling. We used a combination of ancient and modern DNA, radiocarbon dating and predictive habitat modelling to better understand the distribution of gray whales during the Pleistocene and Holocene. Our results reveal that dispersal between the Pacific and Atlantic was climate dependent and occurred both during the Pleistocene prior to the last glacial period and the early Holocene immediately following the opening of the Bering Strait. Genetic diversity in the Atlantic declined over an extended interval that predates the period of intensive commercial whaling, indicating this decline may have been precipitated by Holocene climate or other ecological causes. These first genetic data for Atlantic gray whales, particularly when combined with predictive habitat models for the year 2100, suggest that two recent sightings of gray whales in the Atlantic may represent the beginning of the expansion of this species' habitat beyond its currently realized range.
Widely used diagnostic tools make use of antibodies recognizing targeted molecules, but additional techniques are required in order to alleviate the disadvantages of antibodies. Herein, molecular dynamic calculations are performed for the design of high affinity artificial protein binding surfaces for the recognition of neuron specific enolase (NSE), a known cancer biomarker. Computational simulations are employed to identify particularly stabile secondary structure elements. These epitopes are used for the subsequent molecular imprinting, where surface imprinting approach is applied. The molecular imprints generated with the calculated epitopes of greater stability (Cys-Ep1) show better binding properties than those of lower stability (Cys-Ep5). The average binding strength of imprints created with stabile epitopes is found to be around twofold and fourfold higher for the NSE derived peptide and NSE protein, respectively. The recognition of NSE is investigated in a wide concentration range, where high sensitivity (limit of detection (LOD) = 0.5 ng mL(-1)) and affinity (dissociation constant (K-d) = 5.3 x 10(-11)m) are achieved using Cys-Ep1 imprints reflecting the stable structure of the template molecules. This integrated approach employing stability calculations for the identification of stabile epitopes is expected to have a major impact on the future development of high affinity protein capturing binders.
Genetic studies of the Eurasian brown bear (Ursus arctos) have so far focused on populations from Europe and North America, although the largest distribution area of brown bears is in Asia. In this study, we reveal population genetic parameters for the brown bear population inhabiting the Grand Kackar Mountains (GKM) in the north east of Turkey, western Lesser Caucasus. Using both hair (N = 147) and tissue samples (N = 7) collected between 2008 and 2014, we found substantial levels of genetic variation (10 microsatellite loci). Bear samples (hair) taken from rubbing trees worked better for genotyping than those from power poles, regardless of the year collected. Genotyping also revealed that bears moved between habitat patches, despite ongoing massive habitat alterations and the creation of large water reservoirs. This population has the potential to serve as a genetic reserve for future reintroduction in the Middle East. Due to the importance of the GKM population for on-going and future conservation actions, the impacts of habitat alterations in the region ought to be minimized; e.g., by establishing green bridges or corridors over reservoirs and major roads to maintain habitat connectivity and gene flow among populations in the Lesser Caucasus.
Genetic studies of the Eurasian brown bear (Ursus arctos) have so far focused on populations from Europe and North America, although the largest distribution area of brown bears is in Asia. In this study, we reveal population genetic parameters for the brown bear population inhabiting the Grand Kaçkar Mountains (GKM) in the north east of Turkey, western Lesser Caucasus. Using both hair (N = 147) and tissue samples (N = 7) collected between 2008 and 2014, we found substantial levels of genetic variation (10 microsatellite loci). Bear samples (hair) taken from rubbing trees worked better for genotyping than those from power poles, regardless of the year collected. Genotyping also revealed that bears moved between habitat patches, despite ongoing massive habitat alterations and the creation of large water reservoirs. This population has the potential to serve as a genetic reserve for future reintroductions in the Middle East. Due to the importance of the GKM population for on-going and future conservation actions, the impacts of habitat alterations in the region ought to be minimized; e.g., by establishing green bridges or corridors over reservoirs and major roads to maintain habitat connectivity and gene flow among populations in the Lesser Caucasus.
The African weakly electric fish genus Campylomormyrus includes 15 described species mostly native to the Congo River and its tributaries. They are considered sympatric species, because their distribution area overlaps. These species generate species-specific electric organ discharges (EODs) varying in waveform characteristics, including duration, polarity, and phase number. They exhibit also pronounced divergence in their snout, i.e. the length, thickness, and curvature. The diversifications in these two phenotypical traits (EOD and snout) have been proposed as key factors promoting adaptive radiation in Campylomormyrus. The role of EODs as a pre-zygotic isolation mechanism driving sympatric speciation by promoting assortative mating has been examined using behavioral, genetical, and histological approaches. However, the evolutionary effects of the snout morphology and its link to species divergence have not been closely examined. Hence, the main objective of this study is to investigate the effect of snout morphology diversification and its correlated EOD to better understand their sympatric speciation and evolutionary drivers. Moreover, I aim to utilize the intragenus and intergenus hybrids of Campylomormyrus to better understand trait divergence as well as underlying molecular/genetic mechanisms involved in the radiation scenario. To this end, I utilized three different approaches: feeding behavior analysis, diet assessment, and geometric morphometrics analysis. I performed feeding behavior experiments to evaluate the concept of the phenotype-environment correlation by testing whether Campylomormyrus species show substrate preferences. The behavioral experiments showed that the short snout species exhibits preference to sandy substrate, the long snout species prefers a stone substrate, and the species with intermediate snout size does not exhibit any substrate preference. The experiments suggest that the diverse feeding apparatus in the genus Campylomormyrus may have evolved in adaptation to their microhabitats. I also performed diet assessments of sympatric Campylomormyrus species and a sister genus species (Gnathonemus petersii) with markedly different snout morphologies and EOD using NGS-based DNA metabarcoding of their stomach contents. The diet of each species was documented showing that aquatic insects such as dipterans, coleopterans and trichopterans represent the major diet component. The results showed also that all species are able to exploit diverse food niches in their habitats. However, comparing the diet overlap indices showed that different snout morphologies and the associated divergence in the EOD translated into different prey spectra. These results further support the idea that the EOD could be a ‘magic trait’ triggering both adaptation and reproductive isolation. Geometric morphometrics method was also used to compare the phenotypical shape traits of the F1 intragenus (Campylomormyrus) and intergenus (Campylomormyrus species and Gnathonemus petersii) hybrids relative to their parents. The hybrids of these species were well separated based on the morphological traits, however the hybrid phenotypic traits were closer to the short-snouted species. In addition, the likelihood that the short snout expressed in the hybrids increases with increasing the genetic distance of the parental species. The results confirmed that additive effects produce intermediate phenotypes in F1-hybrids. It seems, therefore, that morphological shape traits in hybrids, unlike the physiological traits, were not expressed straightforward.
Under an ecological speciation scenario, the radiation of African weakly electric fish (genus Campylomormyrus) is caused by an adaptation to different food sources, associated with diversification of the electric organ discharge (EOD). This study experimentally investigates a phenotype-environment correlation to further support this scenario. Our behavioural experiments showed that three sympatric Campylomormyrus species with significantly divergent snout morphology differentially react to variation in substrate structure. While the short snout species (C. tamandua) exhibits preference to sandy substrate, the long snout species (C. rhynchophorus) significantly prefers a stone substrate for feeding. A third species with intermediate snout size (C. compressirostris) does not exhibit any substrate preference. This preference is matched with the observation that long-snouted specimens probe deeper into the stone substrate, presumably enabling them to reach prey more distant to the substrate surface. These findings suggest that the diverse feeding apparatus in the genus Campylomormyrus may have evolved in adaptation to specific microhabitats, i.e., substrate structures where these fish forage. Whether the parallel divergence in EOD is functionally related to this adaptation or solely serves as a prezygotic isolation mechanism remains to be elucidated.
Under an ecological speciation scenario, the radiation of African weakly electric fish (genus Campylomormyrus) is caused by an adaptation to different food sources, associated with diversification of the electric organ discharge (EOD). This study experimentally investigates a phenotype-environment correlation to further support this scenario. Our behavioural experiments showed that three sympatric Campylomormyrus species with significantly divergent snout morphology differentially react to variation in substrate structure. While the short snout species (C. tamandua) exhibits preference to sandy substrate, the long snout species (C. rhynchophorus) significantly prefers a stone substrate for feeding. A third species with intermediate snout size (C. compressirostris) does not exhibit any substrate preference. This preference is matched with the observation that long-snouted specimens probe deeper into the stone substrate, presumably enabling them to reach prey more distant to the substrate surface. These findings suggest that the diverse feeding apparatus in the genus Campylomormyrus may have evolved in adaptation to specific microhabitats, i.e., substrate structures where these fish forage. Whether the parallel divergence in EOD is functionally related to this adaptation or solely serves as a prezygotic isolation mechanism remains to be elucidated.
Resource polymorphism is common across taxa and can result in alternate ecotypes with specific morphologies, feeding modes, and behaviors that increase performance in a specific habitat. This can result in high intraspecific variation in the expression of specific traits and the extent to which these traits are correlated within a single population. Although metabolic rate influences resource acquisition and the overall pace of life of individuals it is not clear how metabolic rate interacts with the larger suite of traits to ultimately determine individual fitness. We examined the relationship between metabolic rates and the major differences (habitat use, morphology, and resource use) between littoral and pelagic ecotypes of European perch (Perca fluviatilis) from a single lake in Central Sweden. Standard metabolic rate (SMR) was significantly higher in pelagic perch but did not correlate with resource use or morphology. Maximum metabolic rate (MMR) was not correlated with any of our explanatory variables or with SMR. Aerobic scope (AS) showed the same pattern as SMR, differing across habitats, but contrary to expectations, was lower in pelagic perch. This study helps to establish a framework for future experiments further exploring the drivers of intraspecific differences in metabolism. In addition, since metabolic rates scale with temperature and determine predator energy requirements, our observed differences in SMR across habitats will help determine ecotype-specific vulnerabilities to climate change and differences in top-down predation pressure across habitats.
A systems biological approach towards the molecular basis of heterosis in Arabidopsis thaliana
(2011)
Heterosis is defined as the superiority in performance of heterozygous genotypes compared to their corresponding genetically different homozygous parents. This phenomenon is already known since the beginning of the last century and it has been widely used in plant breeding, but the underlying genetic and molecular mechanisms are not well understood. In this work, a systems biological approach based on molecular network structures is proposed to contribute to the understanding of heterosis. Hybrids are likely to contain additional regulatory possibilities compared to their homozygous parents and, therefore, they may be able to correctly respond to a higher number of environmental challenges, which leads to a higher adaptability and, thus, the heterosis phenomenon. In the network hypothesis for heterosis, presented in this work, more regulatory interactions are expected in the molecular networks of the hybrids compared to the homozygous parents. Partial correlations were used to assess this difference in the global interaction structure of regulatory networks between the hybrids and the homozygous genotypes. This network hypothesis for heterosis was tested on metabolite profiles as well as gene expression data of the two parental Arabidopsis thaliana accessions C24 and Col-0 and their reciprocal crosses. These plants are known to show a heterosis effect in their biomass phenotype. The hypothesis was confirmed for mid-parent and best-parent heterosis for either hybrid of our experimental metabolite as well as gene expression data. It was shown that this result is influenced by the used cutoffs during the analyses. Too strict filtering resulted in sets of metabolites and genes for which the network hypothesis for heterosis does not hold true for either hybrid regarding mid-parent as well as best-parent heterosis. In an over-representation analysis, the genes that show the largest heterosis effects according to our network hypothesis were compared to genes of heterotic quantitative trait loci (QTL) regions. Separately for either hybrid regarding mid-parent as well as best-parent heterosis, a significantly larger overlap between the resulting gene lists of the two different approaches towards biomass heterosis was detected than expected by chance. This suggests that each heterotic QTL region contains many genes influencing biomass heterosis in the early development of Arabidopsis thaliana. Furthermore, this integrative analysis led to a confinement and an increased confidence in the group of candidate genes for biomass heterosis in Arabidopsis thaliana identified by both approaches.
