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The phase behavior of a dendritic amphiphile containing a Newkome-type dendron as the hydrophilic moiety and a cholesterol unit as the hydrophobic segment is investigated at the air-liquid interface. The amphiphile forms stable monomolecular films at the airliquid interface on different subphases. Furthermore, the mineralization of calcium phosphate beneath the monolayer at different calcium and phosphate concentrations versus mineralization time shows that at low calcium and phosphate concentrations needles form, whereas flakes and spheres dominate at higher concentrations. Energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and electron diffraction confirm the formation of calcium phosphate. High-resolution transmission electron microscopy and electron diffraction confirm the predominant formation of octacalcium phosphate and hydroxyapatite. The data also indicate that the final products form via a complex multistep reaction, including an association step, where nano-needles aggregate into larger flake-like objects.
Strand breaks and conformational changes of DNA have consequences for the physiological role of DNA. The natural protecting molecule ectoine is beneficial to entire bacterial cells and biomolecules such as proteins by mitigating detrimental effects of environmental stresses. It was postulated that ectoine-like molecules bind to negatively charged spheres that mimic DNA surfaces. We investigated the effect of ectoine on DNA and whether ectoine is able to protect DNA from damages caused by ultraviolet radiation (UV-A). In order to determine different isoforms of DNA, agarose gel electrophoresis and atomic force microscopy experiments were carried out with plasmid pUC19 DNA. Our quantitative results revealed that a prolonged incubation of DNA with ectoine leads to an increase in transitions from supercoiled (undamaged) to open circular (single-strand break) conformation at pH 6.6. The effect is pH dependent and no significant changes were observed at physiological pH of 7.5. After UV-A irradiation in ectoine solution, changes in DNA conformation were even more pronounced and this effect was pH dependent. We hypothesize that ectoine is attracted to the negatively charge surface of DNA at lower pH and therefore fails to act as a stabilizing agent for DNA in our in vitro experiments.
Intrinsically Disordered Stress Protein COR15A Resides at the Membrane Surface during Dehydration
(2017)
Plants from temperate climate zones are able to increase their freezing tolerance during exposure to low, above zero temperatures in a process termed cold acclimation. During this process, several cold-regulated (COR) proteins are accumulated in the cells. One of them is COR15A, a small, intrinsically disordered protein that contributes to leaf freezing tolerance by stabilizing cellular membranes. The isolated protein folds into amphipathic a-helices in response to increased crowding conditions, such as high concentrations of glycerol. Although there is evidence for direct COR15A-membrane interactions, the orientation and depth of protein insertion were unknown. In addition, although folding due to high osmolyte concentrations had been established, the folding response of the protein under conditions of gradual dehydration had not been investigated. Here we show, using Fourier transform infrared spectroscopy, that COR15A starts to fold into a-helices already under mild dehydration conditions (97% relative humidity (RH), corresponding to freezing at -3 degrees C) and that folding gradually increases with decreasing RH. Neutron diffraction experiments at 97 and 75% RH established that the presence of COR15A had no significant influence on the structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. However, using deuterated POPC we. could clearly establish that COR15A interacts with the membranes and penetrates below the headgroup region into the upper part of the fatty acyl chain region. This localization is in agreement with our hypothesis that COR15A-membrane interaction is at least, in part, driven by a hydrophobic interaction between the lipids and the hydrophobic face of the amphipathic protein alpha-helix.
Vitamin E inhibits the propagation of lipid peroxidation and helps protecting photosystem II from photoinhibition, but little is known about its possible role in plant response to Pi availability. Here, we aimed at examining the effect of vitamin E deficiency in Arabidopsis thaliana vte mutants on phytohormone contents and the expression of transcription factors in plants exposed to contrasting Pi availability. Plants were subjected to two doses of Pi, either unprimed (controls) or previously exposed to low Pi (primed). In the wild type, alpha-tocopherol contents increased significantly in response to repeated periods of low Pi, which was paralleled by increased growth, indicative of a priming effect. This growth-stimulating effect was, however, abolished in vte mutants. Hormonal profiling revealed significant effects of Pi availability, priming and genotype on the contents of jasmonates and salicylates; remarkably, vte mutants showed enhanced accumulation of both hormones under low Pi. Furthermore, expression profiling of 1,880 transcription factors by qRT-PCR revealed a pronounced effect of priming on the transcript levels of 45 transcription factors mainly associated with growth and stress in wild-type plants in response to low Pi availability; while distinct differences in the transcriptional response were detected in vte mutants. We conclude that alpha-tocopherol plays a major role in the response of plants to Pi availability not only by protecting plants from photo-oxidative stress, but also by exerting a control over growth-and defense-related transcriptional reprogramming and hormonal modulation.