We propose a network structure-based model for heterosis, and investigate it relying on metabolite profiles from Arabidopsis. A simple feed-forward two-layer network model (the Steinbuch matrix) is used in our conceptual approach. It allows for directly relating structural network properties with biological function. Interpreting heterosis as increased adaptability, our model predicts that the biological networks involved show increasing connectivity of regulatory interactions. A detailed analysis of metabolite profile data reveals that the increasing-connectivity prediction is true for graphical Gaussian models in our data from early development. This mirrors properties of observed heterotic Arabidopsis phenotypes. Furthermore, the model predicts a limit for increasing hybrid vigor with increasing heterozygosity—a known phenomenon in the literature.
Non-mycorrhizal fungal endophytes are able to colonize internally roots without causing visible disease symptoms establishing neutral or mutualistic associations with plants. These fungi known as non-clavicipitaceous endophytes have a broad host range of monocot and eudicot plants and are highly diverse. Some of them promote plant growth and confer increased abiotic-stress tolerance and disease resistance. According to such possible effects on host plants, it was aimed to isolate and to characterize native fungal root endophytes from tomato (Lycopersicon esculentum Mill.) and to analyze their effects on plant development, plant resistance and fruit yield and quality together with the model endophyte Piriformospora indica. Fifty one new fungal strains were isolated from desinfected tomato roots of four different crop sites in Colombia. These isolates were roughly characterized and fourteen potential endophytes were further analyzed concerning their taxonomy, their root colonization capacity and their impact on plant growth. Sequencing of the ITS region from the ribosomal RNA gene cluster and in-depth morphological characterisation revealed that they correspond to different phylogenetic groups among the phylum Ascomycota. Nine different morphotypes were described including six dark septate endophytes (DSE) that did not correspond to the Phialocephala group. Detailed confocal microscopy analysis showed various colonization patterns of the endophytes inside the roots ranging from epidermal penetration to hyphal growth through the cortex. Tomato pot experiments under glass house conditions showed that they differentially affect plant growth depending on colonization time and inoculum concentration. Three new isolates (two unknown fungal endophyte DSE48, DSE49 and one identified as Leptodontidium orchidicola) with neutral or positiv effects were selected and tested in several experiments for their influence on vegetative growth, fruit yield and quality and their ability to diminish the impact of the pathogen Verticillium dahliae on tomato plants. Although plant growth promotion by all three fungi was observed in young plants, vegetative growth parameters were not affected after 22 weeks of cultivation except a reproducible increase of root diameter by the endophyte DSE49. Additionally, L. orchidicola increased biomass and glucose content of tomato fruits, but only at an early date of harvest and at a certain level of root colonization. Concerning bioprotective effects, the endophytes DSE49 and L. orchidicola decreased significantly disease symptoms caused by the pathogen V. dahliae, but only at a low dosis of the pathogen. In order to analyze, if the model root endophytic fungus Piriformospora indica could be suitable for application in production systems, its impact on tomato was evaluated. Similarly to the new fungal isolates, significant differences for vegetative growth parameters were only observable in young plants and, but protection against V. dahliae could be seen in one experiment also at high dosage of the pathogen. As the DSE L. orchidicola, P. indica increased the number and biomass of marketable tomatoes only at the beginning of fruit setting, but this did not lead to a significant higher total yield. If the effects on growth are due to a better nutrition of the plant with mineral element was analyzed in barley in comparison to the arbuscular mycorrhizal fungus Glomus mosseae. While the mycorrhizal fungus increased nitrogen and phosphate uptake of the plant, no such effect was observed for P. indica. In summary this work shows that many different fungal endophytes can be also isolated from roots of crops and, that these isolates can have positive effects on early plant development. This does, however, not lead to an increase in total yield or in improvement of fruit quality of tomatoes under greenhouse conditions.
Carbohydrate recognition is a ubiquitous principle underlying many fundamental biological processes like fertilization, embryogenesis and viral infections. But how carbohydrate specificity and affinity induce a molecular event is not well understood. One of these examples is bacteriophage P22 that binds and infects three distinct Salmonella enterica (S.) hosts. It recognizes and depolymerizes repetitive carbohydrate structures of O antigen in its host´s outer membrane lipopolysaccharide molecule. This is mediated by tailspikes, mainly β helical appendages on phage P22 short non contractile tail apparatus (podovirus). The O antigen of all three Salmonella enterica hosts is built from tetrasaccharide repeating units consisting of an identical main chain with a distinguished 3,6 dideoxyhexose substituent that is crucial for P22 tailspike recognition: tyvelose in S. Enteritidis, abequose in S. Typhimurium and paratose in S. Paratyphi. In the first study the complexes of P22 tailspike with its host’s O antigen octasaccharide were characterized. S. Paratyphi octasaccharide binds less tightly (ΔΔG≈7 kJ/mol) to the tailspike than the other two hosts. Crystal structure analysis of P22 tailspike co crystallized with S. Paratyphi octasaccharides revealed different interactions than those observed before in tailspike complexes with S. Enteritidis and S. Typhimurium octasaccharides. These different interactions occur due to a structural rearrangement in the S. Paratyphi octasaccharide. It results in an unfavorable glycosidic bond Φ/Ψ angle combination that also had occurred when the S. Paratyphi octasaccharide conformation was analyzed in an aprotic environment. Contributions of individual protein surface contacts to binding affinity were analyzed showing that conserved structural waters mediate specific recognition of all three different Salmonella host O antigens. Although different O antigen structures possess distinct binding behavior on the tailspike surface, all are recognized and infected by phage P22. Hence, in a second study, binding measurements revealed that multivalent O antigen was able to bind with high avidity to P22 tailspike. Dissociation rates of the polymer were three times slower than for an octasaccharide fragment pointing towards high affinity for O antigen polysaccharide. Furthermore, when phage P22 was incubated with lipopolysaccharide aggregates before plating on S. Typhimurium cells, P22 infectivity became significantly reduced. Therefore, in a third study, the function of carbohydrate recognition on the infection process was characterized. It was shown that large S. Typhimurium lipopolysaccharide aggregates triggered DNA release from the phage capsid in vitro. This provides evidence that phage P22 does not use a second receptor on the Salmonella surface for infection. P22 tailspike binding and cleavage activity modulate DNA egress from the phage capsid. DNA release occurred more slowly when the phage possessed mutant tailspikes with less hydrolytic activity and was not induced if lipopolysaccharides contained tailspike shortened O antigen polymer. Furthermore, the onset of DNA release was delayed by tailspikes with reduced binding affinity. The results suggest a model for P22 infection induced by carbohydrate recognition: tailspikes position the phage on Salmonella enterica and their hydrolytic activity forces a central structural protein of the phage assembly, the plug protein, onto the host´s membrane surface. Upon membrane contact, a conformational change has to occur in the assembly to eject DNA and pilot proteins from the phage to establish infection. Earlier studies had investigated DNA ejection in vitro solely for viruses with long non contractile tails (siphovirus) recognizing protein receptors. Podovirus P22 in this work was therefore the first example for a short tailed phage with an LPS recognition organelle that can trigger DNA ejection in vitro. However, O antigen binding and cleaving tailspikes are widely distributed in the phage biosphere, for example in siphovirus 9NA. Crystal structure analysis of 9NA tailspike revealed a complete similar fold to P22 tailspike although they only share 36 % sequence identity. Moreover, 9NA tailspike possesses similar enzyme activity towards S. Typhimurium O antigen within conserved amino acids. These are responsible for a DNA ejection process from siphovirus 9NA triggered by lipopolysaccharide aggregates. 9NA expelled its DNA 30 times faster than podovirus P22 although the associated conformational change is controlled with a similar high activation barrier. The difference in DNA ejection velocity mirrors different tail morphologies and their efficiency to translate a carbohydrate recognition signal into action.
Die 11beta-HSD1 reguliert intrazellulär die Cortisolkonzentration durch Regeneration von Cortison z.B. aus dem Blutkreislauf, zu Cortisol. Daher stellt diese ein wichtiges Element in der Glucocorticoid-vermittelten Genregulation dar. Die 11beta-HSD1 wird ubiquitär exprimiert, auf hohem Niveau besonders in Leber, Fettgewebe und glatten Muskelzellen. Insbesondere die Bedeutung der 11beta-HSD1 in Leber und Fettgewebe konnte mehrfach nachgewiesen werden. In der Leber führte eine erhöhte Aktivität aufgrund einer Überexpression in Mäusen zu einer verstärkten Gluconeogeneserate. Des Weiteren konnte gezeigt werden, dass eine erhöhte Expression und erhöhte Enzymaktivität der 11beta-HSD1 im subkutanen und viszeralen Fettgewebe assoziiert ist mit Fettleibigkeit, Insulinresistenz und Dyslipidämie. Über die Regulation ist jedoch noch wenig bekannt. Zur Untersuchung der Promotoraktivität wurde der Promotorbereich von -3034 bis +188, vor und nach dem Translations- und Transkriptionsstart, der 11beta-HSD1 kloniert. 8 Promotorfragmente wurden mittels Dual-Luciferase-Assay in humanen HepG2-Zellen sowie undifferenzierten und differenzierten murinen 3T3-L1-Zellen untersucht. Anschließend wurde mittels nicht-radioaktiven EMSA die Bindung des TATA-Binding Proteins (TBP) sowie von CCAAT/Enhancer-Binding-Proteinen (C/EBP) an ausgewählte Promotorregionen analysiert. Nach der Charakterisierung des Promotors wurden spezifische endogene und exogene Regulatoren untersucht. Fettsäuren modifizieren die Entstehung von Adipositas und Insulinresistenz. Ihre Wirkung wird u.a. PPARgamma-abhängig vermittelt und kann durch das Inkretin (Glucose-dependent insulinotropic Peptide) GIP modifiziert werden. So wurden die Effekte von unterschiedlichen Fettsäuren, vom PPARgamma Agonisten Rosiglitazon sowie dem Inkretin GIP auf die Expression und Enzymaktivität der 11beta-HSD1 untersucht. Dies wurde in-vitro-, tierexperimentell und in humanen in-vivo-Studien realisiert. Zuletzt wurden 2 Single Nucleotide Polymorphismen (SNP) im Promotorbereich der 11beta-HSD1 in der Zellkultur im Hinblick auf potentielle Funktionalität analysiert sowie die Assoziation mit Diabetes mellitus Typ 2 und Körpergewicht in der MeSyBePo-Kohorte bei rund 1.800 Personen untersucht. Die Luciferase-Assays zeigten basal eine zell-spezifische Regulation der 11beta-HSD1, wobei in allen 3 untersuchten Zelltypen die Bindung eines Repressors nachgewiesen werden konnte. Zudem konnte eine mögliche Bindung des TBPs sowie von C/EBP-Proteinen an verschiedene Positionen gezeigt werden. Die Transaktivierungsassays mit den C/EBP-Proteinen -alpha, -beta und -delta zeigten eben-falls eine zellspezifische Regulation des 11beta-HSD1-Promotors. Die Aktivität und Expression der 11beta-HSD1 wurde durch die hier untersuchten endogenen und exogenen Faktoren spezifisch modifiziert, was sowohl in-vitro als auch in-vivo in unterschiedlichen Modellsystemen dargestellt werden konnte. Die Charakterisierung der MeSyBePo-Kohorte ergab keine direkten Assoziationen zwischen Polymorphismus und klinischem Phänotyp, jedoch Tendenzen für eine erhöhtes Körper-gewicht und Typ 2 Diabetes mellitus in Abhängigkeit des Genotyps. Der Promotor der 11beta-HSD1 konnte aufgrund der Daten aus den Luciferaseassays sowie den Daten aus den EMSA-Analysen näher charakterisiert werden. Dieser zeigt eine variable und zell-spezifische Regulation. Ein wichtiger Regulator stellen insbesondere in den HepG2-Zellen die C/EBP-Proteine -alpha, -beta und -delta dar. Aus den in-vivo-Studien ergab sich eine Regulation der 11beta-HSD1 durch endogene, exogene und pharmakologische Substanzen, die durch die Zellkulturversuche bestätigt und näher charakterisiert werden konnten.
In der molekularen Diagnostik besteht ein Bedarf an schnellen und spezifischen Testsystemen, die entweder für die Labordiagnostik oder in Point of Care-Umgebungen eingesetzt werden können. Um dieses Ziel zu erreichen, stehen die Miniaturisierung und Parallelisierung im Mittelpunkt des Forschungsinteresses. Die führende Methode im Bereich der DNA-Analytik ist derzeit die Realtime-PCR. Dieser Technologie sind hinsichtlich der Multiplexfähigkeit technologischen Hürden gesetzt, da derzeit nur eine Analyse von maximal vier Parametern parallel in einem Versuchsansatz erfolgen kann. Microarrays stellen hingegen die benötigten Voraussetzungen zur Verfügung, um als Werkzeuge für die Multiparameteranalyse in verschiedensten Anwendungsbereichen zu dienen. Ein Schwerpunkt dieser Arbeit war es, Multiplex-PCRs und diagnostische Microarrays zu entwickeln, die für analytische Fragestellungen eine schnelle und zuverlässige Multiparameteranalytik ermöglichen, um die bisherigen Einschränkungen aktueller Nachweisverfahren zu vermeiden. Als Anwendungen wurden zum einen ein Nachweissystem für acht relevante Geflügelpathogene zur Überwachung in der Geflügelzucht, zum anderen ein Nachweissystem zur Identifikation potentiell allergener Lebensmittelinhaltstoffe entwickelt. Neben der Entwicklung geeigneter PCR und Multiplex-PCR-Verfahren sowie spezifischer Microarrays für die Detektion der gesuchten Zielsequenzen stand auch die weiterführende Integration von DNA-Amplifikation und Microarray-Technologie im Fokus dieser Arbeit. Die OnChip-Amplifikation stellt eine Möglichkeit dar, um DNA-Analytik und Detektion in einem Reaktionsschritt zu integrieren. Entsprechend wurden die in der Arbeit entwickelten PCR- und Multiplex-PCR-Verfahren zum Nachweis potentieller allergener Lebensmittelinhaltsstoffe für die OnChip-Amplifikation adaptiert und Reaktionsbedingungen getestet, die eine Multiparameteranalyse auf dem Chip ermöglichen. Die entwickelten OnChip-PCR-Verfahren zeigten eine hohe Spezifität sowohl in Single- als auch in der Multiplex-OnChip-PCR. Eine Sensitivität von 10 Kopien bzw. <10ppm konnte in Single-OnChip-PCRs für den Nachweis allergener Lebensmittelinhaltsstoffe gezeigt werden. In Multiplex-OnChip-PCRs konnten 10-100ppm allergene Verunreinigungen spezifisch in unterschiedlichen Lebensmitteln nachgewiesen werden. Ein weiterer Schritt in Richtung einer möglichen Verwendung im Point of Care-Bereich stellt der Einsatz eines isothermalen Amplifikationsverfahrens dar. Vorteil eines solchen Verfahrens ist die Möglichkeit, auf das ansonsten benötigte Thermocycling zu verzichten. Dies vereinfacht eine Integration der OnChip-Amplifikation in mobile Analysegeräte oder Lab on Chip-Systeme und qualifiziert das Verfahren für den Einsatz in Point of Care-Umgebungen. In dieser Arbeit wurde eine noch junge isothermale Amplifikationsmethode, die helikase-abhängige Amplifikation (HDA), hinsichtlich ihrer Eignung für die Integration auf einem Microarray getestet. Hierfür konnte die bislang erste OnChip-HDA für Einzel- und Duplex-Nachweise von Pathogenen entwickelt werden.
The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.
Coherent network partitions
(2019)
Graph clustering is widely applied in the analysis of cellular networks reconstructed from large-scale data or obtained from experimental evidence. Here we introduce a new type of graph clustering based on the concept of coherent partition. A coherent partition of a graph G is a partition of the vertices of G that yields only disconnected subgraphs in the complement of G. The coherence number of G is then the size of the smallest edge cut inducing a coherent partition. A coherent partition of G is optimal if the size of the inducing edge cut is the coherence number of G. Given a graph G, we study coherent partitions and the coherence number in connection to (bi)clique partitions and the (bi)clique cover number. We show that the problem of finding the coherence number is NP-hard, but is of polynomial time complexity for trees. We also discuss the relation between coherent partitions and prominent graph clustering quality measures.
Life history theory predicts that experiencing stress during the early period of life will result in accelerated growth and earlier maturation. Indeed, animal and some human studies documented a faster pace of growth in the offspring of stressed mothers. Recent advances in epigenetics suggest that the effects of early developmental stress might be passed across the generations. However, evidence for such intergenerational transmission is scarce, at least in humans. Here we report the results of the study investigating the association between childhood trauma in mothers and physical growth in their children during the first months of life. Anthropometric and psychological data were collected from 99 mothers and their exclusively breastfed children at the age of 5 months. The mothers completed the Early Life Stress Questionnaire to assess childhood trauma. The questionnaire includes questions about the most traumatic events that they had experienced before the age of 12 years. Infant growth was evaluated based on the anthropometric measurements of weight, length, and head circumference. Also, to control for the size of maternal investment, the composition of breast milk samples taken at the time of infant anthropometric measurements was investigated. The children of mothers with higher early life stress tended to have higher weight and bigger head circumference. The association between infant anthropometrics and early maternal stress was not affected by breast milk composition, suggesting that the effect of maternal stress on infant growth was independent of the size of maternal investment. Our results demonstrate that early maternal trauma may affect the pace of growth in the offspring and, in consequence, lead to a faster life history strategy. This effect might be explained via changes in offspring epigenetics.
Seasonal, host sex and age-related variations in helminth egg and coccidian oocyst counts were investigated in a naturally infected wild bushbuck (Tragelaphus scriptus) population in Queen Elizabeth National Park, western Uganda from April 2000 to February 2002. The prevalence and mean intensity quantified as the number of eggs and oocysts per gram of faeces were taken as a measure of parasite burdens. Host sex and age-related differences in prevalence values were not found but the overall prevalence of Eimeria sp. was significantly higher during the rainy season, and peak counts were recorded either during or soon after a peak rainfall. A similar trend was observed for Moniezia spp., although the results were marginally not significant. There were also no significant differences in mean intensity values, relative to host sex, age or season.
The central melanin-concentrating hormone (MCH) system has been intensively studied for its involvement in the regulation of feeding behaviour and body weight regulation. The importance of the neuropeptide MCH in the control of energy balance has been underlined by MCH knock out and Melanin-concentrating hormone receptor subtype 1 (MCHR-1) knock-out animals. The anorectic and anti-obesity effects of selective MCHR-1 antagonists have confirmed the notion that pharmacological blockade of MCHR-1 is a potential therapeutic approach for obesity. First aim of this work is to study the neurochemical “equipment” of MCHR-1 immunoreactive neurons by double-labelling immunohistochemistry within the rat hypothalamus. Of special interest is the neuroanatomical identification of other hypothalamic neuropeptides that are co-distributed with MCHR-1. A second part of this study deals with the examination of neuronal activation patterns after pharmacological or physiological, feeding-related stimuli and was introduced to further understand central regulatory mechanisms of the MCH system. In the first part of work, I wanted to neurochemically characterize MCHR-1 immunoreactive neurons in the rat hypothalamus for colocalisation with neuropeptides of interest. Therefore I performed an immunohistochemical colocalisation study using a specific antibody against MCHR-1 in combination with antibodies against hypothalamic neuropeptides. I showed that MCHR-1 immunoreactivity (IR) was co-localised with orexin A in the lateral hypothalamus, and with adrenocorticotropic hormone and neuropeptide Y in the arcuate nucleus. Additionally, MCHR-1 IR was co-localised with the neuropeptides vasopressin and oxytocin in magnocellular neurons of the supraoptic and paraventricular hypothalamic nucleus and corticotrophin releasing hormone in the parvocellular division of the paraventricular hypothalamic nucleus. Moreover, for the first time MCHR-1 immunoreactivity was found in both the adenohypophyseal and neurohypophyseal part of the rat pituitary. These results provide the neurochemical basis for previously described potential physiological actions of MCH at its target receptor. In particular, the MCHR-1 may be involved not only in food intake regulation, but also in other physiological actions such as fluid regulation, reproduction and stress response, possibly through here examined neuropeptides. Central activation patterns induced by pharmacological or physiological stimulation can be mapped using c-Fos immunohistochemistry. In the first experimental design, central administration (icv) of MCH in the rat brain resulted in acute and significant increase of food and water intake, but this animal treatment did not induce a specific c-Fos induction pattern in hypothalamic nuclei. In contrast, sub-chronic application of MCHR-1 antagonist promoted a significant decrease in food- and water intake during an eight day treatment period. A qualitative analysis of c-Fos immunohistochemistry of sections derived from MCHR-1 antagonist treated animals showed a specific neuronal activation in the paraventricular nucleus, the supraoptic nucleus and the dorsomedial hypothalamus. These results could be substantiated by quantitative evaluation of an automated, software-supported analysis of the c-Fos signal. Additionally, I examined the activation pattern of rats in a restricted feeding schedule (RFS) to identify pathways involved in hunger and satiety. Animals were trained for 9 days to feed during a three hour period. On the last day, food restricted animals was also allowed to feed for the three hours, while food deprived (FD) animals did not receive food. Mapping of neuronal activation showed a clear difference between stareved (FD) and satiated (FR) rats. FD animals showed significant induction of c-Fos in forebrain regions, several hypothalamic nuclei, amygdaloid thalamus and FR animals in the supraoptic nucleus and the paraventricular nucleus of the hypothalamus, and the nucleus of the solitary tract. In the lateral hypothalamus of FD rats, c-Fos IR showed strong colocalisation for Orexin A, but no co-staining for MCH immunoreactivity. However, a large number of c-Fos IR neurons within activated regions of FD and FR animals was co-localised with MCHR-1 within selected regions. To conclude, the experimental set-up of scheduled feeding can be used to induce a specific hunger or satiety activation pattern within the rat brain. My results show a differential activation by hunger signals of MCH neurons and furthermore, demonstrates that MCHR-1 expressing neurons may be essential parts of downstream processing of physiological feeding/hunger stimuli. In the final part of my work, the relevance of here presented studies is discussed with respect to possible introduction of MCHR-1 antagonists as drug candidates for the treatment of obesity.
Starch is an essential biopolymer produced by plants. Starch can be made inside source tissue (such as leaves) and sink tissue (such as fruits and tubers). Nevertheless, understanding how starch metabolism is regulated in source and sink tissues is fundamental for improving crop production.
Despite recent advances in the understanding of starch and its metabolism, there is still a knowledge gap in the source and sink metabolism. Therefore, this study aimed to summarize the state of the art regarding starch structure and metabolism inside plants. In addition, this study aimed to elucidate the regulation of starch metabolism in the source tissue using the leaves of a model organism, Arabidopsis thaliana, and the sink tissue of oil palm (Elaeis guineensis) fruit as a commercial crop.
The research regarding the source tissue will focus on the effect of the blockage of starch degradation on the starch parameter in leaves, especially in those of A. thaliana, which lack both disproportionating enzyme 2 (DPE2) and plastidial glucan phosphorylase 1 (PHS1) (dpe2/phs1). The additional elimination of phosphoglucan water dikinase (PWD), starch excess 4 (SEX4), isoamylase 3 (ISA3), and disproportionating enzyme 1 (DPE1) in the dpe2/phs1 mutant background demonstrates the alteration of starch granule number per chloroplast. This study provides insights into the control mechanism of granule number regulation in the chloroplast.
The research regarding the sink tissue will emphasize the relationship between starch metabolism and the lipid metabolism pathway in oil palm fruits. This study was conducted to observe the alteration of starch parameters, metabolite abundance, and gene expression during oil palm fruit development with different oil yields. This study shows that starch and sucrose can be used as biomarkers for oil yield in oil palms. In addition, it is revealed that the enzyme isoforms related to starch metabolism influence the oil production in oil palm fruit.
Overall, this thesis presents novel information regarding starch metabolism in the source tissue of A.thaliana and the sink tissue of E.guineensis. The results shown in this thesis can be applied to many applications, such as modifying the starch parameter in other plants for specific needs.