Tyramine is an important neurotransmitter, neuromodulator, and neurohormone in insects. In honeybees, it is assumed to have functions in modulating sensory responsiveness and controlling motor behavior. Tyramine can bind to two characterized receptors in honeybees, both of which are coupled to intracellular cAMP pathways. How tyramine acts on neuronal, cellular and circuit levels is unclear. We investigated the spatial brain expression of the tyramine receptor AmTAR1 using a specific antibody. This antibody detects a membrane protein of the expected molecular weight in western blot analysis. In honeybee brains, it labels different structures which process sensory information. Labeling along the antennal nerve, in projections of the dorsal lobe and in the gnathal ganglion suggest that tyramine receptors are involved in modulating gustatory and tactile perception. Furthermore, the ellipsoid body of the central complex and giant synapses in the lateral complex show AmTAR1-like immunoreactivity (AmTAR1-IR), suggesting a role of this receptor in modulating sky-compass information and/or higher sensor-motor control. Additionally, intense signals derive from the mushroom bodies, higher-order integration centers for olfactory, visual, gustatory and tactile information. To investigate whether AmTAR1-expressing brain structures are in vicinity to tyramine releasing sites, a specific tyramine antibody was applied. Tyramine-like labeling was observed in AmTAR1-IR positive structures, although it was sometimes weak and we did not always find a direct match of ligand and receptor. Moreover, tyramine-like immunoreactivity was also found in brain regions without AmTAR1-IR (optic lobes, antennal lobes), indicating that other tyramine-specific receptors may be expressed there.
Photosynthesis and water use efficiency, key factors affecting plant growth, are directly controlled by microscopic and adjustable pores in the leaf-the stomata. The size of the pores is modulated by the guard cells, which rely on molecular mechanisms to sense and respond to environmental changes. It has been shown that the physiology of mesophyll and guard cells differs substantially. However, the implications of these differences to metabolism at a genome-scale level remain unclear. Here, we used constraint-based modeling to predict the differences in metabolic fluxes between the mesophyll and guard cells of Arabidopsis thaliana by exploring the space of fluxes that are most concordant to cell-type-specific transcript profiles. An independent C-13-labeling experiment using isolated mesophyll and guard cells was conducted and provided support for our predictions about the role of the Calvin-Benson cycle in sucrose synthesis in guard cells. The combination of in silico with in vivo analyses indicated that guard cells have higher anaplerotic CO2 fixation via phosphoenolpyruvate carboxylase, which was demonstrated to be an important source of malate. Beyond highlighting the metabolic differences between mesophyll and guard cells, our findings can be used in future integrated modeling of multicellular plant systems and their engineering towards improved growth.
Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes
(2017)
Sulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a “closed” conformation, suitable for internal electron transfer, and an “open” conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization leads to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modeled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization.
The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an L-cysteine desulfurase as an initial sulfur mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human L-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of similar to 60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm(5)s(2)U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present.
Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (alpha- and beta-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify beta-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels. (C) 2017 Published by Elsevier Inc.
The biosynthesis of the molybdenum cofactor (Moco) is a highly conserved pathway in bacteria, archaea and eukaryotes. The molybdenum atom in Moco-containing enzymes is coordinated to the dithiolene group of a tricyclic pyranopterin monophosphate cofactor. The biosynthesis of Moco can be divided into three conserved steps, with a fourth present only in bacteria and archaea: (1) formation of cyclic pyranopterin monophosphate, (2) formation of molybdopterin (MPT), (3) insertion of molybdenum into MPT to form Mo-MPT, and (4) additional modification of Mo-MPT in bacteria with the attachment of a GMP or CMP nucleotide, forming the dinucleotide variants of Moco. While the proteins involved in the catalytic reaction of each step of Moco biosynthesis are highly conserved among the Phyla, a surprising link to other cellular pathways has been identified by recent discoveries. In particular, the pathways for FeS cluster assembly and thio-modifications of tRNA are connected to Moco biosynthesis by sharing the same protein components. Further, proteins involved in Moco biosynthesis are not only shared with other pathways, but additionally have moonlighting roles. This review gives an overview of Moco biosynthesis in bacteria and humans and highlights the shared function and moonlighting roles of the participating proteins.