Transcriptomic dataset for early inflorescence stages of oil palm in response to defoliation stress
(2022)
Oil palm breeding and seed development have been hindered due to the male parent's incapacity to produce male inflorescence as a source of pollen under normal conditions. On the other hand, a young oil palm plantation has a low pollination rate due to a lack of male flowers. These are the common problem of sex ratio in the oil palm industry. Nevertheless, the regulation of sex ratio in oil palm plants is a complex mechanism and remains an open question until now. Researchers have previously used complete defoliation to induce male inflorescences, but the biological and molecular mechanisms underlying this morphological change have yet to be discovered. Here, we present an RNA-seq dataset from three early stages of an oil palm inflorescence under normal conditions and complete defoliation stress. This transcriptomic dataset is a valuable resource to improve our understanding of sex determination mechanisms in oil palm inflorescence.
Starch is a complex carbohydrate polymer produced by plants and especially by crops in huge amounts. It consists of amylose and amylopectin, which have alpha-1,4-and alpha-1,6-linked glucose units. Despite this simple chemistry, the entire starch metabolism is complex, containing various (iso)enzymes/proteins. However, whose interplay is still not yet fully understood. Starch is essential for humans and animals as a source of nutrition and energy. Nowadays, starch is also commonly used in non-food industrial sectors for a variety of purposes. However, native starches do not always satisfy the needs of a wide range of (industrial) applications. This review summarizes the structural properties of starch, analytical methods for starch characterization, and in planta starch modifications.
Elaeidobius kamerunicus Faust. (Coleoptera: Curculionidae) is an essential insect pollinator in oil palm plantations. Recently, researches have been undertaken to improve pollination efficiency using this species. A fundamental understanding of the genes related to this pollinator behavior is necessary to achieve this goal. Here, we present the draft genome sequence, annotation, and simple sequence repeat (SSR) marker data for this pollinator. In total, 34.97 Gb of sequence data from one male individual (monoisolate) were obtained using Illumina short-read platform NextSeq 500. The draft genome assembly was found to be 269.79 Mb and about 59.9% of completeness based on Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment. Functional gene annotation predicted about 26.566 genes. Also, a total of 281.668 putative SSR markers were identified. This draft genome sequence is a valuable resource for understanding the population genetics, phylogenetics, dispersal patterns, and behavior of this species.
The complete mitochondrial genome of oil palm pollinating weevil, Elaeidobius kamerunicus Faust
(2020)
Elaeidobius kamerunicusis the most important insect pollinator in oil palm plantations. In this study, the mitochondrial genome (mitogenome) ofE. kamerunicus(17.729 bp), a member of the Curculionidae family, will be reported. The mitogenome consisted of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a putative control region (CR). Phylogenetic analysis based on 13 protein-coding genes (PCGs) using maximum Likelihood (ML) methods indicated thatE. kamerunicusbelongs to the Curculionidae family. This mitochondrial genome provides essential information for understanding genetic populations, phylogenetics, molecular evolution, and other biological applications in this species.
Biochemical and physiological studies of Arabidopsis thaliana Diacylglycerol Kinase 7 (AtDGK7)
(2006)
A family of diacylglycerol kinases (DGK) phosphorylates the substrate diacylglycerol (DAG) to generate phosphatidic acid (PA) . Both molecules, DAG and PA, are involved in signal transduction pathways. In the model plant Arabidopsis thaliana, seven candidate genes (named AtDGK1 to AtDGK7) code for putative DGK isoforms. Here I report the molecular cloning and characterization of AtDGK7. Biochemical, molecular and physiological experiments of AtDGK7 and their corresponding enzyme are analyzed. Information from Genevestigator says that AtDGK7 gene is expressed in seedlings and adult Arabidopsis plants, especially in flowers. The AtDGK7 gene encodes the smallest functional DGK predicted in higher plants; but also, has an alternative coding sequence containing an extended AtDGK7 open reading frame, confirmed by PCR and submitted to the GenBank database (under the accession number DQ350135). The new cDNA has an extension of 439 nucleotides coding for 118 additional amino acids The former AtDGK7 enzyme has a predicted molecular mass of ~41 kDa and its activity is affected by pH and detergents. The DGK inhibitor R59022 also affects AtDGK7 activity, although at higher concentrations (i.e. IC50 ~380 µM). The AtDGK7 enzyme also shows a Michaelis-Menten type saturation curve for 1,2-DOG. Calculated Km and Vmax were 36 µM 1,2-DOG and 0.18 pmol PA min-1 mg of protein-1, respectively, under the assay conditions. Former protein AtDGK7 are able to phosphorylate different DAG analogs that are typically found in plants. The new deduced AtDGK7 protein harbors the catalytic DGKc and accessory domains DGKa, instead the truncated one as the former AtDGK7 protein (Gomez-Merino et al., 2005).
Plastic pollution is ubiquitous on the planet since several millions of tons of plastic waste enter aquatic ecosystems each year. Furthermore, the amount of plastic produced is expected to increase exponentially shortly. The heterogeneity of materials, additives and physical characteristics of plastics are typical of these emerging contaminants and affect their environmental fate in marine and freshwaters. Consequently, plastics can be found in the water column, sediments or littoral habitats of all aquatic ecosystems. Most of this plastic debris will fragment as a product of physical, chemical and biological forces, producing particles of small size. These particles (< 5mm) are known as “microplastics” (MP). Given their high surface-to-volume ratio, MP stimulate biofouling and the formation of biofilms in aquatic systems.
As a result of their unique structure and composition, the microbial communities in MP biofilms are referred to as the “Plastisphere.” While there is increasing data regarding the distinctive composition and structure of the microbial communities that form part of the plastisphere, scarce information exists regarding the activity of microorganisms in MP biofilms. This surface-attached lifestyle is often associated with the increase in horizontal gene transfer (HGT) among bacteria. Therefore, this type of microbial activity represents a relevant function worth to be analyzed in MP biofilms. The horizontal exchange of mobile genetic elements (MGEs) is an essential feature of bacteria. It accounts for the rapid evolution of these prokaryotes and their adaptation to a wide variety of environments. The process of HGT is also crucial for spreading antibiotic resistance and for the evolution of pathogens, as many MGEs are known to contain antibiotic resistance genes (ARGs) and genetic determinants of pathogenicity.
In general, the research presented in this Ph.D. thesis focuses on the analysis of HGT and heterotrophic activity in MP biofilms in aquatic ecosystems. The primary objective was to analyze the potential of gene exchange between MP bacterial communities vs. that of the surrounding water, including bacteria from natural aggregates. Moreover, the thesis addressed the potential of MP biofilms for the proliferation of biohazardous bacteria and MGEs from wastewater treatment plants (WWTPs) and associated with antibiotic resistance. Finally, it seeks to prove if the physiological profile of MP biofilms under different limnological conditions is divergent from that of the water communities. Accordingly, the thesis is composed of three independent studies published in peer-reviewed journals. The two laboratory studies were performed using both model and environmental microbial communities. In the field experiment, natural communities from freshwater ecosystems were examined.
In Chapter I, the inflow of treated wastewater into a temperate lake was simulated with a concentration gradient of MP particles. The effects of MP on the microbial community structure and the occurrence of integrase 1 (int 1) were followed. The int 1 is a marker associated with mobile genetic elements and known as a proxy for anthropogenic effects on the spread of antimicrobial resistance genes. During the experiment, the abundance of int1 increased in the plastisphere with increasing MP particle concentration, but not in the surrounding water. In addition, the microbial community on MP was more similar to the original wastewater community with increasing microplastic concentrations. Our results show that microplastic particles indeed promote persistence of standard indicators of microbial anthropogenic pollution in natural waters.
In Chapter II, the experiments aimed to compare the permissiveness of aquatic bacteria towards model antibiotic resistance plasmid pKJK5, between communities that form biofilms on MP vs. those that are free-living. The frequency of plasmid transfer in bacteria associated with MP was higher when compared to bacteria that are free-living or in natural aggregates. Moreover, comparison increased gene exchange occurred in a broad range of phylogenetically-diverse bacteria. The results indicate a different activity of HGT in MP biofilms, which could affect the ecology of aquatic microbial communities on a global scale and the spread of antibiotic resistance.
Finally, in Chapter III, physiological measurements were performed to assess whether microorganisms on MP had a different functional diversity from those in water. General heterotrophic activity such as oxygen consumption was compared in microcosm assays with and without MP, while diversity and richness of heterotrophic activities were calculated by using Biolog® EcoPlates. Three lakes with different nutrient statuses presented differences in MP-associated biomass build up. Functional diversity profiles of MP biofilms in all lakes differed from those of the communities in the surrounding water, but only in the oligo-mesotrophic lake MP biofilms had a higher functional richness compared to the ambient water. The results support that MP surfaces act as new niches for aquatic microorganisms and can affect global carbon dynamics of pelagic environments.
Overall, the experimental works presented in Chapters I and II support a scenario where MP pollution affects HGT dynamics among aquatic bacteria. Among the consequences of this alteration is an increase in the mobilization and transfer efficiency of ARGs. Moreover, it supposes that changes in HGT can affect the evolution of bacteria and the processing of organic matter, leading to different catabolic profiles such as demonstrated in Chapter III. The results are discussed in the context of the fate and magnitude of plastic pollution and the importance of HGT for bacterial evolution and the microbial loop, i.e., at the base of aquatic food webs. The thesis supports a relevant role of MP biofilm communities for the changes observed in the aquatic microbiome as a product of intense human intervention.
Heterotrophic microbes with the capability to process considerable amounts of organic matter can colonize microplastic particles (MP) in aquatic ecosystems. Weather colonization of microorganisms on MP will alter ecological niche and functioning of microbial communities remains still unanswered. Therefore, we compared the functional diversity of biofilms on microplastics when incubated in three lakes in northeastern Germany differing in trophy and limnological features. For all lakes, we compared heterotrophic activities of MP biofilms with those of microorganisms in the surrounding water by using Biolog (R) EcoPlates and assessed their oxygen consumption in microcosm assays with and without MP. The present study found that the total biofilm biomass was higher in the oligo-mesotrophic and dystrophic lakes than in the eutrophic lake. In all lakes, functional diversity profiles of MP biofilms consistently differed from those in the surrounding water. However, solely in the oligo-mesotrophic lake MP biofilms had a higher functional richness compared to the ambient water. These results demonstrate that the functionality and hence the ecological role of MP-associated microbial communities are context-dependent, i.e. different environments lead to substantial changes in biomass build up and heterotrophic activities of MP biofilms. We propose that MP surfaces act as new niches for aquatic microorganisms and that the constantly increasing MP pollution has the potential to globally impact carbon dynamics of pelagic environments by altering heterotrophic activities. (C) 2018 Elsevier B.V. All rights reserved.
Pollution by microplastics in aquatic ecosystems is accumulating at an unprecedented scale, emerging as a new surface for biofilm formation and gene exchange. In this study, we determined the permissiveness of aquatic bacteria towards a model antibiotic resistance plasmid, comparing communities that form biofilms on microplastics vs. those that are free-living. We used an exogenous and red-fluorescent E. coli donor strain to introduce the green-fluorescent broad-host-range plasmid pKJKS which encodes for trimethoprim resistance. We demonstrate an increased frequency of plasmid transfer in bacteria associated with microplastics compared to bacteria that are free-living or in natural aggregates. Moreover, comparison of communities grown on polycarbonate filters showed that increased gene exchange occurs in a broad range of phylogenetically-diverse bacteria. Our results indicate horizontal gene transfer in this habitat could distinctly affect the ecology of aquatic microbial communities on a global scale. The spread of antibiotic resistance through microplastics could also have profound consequences for the evolution of aquatic bacteria and poses a neglected hazard for human health.
Microplastics (MP) provide a unique and extensive surface for microbial colonization in aquatic ecosystems. The formation of microorganism-microplastic complexes, such as biofilms, maximizes the degradation of organic matter and horizontal gene transfer. In this context, MP affect the structure and function of microbial communities, which in turn render the physical and chemical fate of MP. This new paradigm generates challenges for microbiology, ecology, and ecotoxicology. Dispersal of MP is concomitant with that of their associated microorganisms and their mobile genetic elements, including antibiotic resistance genes, islands of pathogenicity, and diverse metabolic pathways. Functional changes in aquatic microbiomes can alter carbon metabolism and food webs, with unknown consequences on higher organisms or human microbiomes and hence health. Here, we examine a variety of effects of MP pollution from the microbial ecology perspective, whose repercussions on aquatic ecosystems begin to be unraveled. (C) 2018 Elsevier B.V. All rights reserved.