Arctic and alpine treelines worldwide differ in their reactions to climate change. A northward advance of or densification within the treeline ecotone will likely influence climate-vegetation feedback mechanisms. In our study, which was conducted in the Taimyr Depression in the North Siberian Lowlands, w present a combined field-and model-based approach helping us to better understand the population processes involved in the responses of the whole treeline ecotone, spanning from closed forest to single-tree tundra, to climate warming. Using information on stand structure, tree age, and seed quality and quantity from seven sites, we investigate effects of intra-specific competition and seed availability on the specific impact of recent climate warming on larch stands. Field data show that tree density is highest in the forest-tundra, and average tree size decreases from closed forest to single-tree tundra. Age-structure analyses indicate that the trees in the closed forest and forest-tundra have been present for at least similar to 240 yr. At all sites except the most southerly ones, past establishment is positively correlated with regional temperature increase. In the single-tree tundra, however, a change in growth form from krummholz to erect trees, beginning similar to 130 yr ago, rather than establishment date has been recorded. Seed mass decreases from south to north, while seed quantity increases. Simulations with LAVESI (Larix Vegetation Simulator) further suggest that relative density changes strongly in response to a warming signal in the forest-tundra while intra-specific competition limits densification in the closed forest and seed limitation hinders densification in the single-tree tundra. We find striking differences in strength and timing of responses to recent climate warming. While forest-tundra stands recently densified, recruitment is almost non-existent at the southern and northern end of the ecotone due to autecological processes. Palaeo-treelines may therefore be inappropriate to infer past temperature changes at a fine scale. Moreover, a lagged treeline response to past warming will, via feedback mechanisms, influence climate change in the future.
Behavioral thermoregulation is an important mechanism allowing ectotherms to respond to thermal variations. Its efficiency might become imperative for securing activity budgets under future climate change. For diurnal lizards, thermal microhabitat variability appears to be of high importance, especially in hot deserts where vegetation is highly scattered and sensitive to climatic fluctuations. We investigated the effects of a shading gradient from vegetation on body temperatures and activity timing for two diurnal, terrestrial desert lizards, Ctenotus regius, and Morethia boulengeri, and analyzed their changes under past, present, and future climatic conditions. Both species’ body temperatures and activity timing strongly depended on the shading gradient provided by vegetation heterogeneity. At high temperatures, shaded locations provided cooling temperatures and increased diurnal activity. Conversely, bushes also buffered cold temperature by saving heat. According to future climate change scenarios, cooler microhabitats might become beneficial to warm‐adapted species, such as C. regius, by increasing the duration of daily activity. Contrarily, warmer microhabitats might become unsuitable for less warm‐adapted species such as M. boulengeri for which midsummers might result in a complete restriction of activity irrespective of vegetation. However, total annual activity would still increase provided that individuals would be able to shift their seasonal timing towards spring and autumn. Overall, we highlight the critical importance of thermoregulatory behavior to buffer temperatures and its dependence on vegetation heterogeneity. Whereas studies often neglect ecological processes when anticipating species’ responses to future climate change the strongest impact of a changing climate on terrestrial ectotherms in hot deserts is likely to be the loss of shaded microhabitats rather than the rise in temperature itself. We argue that conservation strategies aiming at addressing future climate changes should focus more on the cascading effects of vegetation rather than on shifts of species distributions predicted solely by climatic envelopes.
Pregnancy termination after encountering a strange male, the Bruce effect, is regarded as a counterstrategy of female mammals towards anticipated infanticide. While confirmed in caged rodent pairs, no verification for the Bruce effect existed from experimental field populations of small rodents. We suggest that the effect may be adaptive for breeding rodent females only under specific conditions related to populations with cyclically fluctuating densities. We investigated the occurrence of delay in birth date after experimental turnover of the breeding male under different population composition in bank voles (Myodes glareolus) in large outdoor enclosures: one-male-multiple-females (n = 6 populations/18 females), multiple-males-multiplefemales (n = 15/45), and single-male-single-female (MF treatment, n = 74/74). Most delays were observed in the MF treatment after turnover. Parallel we showed in a laboratory experiment (n = 205 females) that overwintered and primiparous females, the most abundant cohort during population lows in the increase phase of cyclic rodent populations, were more likely to delay births after turnover of the male than year-born and multiparous females. Taken together, our results suggest that the Bruce effect may be an adaptive breeding strategy for rodent females in cyclic populations specifically at low densities in the increase phase, when isolated, overwintered animals associate in MF pairs. During population lows infanticide risk and inbreeding risk may then be higher than during population highs, while also the fitness value of a litter in an increasing population is higher. Therefore, the Bruce effect may be adaptive for females during annual population lows in the increase phases, even at the costs of delaying reproduction.
Inorganic phosphorus (P-i) is often the primary limiting nutrient in freshwater ecosystems. Since P(i-)limitation affects energy transduction, and inorganic carbon (C-i) acquisition can be energy demanding, C(i-)acquisition strategies were compared in four species of green algae grown under P-i-replete and P-i-limited conditions predominantly at low and partly at high CO2. Although P-i-limitation was evident by the 10-fold higher cellular C:P ratio and enhanced phosphatase activity, it only decreased C-i-acquisition to a small extent. Nonetheless, the effects of Pi-limitation on both CO2 and HCO3- acquisition were demonstrated. Decreased CO2 acquisition under conditions of Pi limitation was mainly visible in the maximum uptake rate (V-max) and, for the neutrophile Scenedesmus vacuolatus, in the affinity for CO2 acquisition. Discrimination against C-13 was higher under P-i-limited, high CO2 conditions, compared with P-i-replete, highCO(2) conditions, in Chlamydomonas acidophila and S. vacuolatus. In the pH-drift experiments, HCO3- acquisition was reduced in P-i-limited C. reinhardtii. In general, energy demanding bicarbonate uptake was indicated by the less strong discrimination against (13)Cunder lowCO(2) conditions in the neutrophiles (HCO3- users), separating them from the acidophilic or acidotolerant species (CO2 users). The high variability of the influence of Pi supply among different green algal species is linked to their species-specific C(i-)acquisition strategies.
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.
Even though recent years have shown a slow recovery of the Eurasian otter (Lutra lutra) populations from their previous lows, the species is still highly endangered in most parts of its European distribution range. Surprisingly, only a few studies have so far assessed the species’ genetic variability and population density, and they have mostly been carried out only in small territories. In Germany, most otter populations live in protected areas whose management urgently needs data on population sizes and densities as well as on genetic variability of the species under their custody. Thus, we analyzed genetic variability and assessed size and density of the otter population in the Bohemian Forest Ecosystem, an area that had not been included in the few previous molecular studies. The study area comprised of 1500 km2, divided into fifteen squares of 10 × 10 km2, each of which was sampled in two collection periods. Overall we collected 261 fecal samples (spraints), of which 60 (23%) could be genotyped at least at eight microsatellite loci, yielding 38 distinct otter genotypes. The low genotyping success rate was the result of high ambient temperature at the time of sampling rather than that of high humidity. The population did not show signs of a past bottleneck, indicating a small yet stable population size. Population size was estimated to be 118 (CI95% 64–163) individuals, with a mean density of 1 animal per 8.5 km2 or 3.1 km river length. Our results imply that hunting, requested by local fishpond owners, should remain banned to avoid a decline in (effective) population size.
Nutrition bioassays in which polyunsaturated fatty acids (PUFA)-deficient diets were supplemented with free long-chain PUFA (>= C20) consistently revealed positive effects on somatic growth and fecundity of Daphnia. However, free PUFA are hardly available in natural diets. In general, PUFA are bound to other lipids, especially to phospholipids and triglycerides. Here, we evaluate the potential of free and phospholipid-bound dietary eicosapentaenoic acid (EPA) to support somatic growth and fecundity of Daphnia magna. In a growth experiment, supplementation of a C20 PUFA-deficient diet with free or phospholipid-bound EPA improved somatic growth rates of D. magna equally. However, the increase in fecundity was significantly more pronounced when phospholipid-bound EPA was provided. Free and phospholipid-bound EPA were provided in the same concentrations in our experiment, suggesting that the allocation to reproduction-related processes is affected differently by phospholipid-bound PUFA and free PUFA. Our finding stresses the need to consider the distribution of dietary PUFA in different lipid classes to gain a better understanding of how PUFA influence life history traits of Daphnids in the field.