From dark to light
(2016)
Mathematical modeling of biological systems is a powerful tool to systematically investigate the functions of biological processes and their relationship with the environment. To obtain accurate and biologically interpretable predictions, a modeling framework has to be devised whose assumptions best approximate the examined scenario and which copes with the trade-off of complexity of the underlying mathematical description: with attention to detail or high coverage. Correspondingly, the system can be examined in detail on a smaller scale or in a simplified manner on a larger scale. In this thesis, the role of photosynthesis and its related biochemical processes in the context of plant metabolism was dissected by employing modeling approaches ranging from kinetic to stoichiometric models. The Calvin-Benson cycle, as primary pathway of carbon fixation in C3 plants, is the initial step for producing starch and sucrose, necessary for plant growth. Based on an integrative analysis for model ranking applied on the largest compendium of (kinetic) models for the Calvin-Benson cycle, those suitable for development of metabolic engineering strategies were identified. Driven by the question why starch rather than sucrose is the predominant transitory carbon storage in higher plants, the metabolic costs for their synthesis were examined. The incorporation of the maintenance costs for the involved enzymes provided a model-based support for the preference of starch as transitory carbon storage, by only exploiting the stoichiometry of synthesis pathways. Many photosynthetic organisms have to cope with processes which compete with carbon fixation, such as photorespiration whose impact on plant metabolism is still controversial. A systematic model-oriented review provided a detailed assessment for the role of this pathway in inhibiting the rate of carbon fixation, bridging carbon and nitrogen metabolism, shaping the C1 metabolism, and influencing redox signal transduction. The demand of understanding photosynthesis in its metabolic context calls for the examination of the related processes of the primary carbon metabolism. To this end, the Arabidopsis core model was assembled via a bottom-up approach. This large-scale model can be used to simulate photoautotrophic biomass production, as an indicator for plant growth, under so-called optimal, carbon-limiting and nitrogen-limiting growth conditions. Finally, the introduced model was employed to investigate the effects of the environment, in particular, nitrogen, carbon and energy sources, on the metabolic behavior. This resulted in a purely stoichiometry-based explanation for the experimental evidence for preferred simultaneous acquisition of nitrogen in both forms, as nitrate and ammonium, for optimal growth in various plant species. The findings presented in this thesis provide new insights into plant system's behavior, further support existing opinions for which mounting experimental evidences arise, and posit novel hypotheses for further directed large-scale experiments.
The mammalian neck adopts a variety of postures during daily life and generates numerous head trajectories. Despite its functional diversity, the neck is constrained to seven cervical vertebrae in (almost) all mammals. Given this low number, an unexpectedly high degree of modularity of the mammalian neck has more recently been uncovered. This work aims to review neck modularity in mammals from a developmental, morpho-functional, and paleontological perspective and how high functional diversity evolved in the mammalian neck after the occurrence of meristic limitations. The fixed number of cervical vertebrae and the developmental modularity of the mammalian neck are closely linked to anterior Hox genes expression and strong developmental integration between the neck and other body regions. In addition, basic neck biomechanics promote morpho-functional modularity due to preferred motion axes in the cranio-cervical and cervico-thoracic junction. These developmental and biomechanical determinants result in the characteristic and highly conserved shape variation among the vertebrae that delimits morphological modules. The step-wise acquisition of these unique cervical traits can be traced in the fossil record. The increasing functional specialization of neck modules, however, did not evolve all at once but started much earlier in the upper than in the lower neck. Overall, the strongly conserved modularity in the mammalian neck represents an evolutionary trade-off between the meristic constraints and functional diversity. Although a morpho-functional partition of the neck is common among amniotes, the degree of modularity and the way neck disparity is realized is unique in mammals.
In a very simplified view, the plant leaf growth can be reduced to two processes, cell division and cell expansion, accompanied by expansion of their surrounding cell walls. The vacuole, as being the largest compartment of the plant cell, plays a major role in controlling the water balance of the plant. This is achieved by regulating the osmotic pressure, through import and export of solutes over the vacuolar membrane (the tonoplast) and by controlling the water channels, the aquaporins. Together with the control of cell wall relaxation, vacuolar osmotic pressure regulation is thought to play an important role in cell expansion, directly by providing cell volume and indirectly by providing ion and pH homestasis for the cytosoplasm. In this thesis the role of tonoplast protein coding genes in cell expansion in the model plant Arabidopsis thaliana is studied and genes which play a putative role in growth are identified. Since there is, to date, no clearly identified protein localization signal for the tonoplast, there is no possibility to perform genome-wide prediction of proteins localized to this compartment. Thus, a series of recent proteomic studies of the tonoplast were used to compile a list of cross-membrane tonoplast protein coding genes (117 genes), and other growth-related genes from notably the growth regulating factor (GRF) and expansin families were included (26 genes). For these genes a platform for high-throughput reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) was developed by selecting specific primer pairs. To this end, a software tool (called QuantPrime, see http://www.quantprime.de) was developed that automatically designs such primers and tests their specificity in silico against whole transcriptomes and genomes, to avoid cross-hybridizations causing unspecific amplification. The RT-qPCR platform was used in an expression study in order to identify candidate growth related genes. Here, a growth-associative spatio-temporal leaf sampling strategy was used, targeting growing regions at high expansion developmental stages and comparing them to samples taken from non-expanding regions or stages of low expansion. Candidate growth related genes were identified after applying a template-based scoring analysis on the expression data, ranking the genes according to their association with leaf expansion. To analyze the functional involvement of these genes in leaf growth on a macroscopic scale, knockout mutants of the candidate growth related genes were screened for growth phenotypes. To this end, a system for non-invasive automated leaf growth phenotyping was established, based on a commercially available image capture and analysis system. A software package was developed for detailed developmental stage annotation of the images captured with the system, and an analysis pipeline was constructed for automated data pre-processing and statistical testing, including modeling and graph generation, for various growth-related phenotypes. Using this system, 24 knockout mutant lines were analyzed, and significant growth phenotypes were found for five different genes.
QuantPrime
(2008)
Background
Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays.
Results
Here we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale qPCR analyses. QuantPrime can be used online through the internet http://www.quantprime.de/ or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant Arabidopsis thaliana, the crop Hordeum vulgare and the model green alga Chlamydomonas reinhardtii show success rates of designed primer pairs exceeding 96%.
Conclusion
QuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative in situ hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broad-range platform for the design of RNA expression assays.
Due to continuously intensifying human usage of the marine environment worldwide ranging cetaceans face an increasing number of threats. Besides whaling, overfishing and by-catch, new technical developments increase the water and noise pollution, which can negatively affect marine species. Cetaceans are especially prone to these influences, being at the top of the food chain and therefore accumulating toxins and contaminants. Furthermore, they are extremely noise sensitive due to their highly developed hearing sense and echolocation ability. As a result, several cetacean species were brought to extinction during the last century or are now classified as critically endangered. This work focuses on two odontocetes. It applies and compares different molecular methods for inference of population status and adaptation, with implications for conservation. The worldwide distributed sperm whale (Physeter macrocephalus) shows a matrilineal population structure with predominant male dispersal. A recently stranded group of male sperm whales provided a unique opportunity to investigate male grouping for the first time. Based on the mitochondrial control region, I was able to infer that male bachelor groups comprise multiple matrilines, hence derive from different social groups, and that they represent the genetic variability of the entire North Atlantic. The harbor porpoise (Phocoena phocoena) occurs only in the northern hemisphere. By being small and occurring mostly in coastal habitats it is especially prone to human disturbance. Since some subspecies and subpopulations are critically endangered, it is important to generate and provide genetic markers with high resolution to facilitate population assignment and subsequent protection measurements. Here, I provide the first harbour porpoise whole genome, in high quality and including a draft annotation. Using it for mapping ddRAD seq data, I identify genome wide SNPs and, together with a fragment of the mitochondrial control region, inferred the population structure of its North Atlantic distribution range. The Belt Sea harbors a distinct subpopulation oppose to the North Atlantic, with a transition zone in the Kattegat. Within the North Atlantic I could detect subtle genetic differentiation between western (Canada-Iceland) and eastern (North Sea) regions, with support for a German North Sea breading ground around the Isle of Sylt. Further, I was able to detect six outlier loci which show isolation by distance across the investigated sampling areas. In employing different markers, I could show that single maker systems as well as genome wide data can unravel new information about population affinities of odontocetes. Genome wide data can facilitate investigation of adaptations and evolutionary history of the species and its populations. Moreover, they facilitate population genetic investigations, providing a high resolution, and hence allowing for detection of subtle population structuring especially important for highly mobile cetaceans.
The globally distributed sperm whale (Physeter macrocephalus) has a partly matrilineal social structure with predominant male dispersal. At the beginning of 2016, a total of 30 male sperm whales stranded in five different countries bordering the southern North Sea. It has been postulated that these individuals were on a migration route from the north to warmer temperate and tropical waters where females live in social groups. By including samples from four countries (n = 27), this event provided a unique chance to genetically investigate the maternal relatedness and the putative origin of these temporally and spatially co-occuring male sperm whales. To utilize existing genetic resources, we sequenced 422 bp of the mitochondrial control region, a molecular marker for which sperm whale data are readily available from the entire distribution range. Based on four single nucleotide polymorphisms (SNPs) within the mitochondrial control region, five matrilines could be distinguished within the stranded specimens, four of which matched published haplotypes previously described in the Atlantic. Among these male sperm whales, multiple matrilineal lineages co-occur. We analyzed the population differentiation and could show that the genetic diversity of these male sperm whales is comparable to the genetic diversity in sperm whales from the entire Atlantic Ocean. We confirm that within this stranding event, males do not comprise maternally related individuals and apparently include assemblages of individuals from different geographic regions. (c) 2017 Deutsche Gesellschaft fur Saugetierkunde. Published by Elsevier GmbH. All rights reserved.
The harbour porpoise (Phocoena phocoena) is a highly mobile cetacean found across the Northern hemisphere. It occurs in coastal waters and inhabits basins that vary broadly in salinity, temperature and food availability. These diverse habitats could drive subtle differentiation among populations, but examination of this would be best conducted with a robust reference genome. Here, we report the first harbour porpoise genome, assembled de novo from an individual originating in the Kattegat Sea (Sweden). The genome is one of the most complete cetacean genomes currently available, with a total size of 2.39 Gb and 50% of the total length found in just 34 scaffolds. Using 122 of the longest scaffolds, we were able to show high levels of synteny with the genome of the domestic cattle (Bos taurus). Our draft annotation comprises 22,154 predicted genes, which we further annotated through matches to the NCBI nucleotide database, GO categorization and motif prediction. Within the predicted genes, we have confirmed the presence of >20 genes or gene families that have been associated with adaptive evolution in other cetaceans. Overall, this genome assembly and draft annotation represent a crucial addition to the genomic resources currently available for the study of porpoises and Phocoenidae evolution, phylogeny and conservation.
Multidrug resistant (MDR) Pseudomonas aeruginosa having strong biofilm potential and virulence factors are a serious threat for hospitalized patients having compromised immunity In this study, 34 P. aeruginosa isolates of human origin (17 MDR and 17 non-MDR clinical isolates) were checked for biofilm formation potential in enriched and minimal media. The biofilms were detected using crystal violet method and a modified software package of the automated VideoScan screening method. Cytotoxic potential of the isolates was also investigated on HepG2, LoVo and T24 cell lines using automated VideoScan technology. Pulse field gel electrophoresis revealed 10 PFGE types in MDR and 8 in non-MDR isolates. Although all isolates showed biofilm formation potential, strong biofilm formation was found more in enriched media than in minimal media. Eight MDR isolates showed strong biofilm potential in both enriched and minimal media by both detection methods. Strong direct correlation between crystal violet and VideoScan methods was observed in identifying strong biofilm forming isolates. High cytotoxic effect was observed by 4 isolates in all cell lines used while 6 other isolates showed high cytotoxic effect on T24 cell line only. Strong association of multidrug resistance was found with biofilm formation as strong biofilms were observed significantly higher in MDR isolates (p-value < 0.05) than non-MDR isolates. No significant association of cytotoxic potential with multidrug resistance or biofilm formation was found (p-value > 0.05). The MDR isolates showing significant cytotoxic effects and strong biofilm formation impose a serious threat for hospitalized patients with weak immune system.