Human hair follicle (HF) growth and hair shaft formation require terminal differentiation-associated cell cycle arrest of highly proliferative matrix keratinocytes. However, the regulation of this complex event remains unknown. CIP/KIP family member proteins (p21(CIP1), p27(KIP1) and p57(KIP2)) regulate cell cycle progression/arrest, endoreplication, differentiation and apoptosis. Since they have not yet been adequately characterized in the human HF, we asked whether and where CIP/KIP proteins localise in the human hair matrix and pre-cortex in relation to cell cycle activity and HF-specific epithelial cell differentiation that is marked by keratin 85 (K85) protein expression. K85 expression coincided with loss or reduction in cell cycle activity markers, including in situ DNA synthesis (EdU incorporation), Ki-67, phospho-histone H3 and cyclins A and B1, affirming a post-mitotic state of pre-cortical HF keratinocytes. Expression of CIP/KIP proteins was found abundantly within the proliferative hair matrix, concomitant with a role in cell cycle checkpoint control. p21(CIP1), p27(Klp1) and cyclin E persisted within post-mitotic keratinocytes of the pre-cortex, whereas p57(MP2) protein decreased but became nuclear. These data imply a supportive role for CIP/KIP proteins in maintaining proliferative arrest, differentiation and anti-apoptotic pathways, promoting continuous hair bulb growth and hair shaft formation in anagen VI. Moreover, post-mitotic hair matrix regions contained cells with enlarged nuclei, and DNA in situ hybridisation showed cells that w ere >2N in the pre-cortex. This suggests that CIP/KIP proteins might counterbalance cyclin E to control further rounds o f DNA replication in a cell population that has a propensity to become tetraploid. These data shed new light on the in situ-biography of human hair matrix keratinocytes on their path of active cell cycling, arrest and terminal differentiation, and showcase the human HF as an excellent, clinically relevant model system for cell cycle physiology research of human epithelial cells within their natural tissue habitat. Crown Copyright (C) 2017 Published by Elsevier GmbH. All rights reserved.
Linking primary producer diversity and food quality effects on herbivores: A biochemical perspective
(2017)
Biodiversity can strongly influence trophic interactions. The nutritional quality of prey communities and how it is related to the prey diversity is suspected to be a major driver of biodiversity effects. As consumer growth can be co-limited by the supply of several biochemical components, biochemically diverse prey communities should promote consumer growth. Yet, there is no clear consensus on how prey specific diversity is linked to community biochemical diversity since previous studies have considered only single nutritional quality traits. Here, we demonstrate that phytoplankton biochemical traits (fatty acids and sterols) can to a large extent explain Daphnia magna growth and its apparent dependence on phytoplankton species diversity. We find strong correlative evidence between phytoplankton species diversity, biochemical diversity, and growth. The relationship between species diversity and growth was partially explained by the fact that in many communities Daphnia was co-limited by long chained polyunsaturated fatty acids and sterols, which was driven by different prey taxa. We suggest that biochemical diversity is a good proxy for the presence of high food quality taxa, and a careful consideration of the distribution of the different biochemical traits among species is necessary before concluding about causal links between species diversity and consumer performance.
The El Nino-Southern Oscillation (ENSO) is the main driver of the interannual variability in eastern African rainfall, with a significant impact on vegetation and agriculture and dire consequences for food and social security. In this study, we identify and quantify the ENSO contribution to the eastern African rainfall variability to forecast future eastern African vegetation response to rainfall variability related to a predicted intensified ENSO. To differentiate the vegetation variability due to ENSO, we removed the ENSO signal from the climate data using empirical orthogonal teleconnection (EOT) analysis. Then, we simulated the ecosystem carbon and water fluxes under the historical climate without components related to ENSO teleconnections. We found ENSO-driven patterns in vegetation response and confirmed that EOT analysis can successfully produce coupled tropical Pacific sea surface temperature-eastern African rainfall teleconnection from observed datasets. We further simulated eastern African vegetation response under future climate change as it is projected by climate models and under future climate change combined with a predicted increased ENSO intensity. Our EOT analysis highlights that climate simulations are still not good at capturing rainfall variability due to ENSO, and as we show here the future vegetation would be different from what is simulated under these climate model outputs lacking accurate ENSO contribution. We simulated considerable differences in eastern African vegetation growth under the influence of an intensified ENSO regime which will bring further environmental stress to a region with a reduced capacity to adapt effects of global climate change and food security.
Sediment resuspension represents a key process in all natural aquatic systems, owing to its role in nutrient cycling and transport of potential contaminants. Although suspended solids are generally accepted as an important quality parameter, current monitoring programs cover quantitative aspects only. Established methodologies do not provide information on origin, fate, and risks associated with uncontrolled inputs of solids in waters. Here we discuss the analytical approaches to assess the occurrence and ecological relevance of resuspended particulate matter in freshwaters, with a focus on the dynamics of associated contaminants and microorganisms. Triggered by the identification of specific physical-chemical traits and community structure of particle-associated microorganisms, recent findings suggest that a quantitative determination of microorganisms can be reasonably used to trace the origin of particulate matter by means of nucleic acid-based assays in different aquatic systems.
Hypolimnetic oxygen demand in lakes is often assumed to be driven mainly by sediment microbial processes, while the role of Chaoborus larvae, which are prevalent in eutrophic lakes with hypoxic to anoxic bottoms, has been overlooked. We experimentally measured the respiration rates of C flavicans at different temperatures yielding a Q(10) of 1.44-1.71 and a respiratory quotient of 0.84-0.98. Applying the experimental data in a system analytical approach, we showed that migrating Chaoborus larvae can significantly add to the water column and sediment oxygen demand, and contribute to the observed linear relationship between water column respiration and depth. The estimated phosphorus excretion by Chaoborus in sediment is comparable in magnitude to the required phosphorus loading for eutrophication. Migrating Chaoborus larvae thereby essentially trap nutrients between the water column and the sediment, and this continuous internal loading of nutrients would delay lake remediation even when external inputs are stopped. (C) 2017 Elsevier Ltd. All rights reserved.
Highly porous poly(ether imide) (PEI) microparticles prepared by a spraying/coagulation process are discussed as candidate adsorber materials for apheresis applications, i.e. removal of uremic toxins from the blood of renal failure patients. PEI particles obtained by the aforementioned procedure can have a broad size distribution with particle diameters ranging from 20 to 800 mu m. In order to further estimate the adsorption behavior of PEI microparticles packed in application relevant apheresis modules, a quantitative information about the relation between particle size and pore morphology is required. In this study, we explored whether the intraparticle porosity of PEI microparticles varies with altering the diameter of the particulate adsorbers. By an analytical wet sieving procedure, the obtained PEI microparticles were separated into five size fractions, which were analyzed by mercury intrusion porosimetry, nitrogen adsorption, and scanning electron microscopy. Mercury intrusion porosimetry revealed for all size fractions high porosity values in the range from 78% to 84% with pore diameters in the range from 10 to 1000nm. A bimodal pore size distribution was found having a first peak at around 100nm, while a second pronounced peak maximum was found at higher pore sizes that increased with raising particle diameter from 300nm for the smallest particle size fraction (50-100 mu m) to 700nm for particles with a diameter of 200 to 250 mu m. Based on these findings, it can be assumed that the main PEI particle size fraction (200-250 mu m) should exhibit the highest adsorption capacity in an apheresis module. Copyright (c) 2016 John Wiley & Sons, Ltd.
While branched polyglycerol (PG)-based molecules are well established as hydrophilic particles, the capacity of utilizing PG in bulk materials and opportunities arising by their further surface functionalization have only recently been considered. Here we investigated how the mold used in PG network synthesis may affect surface composition and how the permeability of substances through PG can be controlled by altering network structure, i.e. introducing 20mol% oligoethylene glycol (OEG) bifunctional spacer molecules. Overall, PG-based bulk network materials were shown to be tailorable, hydrophilic, low swelling and relatively stiff polyether-based materials, with low impact of salt onto material properties. Based on these features, but also on the principal capacity of free hydroxyl groups to be used for functionalization reactions, these materials may be an interesting platform for medical and technical applications, e.g. as diffusion-rate controlling membrane in aqueous environment. Copyright (c) 2016 John Wiley & Sons, Ltd.