MHC genes encode proteins that are responsible for the recognition of foreign antigens and the triggering of a subsequent, adequate immune response of the organism. Thus they hold a key position in the immune system of vertebrates. It is believed that the extraordinary genetic diversity of MHC genes is shaped by adaptive selectional processes in response to the reoccurring adaptations of parasites and pathogens. A large number of MHC studies were performed in a wide range of wildlife species aiming to understand the role of immune gene diversity in parasite resistance under natural selection conditions. Methodically, most of this work with very few exceptions has focussed only upon the structural, i.e. sequence diversity of regions responsible for antigen binding and presentation. Most of these studies found evidence that MHC gene variation did indeed underlie adaptive processes and that an individual’s allelic diversity explains parasite and pathogen resistance to a large extent. Nevertheless, our understanding of the effective mechanisms is incomplete. A neglected, but potentially highly relevant component concerns the transcriptional differences of MHC alleles. Indeed, differences in the expression levels MHC alleles and their potential functional importance have remained unstudied. The idea that also transcriptional differences might play an important role relies on the fact that lower MHC gene expression is tantamount with reduced induction of CD4+ T helper cells and thus with a reduced immune response. Hence, I studied the expression of MHC genes and of immune regulative cytokines as additional factors to reveal the functional importance of MHC diversity in two free-ranging rodent species (Delomys sublineatus, Apodemus flavicollis) in association with their gastrointestinal helminths under natural selection conditions. I established the method of relative quantification of mRNA on liver and spleen samples of both species in our laboratory. As there was no available information on nucleic sequences of potential reference genes in both species, PCR primer systems that were established in laboratory mice have to be tested and adapted for both non-model organisms. In the due course, sets of stable reference genes for both species were found and thus the preconditions for reliable measurements of mRNA levels established. For D. sublineatus it could be demonstrated that helminth infection elicits aspects of a typical Th2 immune response. Whereas mRNA levels of the cytokine interleukin Il4 increased with infection intensity by strongyle nematodes neither MHC nor cytokine expression played a significant role in D. sublineatus. For A. flavicollis I found a negative association between the parasitic nematode Heligmosomoides polygyrus and hepatic MHC mRNA levels. As a lower MHC expression entails a lower immune response, this could be evidence for an immune evasive strategy of the nematode, as it has been suggested for many micro-parasites. This implies that H. polygyrus is capable to interfere actively with the MHC transcription. Indeed, this parasite species has long been suspected to be immunosuppressive, e.g. by induction of regulatory T-helper cells that respond with a higher interleukin Il10 and tumor necrosis factor Tgfb production. Both cytokines in turn cause an abated MHC expression. By disabling recognition by the MHC molecule H. polygyrus might be able to prevent an activation of the immune system. Indeed, I found a strong tendency in animals carrying the allele Apfl-DRB*23 to have an increased infection intensity with H. polygyrus. Furthermore, I found positive and negative associations between specific MHC alleles and other helminth species, as well as typical signs of positive selection acting on the nucleic sequences of the MHC. The latter was evident by an elevated rate of non-synonymous to synonymous substitutions in the MHC sequences of exon 2 encoding the functionally important antigen binding sites whereas the first and third exons of the MHC DRB gene were highly conserved. In conclusion, the studies in this thesis demonstrate that valid procedures to quantify expression of immune relevant genes are also feasible in non-model wildlife organisms. In addition to structural MHC diversity, also MHC gene expression should be considered to obtain a more complete picture on host-pathogen coevolutionary selection processes. This is especially true if parasites are able to interfere with systemic MHC expression. In this case advantageous or disadvantageous effects of allelic binding motifs are abated. The studies could not define the role of MHC gene expression in antagonistic coevolution as such but the results suggest that it depends strongly on the specific parasite species that is involved.
Terrestrial environmental systems are characterised by numerous feedback links between their different compartments. However, scientific research is organized into disciplines that focus on processes within the respective compartments rather than on interdisciplinary links. Major feedback mechanisms between compartments might therefore have been systematically overlooked so far. Without identifying these gaps, initiatives on future comprehensive environmental monitoring schemes and experimental platforms might fail. We performed a comprehensive overview of feedbacks between compartments currently represented in environmental sciences and explores to what degree missing links have already been acknowledged in the literature. We focused on process models as they can be regarded as repositories of scientific knowledge that compile findings of numerous single studies. In total, 118 simulation models from 23 model types were analysed. Missing processes linking different environmental compartments were identified based on a meta-review of 346 published reviews, model inter-comparison studies, and model descriptions. Eight disciplines of environmental sciences were considered and 396 linking processes were identified and ascribed to the physical, chemical or biological domain. There were significant differences between model types and scientific disciplines regarding implemented interdisciplinary links. The most wide-spread interdisciplinary links were between physical processes in meteorology, hydrology and soil science that drive or set the boundary conditions for other processes (e.g., ecological processes). In contrast, most chemical and biological processes were restricted to links within the same compartment. Integration of multiple environmental compartments and interdisciplinary knowledge was scarce in most model types. There was a strong bias of suggested future research foci and model extensions towards reinforcing existing interdisciplinary knowledge rather than to open up new interdisciplinary pathways. No clear pattern across disciplines exists with respect to suggested future research efforts. There is no evidence that environmental research would clearly converge towards more integrated approaches or towards an overarching environmental systems theory. (c) 2017 Elsevier B.V. All rights reserved.
Development of birthweight and length for gestational age and sex references in Yucatan, Mexico
(2022)
Objective To develop sex- and gestational age specific reference percentiles and curves for birth weight and length for Yucatec neonates using data from birth registers of infants born during 2015-2019. Material and methods Observational, descriptive, epidemiologic study in a 5-year period including every registered birth in the state of Yucatan, Mexico using birth registries. A total of 158 432 live, physically healthy singletons (76 442 females and 81 990 males) between 25 and 42 weeks of gestation were included in the analysis. We used the LMS method to construct smoothed reference centiles (3rd, 10th, 25th, 50th, 75th, 95th, and 97th) and curves for males and females separately. Results Mean maternal age was 26 (SD = 6.22) years. Fifty-two percent of births occurred by vaginal delivery, 37% were firstborn and similar proportions were second (33%) and third or more (30%) born. 5.5% of newborns included in the references corresponds to neonates born before 37 weeks of gestation (5.9% boys and 5.1% girls). In both sexes, the percentage of infants with a birthweight less than 2500 g was 6.7%. The birthweight at the 50th percentile for males and females at 40 weeks of gestation in this cohort was 3256 and 3167 g, respectively, and the corresponding values for birth length were 50.23 and 49.84 cm (mean differences between sexes: 89 g and 0.40 cm, respectively). Conclusion The reference percentile and curves developed in this study are useful for research purposes and can help health practitioners to assess the biological status of infants born in Yucatan.
Saharan dust input and seasonal upwelling along North-West Africa provide a model system for studying microbial processes related to the export and recycling of nutrients. This study offers the first molecular characterization of prokaryotic particle-attached (PA; > 3.0 mu m) and free-living (FL; 0.2-3.0 mu m) players in this important ecosystem during August 2016. Environmental drivers for alpha-diversity, bacterial community composition, and differences between FL and PA fractions were identified. The ultra-oligotrophic waters off Senegal were dominated by Cyanobacteria while higher relative abundances of Alphaproteobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes (known particle-degraders) occurred in the upwelling area. Temperature, proxy for different water masses, was the best predictor for changes in FL communities. PA community variation was best explained by temperature and ammonium. Bray Curtis dissimilarities between FL and PA were generally very high and correlated with temperature and salinity in surface waters. Greatest similarities between FL and PA occurred at the deep chlorophyll maximum, where bacterial substrate availability was likely highest. This indicates that environmental drivers do not only influence changes among FL and PA communities but also differences between them. This could provide an explanation for contradicting results obtained by different studies regarding the dissimilarity/similarity between FL and PA communities and their biogeochemical functions.
Biosensors for the detection of benzaldehyde and g-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below −0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer.
Biosensors for the detection of benzaldehyde and g-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below −0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer.
While the underlying mechanisms of Parkinson’s disease (PD) are still insufficiently studied, a complex interaction between genetic and environmental factors is emphasized. Nevertheless, the role of the essential trace element zinc (Zn) in this regard remains controversial. In this study we altered Zn balance within PD models of the versatile model organism Caenorhabditis elegans (C. elegans) in order to examine whether a genetic predisposition in selected genes with relevance for PD affects Zn homeostasis. Protein-bound and labile Zn species act in various areas, such as enzymatic catalysis, protein stabilization pathways and cell signaling. Therefore, total Zn and labile Zn were quantitatively determined in living nematodes as individual biomarkers of Zn uptake and bioavailability with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) or a multi-well method using the fluorescent probe ZinPyr-1. Young and middle-aged deletion mutants of catp-6 and pdr-1, which are orthologues of mammalian ATP13A2 (PARK9) and parkin (PARK2), showed altered Zn homeostasis following Zn exposure compared to wildtype worms. Furthermore, age-specific differences in Zn uptake were observed in wildtype worms for total as well as labile Zn species. These data emphasize the importance of differentiation between Zn species as meaningful biomarkers of Zn uptake as well as the need for further studies investigating the role of dysregulated Zn homeostasis in the etiology of PD.
Manganese (Mn) and zinc (Zn) are not only essential trace elements, but also potential exogenous risk factors for various diseases. Since the disturbed homeostasis of single metals can result in detrimental health effects, concerns have emerged regarding the consequences of excessive exposures to multiple metals, either via nutritional supplementation or parenteral nutrition. This study focuses on Mn-Zn-interactions in the nematode Caenorhabditis elegans (C. elegans) model, taking into account aspects related to aging and age-dependent neurodegeneration.
Manganese (Mn) and zinc (Zn) are not only essential trace elements, but also potential exogenous risk factors for various diseases. Since the disturbed homeostasis of single metals can result in detrimental health effects, concerns have emerged regarding the consequences of excessive exposures to multiple metals, either via nutritional supplementation or parenteral nutrition. This study focuses on Mn-Zn-interactions in the nematode Caenorhabditis elegans (C. elegans) model, taking into account aspects related to aging and age-dependent neurodegeneration.
A balance to death
(2018)
Leaf senescence plays a crucial role in nutrient recovery in late-stage plant development and requires vast transcriptional reprogramming by transcription factors such as ORESARA1 (ORE1). A proteolytic mechanism is now found to control ORE1 degradation, and thus senescence, during nitrogen starvation.
Coarse-grained molecular model for the Glycosylphosphatidylinositol anchor with and without protein
(2020)
Glycosylphosphatidylinositol (GPI) anchors are a unique class of complex glycolipids that anchor a great variety of proteins to the extracellular leaflet of plasma membranes of eukaryotic cells. These anchors can exist either with or without an attached protein called GPI-anchored protein (GPI-AP) both in vitro and in vivo. Although GPIs are known to participate in a broad range of cellular functions, it is to a large extent unknown how these are related to GPI structure and composition. Their conformational flexibility and microheterogeneity make it difficult to study them experimentally. Simplified atomistic models are amenable to all-atom computer simulations in small lipid bilayer patches but not suitable for studying their partitioning and trafficking in complex and heterogeneous membranes. Here, we present a coarse-grained model of the GPI anchor constructed with a modified version of the MARTINI force field that is suited for modeling carbohydrates, proteins, and lipids in an aqueous environment using MARTINI's polarizable water. The nonbonded interactions for sugars were reparametrized by calculating their partitioning free energies between polar and apolar phases. In addition, sugar-sugar interactions were optimized by adjusting the second virial coefficients of osmotic pressures for solutions of glucose, sucrose, and trehalose to match with experimental data. With respect to the conformational dynamics of GPI-anchored green fluorescent protein, the accessible time scales are now at least an order of magnitude larger than for the all-atom system. This is particularly important for fine-tuning the mutual interactions of lipids, carbohydrates, and amino acids when comparing to experimental results. We discuss the prospective use of the coarse-grained GPI model for studying protein-sorting and trafficking in membrane models.