Gelatin-based hydrogels offer various biochemical cues that support encapsulated cells and are therefore suitable as cell delivery vehicles in regenerative medicine. However, besides the biochemical signals, biomechanical cues are crucial to ensure an optimal support of encapsulated cells. Hence, we aimed to correlate the cellular response of encapsulated cells to macroscopic and microscopic elastic properties of glycidylmethacrylate (GMA)-functionalized gelatin-based hydrogels. To ensure that different observations in cellular behavior could be attributed to differences in elastic properties, an identical concentration as well as degree of functionalization of biopolymers was utilized to form covalently crosslinked hydrogels. Elastic properties were merely altered by varying the average gelatin-chain length. Hydrogels exhibited an increased degree of swelling and a decreased bulk elastic modulus G with prolonged autoclaving of the starting solution. This was accompanied by an increase of hydrogel mesh size and thus by a reduction of crosslinking density. Tougher hydrogels retained the largest amount of cells; however, they also interfered with cell viability. Softer gels contained a lower cell density, but supported cell elongation and viability. Observed differences could be partially attributed to differences in bulk properties, as high crosslinking densities interfere with diffusion and cell spreading and thus can impede cell viability. Interestingly, a microscopic elastic modulus in the range of native soft tissue supported cell viability and elongation best while ensuring a good cell entrapment. In conclusion, gelatin-based hydrogels providing a soft tissue-like microenvironment represent adequate cell delivery vehicles for tissue engineering approaches. Copyright (c) 2016 John Wiley & Sons, Ltd.
Aim: Poverty has often been associated with malnutrition, stunted growth, impaired cognitive development and poor earnings. We studied whether these associations were found in German men born and raised shortly after World War II during severe and long-standing nationwide malnutrition. Methods: We analysed German old-age pension payments, as a rough measure of lifetime earnings, in German men born from 1932 to 1960 and compared the at-risk-of-poverty rates of German men born in 1945-1948 versus 1935-1938 and 1955-1958. Results: Substantially fewer women worked during this period and their longer life expectancy makes their pension payments difficult to interpret. We therefore limited our analysis to men. Men born in the 1930s received the highest monthly old-age pensions and these declined slightly in men born from 1945 to 1948, indicating a minute impairment in work-related income in cohorts born shortly after the war. We also found that there was no evidence for increased at-risk-of-poverty rates in men born in 1945-1948 versus those born in 1935-1938 and in 1955-1958. Conclusion: Being born and raised following World War II was associated with a minute work and pension impairment that was not visible in the at-risk-of-poverty rates. These findings question statements associating early childhood nutrition and future lifetime earnings.
Glycoproteins adsorbing on an implant upon contact with body fluids can affect the biological response in vitro and in vivo, depending on the type and conformation of the adsorbed biomacromolecules. However, this process is poorly characterized and so far not controllable. Here, protein monolayers of high molecular cohesion with defined density are transferred onto polymeric substrates by the Langmuir-Schaefer (LS) technique and were compared with solution deposition (SO) method. It is hypothesized that on polydimethylsiloxane (PDMS), a substrate with poor cell adhesion capacity, the fibronectin (FN) layers generated by the LS and SO methods will differ in their organization, subsequently facilitating differential stem cell adhesion behavior. Indeed, atomic force microscopy visualization and immunofluorescence images indicated that organization of the FN layer immobilized on PDMS was uniform and homogeneous. In contrast, FN deposited by SO method was rather heterogeneous with appearance of structures resembling protein aggregates. Human mesenchymal stem cells showed reduced absolute numbers of adherent cells, and the vinculin expression seemed to be higher and more homogenously distributed after seeding on PDMS equipped with FN by LS in comparison with PDMS equipped with FN by SO. These divergent responses could be attributed to differences in the availability of adhesion molecule ligands such as the Arg-Gly-Asp (RGD) peptide sequence presented at the interface. The LS method allows to control the protein layer characteristics, including the thickness and the protein orientation or conformation, which can be harnessed to direct stem cell responses to defined outcomes, including migration and differentiation. Copyright (c) 2016 John Wiley & Sons, Ltd.