Coarse-grained molecular model for the Glycosylphosphatidylinositol anchor with and without protein
(2020)
Glycosylphosphatidylinositol (GPI) anchors are a unique class of complex glycolipids that anchor a great variety of proteins to the extracellular leaflet of plasma membranes of eukaryotic cells. These anchors can exist either with or without an attached protein called GPI-anchored protein (GPI-AP) both in vitro and in vivo. Although GPIs are known to participate in a broad range of cellular functions, it is to a large extent unknown how these are related to GPI structure and composition. Their conformational flexibility and microheterogeneity make it difficult to study them experimentally. Simplified atomistic models are amenable to all-atom computer simulations in small lipid bilayer patches but not suitable for studying their partitioning and trafficking in complex and heterogeneous membranes. Here, we present a coarse-grained model of the GPI anchor constructed with a modified version of the MARTINI force field that is suited for modeling carbohydrates, proteins, and lipids in an aqueous environment using MARTINI's polarizable water. The nonbonded interactions for sugars were reparametrized by calculating their partitioning free energies between polar and apolar phases. In addition, sugar-sugar interactions were optimized by adjusting the second virial coefficients of osmotic pressures for solutions of glucose, sucrose, and trehalose to match with experimental data. With respect to the conformational dynamics of GPI-anchored green fluorescent protein, the accessible time scales are now at least an order of magnitude larger than for the all-atom system. This is particularly important for fine-tuning the mutual interactions of lipids, carbohydrates, and amino acids when comparing to experimental results. We discuss the prospective use of the coarse-grained GPI model for studying protein-sorting and trafficking in membrane models.
Das 1817 erstmals schriftlich erwähnte Selen galt lange Zeit nur als toxisch und sogar als procancerogen, bis es 1957 von Schwarz und Foltz als essentielles Spurenelement erkannt wurde, dessen biologische Funktionen in Säugern durch Selenoproteine vermittelt werden. Die Familie der Glutathionperoxidasen nimmt hierbei eine wichtige Stellung ein. Für diese sind konkrete Funktionen und die dazugehörigen molekularen Mechanismen, welche über die von ihnen katalysierte Hydroperoxidreduktion und damit verbundene antioxidative Kapazität hinausgehen, bislang nur unzureichend beschrieben worden. Die Funktion der gastrointestinalen Glutathionperoxidase (GI-GPx) wird als Barriere gegen eine Hydroperoxidabsorption im Gastrointestinaltrakt definiert. Neuen Erkenntnissen zufolge wird die GI-GPx aber auch in verschiedenen Tumoren verstärkt exprimiert, was weitere, bis dato unbekannte, Funktionen dieses Enzymes wahrscheinlich macht. Um mögliche neue Funktionen der GI-GPx, vor allem während der Cancerogenese, abzuleiten, wurde hier die transkriptionale Regulation der GI-GPx detaillierter untersucht. Die Sequenzanalyse des humanen GI-GPx-Promotors ergab das Vorhandensein von zwei möglichen "antioxidant response elements" (ARE), bei welchen es sich um Erkennungssequenzen des Transkriptionsfaktors Nrf2 handelt. Die meisten der bekannten Nrf2-Zielgene gehören in die Gruppe der Phase-II-Enzyme und verfügen über antioxidative und/oder detoxifizierende Eigenschaften. Sowohl auf Promotorebene als auch auf mRNA- und Proteinebene konnte die Expression der GI-GPx durch typische, in der Nahrung enthaltene, Nrf2-Aktivatoren wie z.B. Sulforaphan oder Curcumin induziert werden. Eine direkte Beteiligung von Nrf2 wurde durch Cotransfektion von Nrf2 selbst bzw. von Keap1, das Nrf2 im Cytoplasma festhält, demonstriert. Somit konnte die GI-GPx eindeutig als Nrf2-Zielgen identifiziert werden. Ob sich die GI-GPx in die Gruppe der antiinflammatorischen und anticancerogenen Phase-II-Enzyme einordnen lässt, bleibt noch zu untersuchen. Die Phospholipidhydroperoxid Glutathionperoxidase (PHGPx) nimmt aufgrund ihres breiten Substratspektrums, ihrer hohen Lipophilie und ihrer Fähigkeit, Thiole zu modifizieren, eine Sonderstellung innerhalb der Familie der Glutathionperoxidasen ein. Mit Hilfe eines PHGPx-überexprimierenden Zellmodells wurden deshalb Beeinflussungen des zellulären Redoxstatus und daraus resultierende Veränderungen in der Aktivität redoxsensitiver Transkriptionsfaktorsysteme und in der Expression atheroskleroserelevanter Adhäsionsmoleküle untersucht. Als Transkriptionsfaktoren wurden NF-kB und Nrf2 ausgewählt. Die Bindung von NF-kB an sein entsprechendes responsives Element in der DNA erfordert das Vorhandensein freier Thiole, wohingegen Nrf2 durch Thiolmodifikation von Keap1 freigesetzt wird und in den Kern transloziert. Eine erhöhte Aktivität der PHGPx resultierte in einer Erhöhung des Verhältnisses von GSH zu GSSG, andererseits aber in einer verminderten Markierbarkeit freier Proteinthiole. PHGPx-Überexpression reduzierte die IL-1-induzierte NF-kB-Aktivität, die sich in einer verminderten NF-kB-DNA-Bindefähigkeit und Transaktivierungsaktivität ausdrückte. Auch war die Proliferationsrate der Zellen vermindert. Die Expression des NF-kB-regulierten vaskulären Zelladhäsionsmoleküls, VCAM-1, war ebenfalls deutlich verringert. Umgekehrt war in PHGPx-überexprimierenden Zellen eine erhöhte Nrf2-Aktivität und Expression der Nrf2-abhängigen Hämoxygenase-1 zu verzeichnen. Letzte kann für die meisten der beobachteten Effekte verantwortlich gemacht werden. Die hier dargestellten Ergebnisse verdeutlichen, dass eine Modifizierung von Proteinthiolen als wichtige Determinante für die Regulation der Expression und Funktion von Glutathionperoxidasen angesehen werden kann. Entgegen früheren Vermutungen, welche oxidative Vorgänge generell mit pathologischen Veränderungen assoziierten, scheint ein moderater oxidativer Stress, bedingt durch eine transiente Thiolmodifikation, durchaus günstige Auswirkungen zu haben, da, wie hier dargelegt, verschiedene, miteinander interagierende, cytoprotektive Mechanismen ausgelöst werden. Hieran wird deutlich, dass sich "antioxidative Wirkung" oder "oxidativer Stress" keineswegs nur auf "gute" oder "schlechte" Vorgänge beschränken lassen, sondern im Zusammenhang mit den beeinflussten (patho)physiologischen Prozessen und dem Ausmaß der "Störung" des physiologischen Redoxgleichgewichtes betrachtet werden müssen.
Ciboria ploettneriana, Schroeteria decaisneana, and S. poeltii produce morphologically very similar apothecia emerging from fallen stromatized seeds of Veronica spp., the former two on V. hederifolia agg. in temperate central Europe and S. poeltii on V. cymbalaria in mediterranean southern Europe. They are described and illustrated in detail based on fresh collections or moist chamber cultures of infected seeds. A key is provided to differentiate the three species from their teleomorphs. For the first time, connections between two teleomorphs and two Schroeteria anamorphs are reported. Members of the anamorph-typified genus Schroeteria are known as host-specific plant parasites that infect seeds of different Veronica spp. In earlier times, they were classified in the Ustilaginales (Basidiomycota), but since more than 30 years, they are referred to as false smut fungi producing smut-like chlamydospores, based on light microscopic and ultrastructural studies which referred them to the Sclerotiniaceae (Helotiales). During the present study, rDNA sequences were obtained for the first time from chlamydospores of Schroeteria bornmuelleri (on V. rubrifolia), S. decaisneana (on V. hederifolia), S. delastrina (generic type, on V. arvensis), and S. poeltii (on V. cymbalaria) and from apothecia of C. ploettneriana, S. decaisneana, and S. poeltii. As a result, the anamorph-teleomorph connection could be established for S. decaisneana and S. poeltii by a 100% ITS similarity, whereas C. ploettneriana could not be connected to a smut-like anamorph. Ciboria ploettneriana in the here-redefined sense clustered in our combined phylogenetic analyses of ITS and LSU in relationship of Sclerotinia s.l., Botrytis, and Myriosclerotinia rather than Ciboria, but its placement was not supported. Its affiliation in Ciboria was retained until a better solution is found. Also Schroeteria poeltii clustered unresolved in this relationship but with a much higher molecular distance. The remaining three Schroeteria spp. formed a strongly supported monophyletic group, here referred to as "Schroeteria core clade", which clustered with medium to high support as a sister clade of Monilinia jezoensis, a member of the Monilinia alpina group of section Disjunctoriae. We observed ITS distances of 5-6.3% among members of the Schroeteria core clade, but 13.8-14.7% between this clade and S. poeltii, which appears to be correlated with the deviating chlamydospore morphology of S. poeltii. Despite its apparent paraphyly, Schroeteria is accepted here in a wide sense as a genus distinct from Monilinia, particularly because of its very special anamorphs. A comparable heterogeneity in rDNA analyses was observed in Monilinia and other genera of Sclerotiniaceae. Such apparent heterogeneity should be met with skepticism, however, because the inclusion of protein-coding genes in phylogenetic analyses resulted in a monophyletic genus Monilinia. More sclerotiniaceous taxa should be analysed for protein-coding genes in the future, including Schroeteria. Four syntype specimens of Ciboria ploettneriana in B were reexamined in the present study, revealing a mixture of the two species growing on V. hederifolia agg. Based on its larger ascospores in comparison with S. decaisneana, a lectotype is proposed for C. ploettneriana.
Die Tailspike Proteine (TSP) der Bakteriophagen P22, Sf6 und HK620 dienen der Erkennung von Kohlenhydratstrukturen auf ihren gram-negativen Wirtsbakterien und zeigen, von den ersten 110 Aminosäuren des N-Terminus abgesehen, keine Sequenzübereinstimmung. Mit Röntgenkristallstrukturanalyse konnte gezeigt werden, dass HK620TSP und Sf6TSP ebenfalls zu einer parallelen, rechtsgängigen beta-Helix falten, wie dies schon für P22TSP bekannt war. Die Kohlenhydratbindestelle ist bei Sf6TSP im Vergleich zu P22TSP zwischen die Untereinheiten verschoben.
Actin-based directional motility is important for embryonic development, wound healing, immune responses, and development of tissues. Actin and myosin are essential players in this process that can be subdivided into protrusion, adhesion, and traction. Protrusion is the forward movement of the membrane at the leading edge of the cell. Adhesion is required to enable movement along a substrate, and traction finally leads to the forward movement of the entire cell body, including its organelles. While actin polymerization is the main driving force in cell protrusions, myosin motors lead to the contraction of the cell body. The goal of this work was to study the regulatory mechanisms of the motile machinery by selecting a representative key player for each stage of the signaling process: the regulation of Arp2/3 activity by WASP (actin system), the role of cGMP in myosin II assembly (myosin system), and the influence of phosphoinositide signaling (upstream receptor pathway). The model organism chosen for this work was the social ameba Dictyostelium discoideum, due to the well-established knowledge of its cytoskeletal machinery, the easy handling, and the high motility of its vegetative and starvation developed cells. First, I focused on the dynamics of the actin cytoskeleton by modulating the activity of one of its key players, the Arp2/3 complex. This was achieved using the carbazole derivative Wiskostatin, an inhibitor of the Arp2/3 activator WASP. Cells treated with Wiskostatin adopted a round shape, with no of few pseudopodia. With the help of a microfluidic cell squeezer device, I could show that Wiskostatin treated cells display a reduced mechanical stability, comparable to cells treated with the actin disrupting agent Latrunculin A. Furthermore, the WASP inhibited cells adhere stronger to a surface and show a reduced motility and chemotactic performance. However, the overall F-actin content in the cells was not changed. Confocal microscopy and TIRF microscopy imaging showed that the cells maintained an intact actin cortex. Localized dynamic patches of increased actin polymerization were observed that, however, did not lead to membrane deformation. This indicated that the mechanisms of actin-driven force generation were impaired in Wiskostatin treated cells. It is concluded that in these cells, an altered architecture of the cortical network leads to a reduced overall stiffness of the cell, which is insufficient to support the force generation required for membrane deformation and pseudopod formation. Second, the role of cGMP in myosin II dynamics was investigated. Cyclic GMP is known to regulate the association of myosin II with the cytoskeleton. In Dictyostelium, intracellular cGMP levels increase when cells are exposed to chemoattractants, but also in response to osmotic stress. To study the influence of cyclic GMP on actin and myosin II dynamics, I used the laser-induced photoactivation of a DMACM-caged-Br-cGMP to locally release cGMP inside the cell. My results show that cGMP directly activates the myosin II machinery, but is also able to induce an actin response independently of cAMP receptor activation and signaling. The actin response was observed in both vegetative and developed cells. Possible explanations include cGMP-induced actin polymerization through VASP (vasodilator-stimulated phosphoprotein) or through binding of cGMP to cyclic nucleotide-dependent kinases. Finally, I investigated the role of phosphoinositide signaling using the Polyphosphoinositide-Binding Peptide (PBP10) that binds preferentially to PIP2. Phosphoinositides can recruit actin-binding proteins to defined subcellular sites and alter their activity. Neutrophils, as well as developed Dictyostelium cells produce PIP3 in the plasma membrane at their leading edge in response to an external chemotactic gradient. Although not essential for chemotaxis, phosphoinositides are proposed to act as an internal compass in the cell. When treated with the peptide PBP10, cells became round, with fewer or no pseudopods. PH-CRAC translocation to the membrane still occurs, even at low cAMP stimuli, but cell motility (random and directional) was reduced. My data revealed that the decrease in the pool of available PIP2 in the cell is sufficient to impair cell motility, but enough PIP2 remains so that PIP3 is formed in response to chemoattractant stimuli. My data thus highlights how sensitive cell motility and morphology are to changes in the phosphoinositide signaling. In summary, I have analyzed representative regulatory mechanisms that govern key parts of the motile machinery and characterized their impact on cellular properties including mechanical stability, adhesion and chemotaxis.
Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells
(2013)
Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system.
The frequent production of the hepatotoxin microcystin (MC) and its impact on the lifestyle of bloom-forming cyanobacteria are poorly understood. Here, we report that MC interferes with the assembly and the subcellular localization of RubisCO, in Microcystis aeruginosa PCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes in M. aeruginosa and relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of MC to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2 fixation product 3-phosphoglycerate are significantly higher in the MC-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction of M. aeruginosa with its heterotrophic microbial community.
Although many large mammal species went extinct at the end of the Pleistocene epoch, their DNA may persist due to past episodes of interspecies admixture. However, direct empirical evidence of the persistence of ancient alleles remains scarce. Here, we present multifold coverage genomic data from four Late Pleistocene cave bears (Ursus spelaeus complex) and show that cave bears hybridized with brown bears (Ursus arctos) during the Pleistocene. We develop an approach to assess both the directionality and relative timing of gene flow. We find that segments of cave bear DNA still persist in the genomes of living brown bears, with cave bears contributing 0.9 to 2.4% of the genomes of all brown bears investigated. Our results show that even though extinction is typically considered as absolute, following admixture, fragments of the gene pool of extinct species can survive for tens of thousands of years in the genomes of extant recipient species.
Consensify
(2020)
A standard practise in palaeogenome analysis is the conversion of mapped short read data into pseudohaploid sequences, frequently by selecting a single high-quality nucleotide at random from the stack of mapped reads. This controls for biases due to differential sequencing coverage, but it does not control for differential rates and types of sequencing error, which are frequently large and variable in datasets obtained from ancient samples. These errors have the potential to distort phylogenetic and population clustering analyses, and to mislead tests of admixture using D statistics. We introduce Consensify, a method for generating pseudohaploid sequences, which controls for biases resulting from differential sequencing coverage while greatly reducing error rates. The error correction is derived directly from the data itself, without the requirement for additional genomic resources or simplifying assumptions such as contemporaneous sampling. For phylogenetic and population clustering analysis, we find that Consensify is less affected by artefacts than methods based on single read sampling. For D statistics, Consensify is more resistant to false positives and appears to be less affected by biases resulting from different laboratory protocols than other frequently used methods. Although Consensify is developed with palaeogenomic data in mind, it is applicable for any low to medium coverage short read datasets. We predict that Consensify will be a useful tool for future studies of palaeogenomes.
Consensify
(2020)
A standard practise in palaeogenome analysis is the conversion of mapped short read data into pseudohaploid sequences, frequently by selecting a single high-quality nucleotide at random from the stack of mapped reads. This controls for biases due to differential sequencing coverage, but it does not control for differential rates and types of sequencing error, which are frequently large and variable in datasets obtained from ancient samples. These errors have the potential to distort phylogenetic and population clustering analyses, and to mislead tests of admixture using D statistics. We introduce Consensify, a method for generating pseudohaploid sequences, which controls for biases resulting from differential sequencing coverage while greatly reducing error rates. The error correction is derived directly from the data itself, without the requirement for additional genomic resources or simplifying assumptions such as contemporaneous sampling. For phylogenetic and population clustering analysis, we find that Consensify is less affected by artefacts than methods based on single read sampling. For D statistics, Consensify is more resistant to false positives and appears to be less affected by biases resulting from different laboratory protocols than other frequently used methods. Although Consensify is developed with palaeogenomic data in mind, it is applicable for any low to medium coverage short read datasets. We predict that Consensify will be a useful tool for future studies of palaeogenomes.
Homotherium was a genus of large-bodied scimitar-toothed cats, morphologically distinct from any extant felid species, that went extinct at the end of the Pleistocene [1-4]. They possessed large, saber-form serrated canine teeth, powerful forelimbs, a sloping back, and an enlarged optic bulb, all of which were key characteristics for predation on Pleistocene megafauna [5]. Previous mitochondrial DNA phylogenies suggested that it was a highly divergent sister lineage to all extant cat species [6-8]. However, mitochondrial phylogenies can be misled by hybridization [9], incomplete lineage sorting (ILS), or sex-biased dispersal patterns [10], which might be especially relevant for Homotherium since widespread mito-nuclear discrepancies have been uncovered in modern cats [10]. To examine the evolutionary history of Homotherium, we generated a -7x nuclear genome and a similar to 38x exome from H. latidens using shotgun and target-capture sequencing approaches. Phylogenetic analyses reveal Homotherium as highly divergent (similar to 22.5 Ma) from living cat species, with no detectable signs of gene flow. Comparative genomic analyses found signatures of positive selection in several genes, including those involved in vision, cognitive function, and energy consumption, putatively consistent with diurnal activity, well-developed social behavior, and cursorial hunting [5]. Finally, we uncover relatively high levels of genetic diversity, suggesting that Homotherium may have been more abundant than the limited fossil record suggests [3, 4, 11-14]. Our findings complement and extend previous inferences from both the fossil record and initial molecular studies, enhancing our understanding of the evolution and ecology of this remarkable lineage.
In self-incompatible plants the female style rejects self pollen, yet the extent to which the female style in the many self-compatible species can still select between different pollen genotypes and thus bias fertilization success is unclear. A new study identifies the molecular basis for how styles of the self-compatible coyote tobacco bias the fertilization success of pollen genotypes using matching gene expression patterns in a manner analogous to cryptic female choice in animals.
Complex networks have been successfully employed to represent different levels of biological systems, ranging from gene regulation to protein-protein interactions and metabolism. Network-based research has mainly focused on identifying unifying structural properties, including small average path length, large clustering coefficient, heavy-tail degree distribution, and hierarchical organization, viewed as requirements for efficient and robust system architectures. Existing studies estimate the significance of network properties using a generic randomization scheme - a Markov-chain switching algorithm - which generates unrealistic reactions in metabolic networks, as it does not account for the physical principles underlying metabolism. Therefore, it is unclear whether the properties identified with this generic approach are related to the functions of metabolic networks. Within this doctoral thesis, I have developed an algorithm for mass-balanced randomization of metabolic networks, which runs in polynomial time and samples networks almost uniformly at random. The properties of biological systems result from two fundamental origins: ubiquitous physical principles and a complex history of evolutionary pressure. The latter determines the cellular functions and abilities required for an organism’s survival. Consequently, the functionally important properties of biological systems result from evolutionary pressure. By employing randomization under physical constraints, the salient structural properties, i.e., the smallworld property, degree distributions, and biosynthetic capabilities of six metabolic networks from all kingdoms of life are shown to be independent of physical constraints, and thus likely to be related to evolution and functional organization of metabolism. This stands in stark contrast to the results obtained from the commonly applied switching algorithm. In addition, a novel network property is devised to quantify the importance of reactions by simulating the impact of their knockout. The relevance of the identified reactions is verified by the findings of existing experimental studies demonstrating the severity of the respective knockouts. The results suggest that the novel property may be used to determine the reactions important for viability of organisms. Next, the algorithm is employed to analyze the dependence between mass balance and thermodynamic properties of Escherichia coli metabolism. The thermodynamic landscape in the vicinity of the metabolic network reveals two regimes of randomized networks: those with thermodynamically favorable reactions, similar to the original network, and those with less favorable reactions. The results suggest that there is an intrinsic dependency between thermodynamic favorability and evolutionary optimization. The method is further extended to optimizing metabolic pathways by introducing novel chemically feasibly reactions. The results suggest that, in three organisms of biotechnological importance, introduction of the identified reactions may allow for optimizing their growth. The approach is general and allows identifying chemical reactions which modulate the performance with respect to any given objective function, such as the production of valuable compounds or the targeted suppression of pathway activity. These theoretical developments can find applications in metabolic engineering or disease treatment. The developed randomization method proposes a novel approach to measuring the significance of biological network properties, and establishes a connection between large-scale approaches and biological function. The results may provide important insights into the functional principles of metabolic networks, and open up new possibilities for their engineering.
Methodological and technological advances have recently paved the way for metabolic flux profiling in higher organisms, like plants. However, in comparison with omics technologies, flux profiling has yet to provide comprehensive differential flux maps at a genome-scale and in different cell types, tissues, and organs. Here we highlight the recent advances in technologies to gather metabolic labeling patterns and flux profiling approaches. We provide an opinion of how recent local flux profiling approaches can be used in conjunction with the constraint-based modeling framework to arrive at genome-scale flux maps. In addition, we point at approaches which use metabolomics data without introduction of label to predict either non-steady state fluxes in a time-series experiment or flux changes in different experimental scenarios. The combination of these developments allows an experimentally feasible approach for flux-based large-scale systems biology studies.
Objective: A key challenge in ancient DNA research is massive microbial DNA contamination from the deposition site which accumulates post mortem in the study organism’s remains. Two simple and cost-effective methods to enrich the relative endogenous fraction of DNA in ancient samples involve treatment of sample powder with either bleach or Proteinase K pre-digestion prior to DNA extraction. Both approaches have yielded promising but vary-ing results in other studies. Here, we contribute data on the performance of these methods using a comprehensive and systematic series of experiments applied to a single ancient bone fragment from a giant panda (Ailuropoda melanoleuca).Results: Bleach and pre-digestion treatments increased the endogenous DNA content up to ninefold. However, the absolute amount of DNA retrieved was dramatically reduced by all treatments. We also observed reduced DNA damage patterns in pre-treated libraries compared to untreated ones, resulting in longer mean fragment lengths and reduced thymine over-representation at fragment ends. Guanine–cytosine (GC) contents of both mapped and total reads are consistent between treatments and conform to general expectations, indicating no obvious biasing effect of the applied methods. Our results therefore confirm the value of bleach and pre-digestion as tools in palaeog-enomic studies, providing sufficient material is available.