The sum of benthic autotrophic and bacterial production often exceeds the sum of pelagic autotrophic and bacterial production, and hence may contribute substantially to whole-lake carbon fluxes, especially in shallow lakes. Furthermore, both benthic and pelagic autotrophic and bacterial production are highly edible and of sufficient nutritional quality for animal consumers. We thus hypothesised that pelagic and benthic transfer efficiencies (ratios of production at adjacent trophic levels) in shallow lakes should be similar. We performed whole ecosystem studies in two shallow lakes (3.5ha, mean depth 2m), one with and one without submerged macrophytes, and quantified pelagic and benthic biomass, production and transfer efficiencies for bacteria, phytoplankton, epipelon, epiphyton, macrophytes, zooplankton, macrozoobenthos and fish. We expected higher transfer efficiencies in the lake with macrophytes, because these provide shelter and food for macrozoobenthos and may thus enable a more efficient conversion of basal production to consumer production. In both lakes, the majority of the whole-lake autotrophic and bacterial production was provided by benthic organisms, but whole-lake primary consumer production mostly relied on pelagic autotrophic and bacterial production. Consequently, transfer efficiency of benthic autotrophic and bacterial production to macrozoobenthos production was an order of magnitude lower than the transfer efficiency of pelagic autotrophic and bacterial production to rotifer and crustacean production. Between-lake differences in transfer efficiencies were minor. We discuss several aspects potentially causing the unexpectedly low benthic transfer efficiencies, such as the food quality of producers, pelagic-benthic links, oxygen concentrations in the deeper lake areas and additional unaccounted consumer production by pelagic and benthic protozoa and meiobenthos at intermediate or top trophic levels. None of these processes convincingly explain the large differences between benthic and pelagic transfer efficiencies. Our data indicate that shallow eutrophic lakes, even with a major share of autotrophic and bacterial production in the benthic zone, can function as pelagic systems with respect to primary consumer production. We suggest that the benthic autotrophic production was mostly transferred to benthic bacterial production, which remained in the sediments, potentially cycling internally in a similar way to what has previously been described for the microbial loop in pelagic habitats. Understanding the energetics of whole-lake food webs, including the fate of the substantial benthic bacterial production, which is either mineralised at the sediment surface or permanently buried, has important implications for regional and global carbon cycling.
Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems.
The mineral and biochemical food quality of prey may limit predator production. This well-studied direct bottom-up effect is especially prominent for herbivore-plant interactions. Low-quality prey species, particularly when defended, are generally considered to be less prone to predator-driven extinction. Undefended high-quality prey species sustain high predator production thereby potentially increasing their own extinction risk. The food quality of primary producers is highly species-specific. In communities of competing prey species, predators thus may supplement their diets of low-quality prey with high-quality prey, leading to indirect horizontal interactions between prey species of different food quality. We explore how these predator-mediated indirect interactions affect species coexistence in a general predator-prey model that is parametrized for an experimental algae-rotifer system. To cover a broad range of three essential functional traits that shape many plant-herbivore interactions we consider differences in 1) the food quality of the prey species, 2) their competitive ability for nutrient uptake and 3) their defence against predation. As expected, low food quality of prey can, similarly to defence, provide protection against extinction by predation. Counterintuitively, our simulations demonstrate that being of high food quality also prevents extinction of that prey species and additionally promotes coexistence with a competing, low-quality prey. The persistence of the high-quality prey enables a high conversion efficiency and control of the low-quality prey by the predator and allows for re-allocation of nutrients to the high-quality competitor. Our results show that high food quality is not necessarily detrimental for a prey species but instead can protect against extinction and promote species richness and functional biodiversity.
Selenium increases hepatic DNA methylation and modulates one-carbon metabolism in the liver of mice
(2017)
The average intake of the essential trace element selenium (Se) is below the recommendation in most European countries, possibly causing sub-optimal expression of selenoproteins. It is still unclear how a suboptimal Se status may affect health. To mimic this situation, mice were fed one of three physiologically relevant amounts of Se. We focused on the liver, the organ most sensitive to changes in the Se supply indicated by hepatic glutathione peroxidase activity. In addition, liver is the main organ for synthesis of methyl groups and glutathione via one-carbon metabolism. Accordingly, the impact of Se on global DNA methylation, methylation capacity, and gene expression was assessed. We observed higher global DNA methylation indicated by LINE1 methylation, and an increase of the methylation potential as indicated by higher S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio and by elevated mRNA expression of serine hydroxymethyltransferase in both or either of the Se groups. Furthermore, increasing the Se supply resulted in higher plasma concentrations of triglycerides. Hepatic expression of glycolytic and lipogenic genes revealed consistent Se dependent up-regulation of glucokinase. The sterol regulatory element-binding transcription factor 1 (Srebf1) was also up-regulated by Se. Both effects were confirmed in primary hepatocytes. In contrast to the overall Se-dependent increase of methylation capacity, the up-regulation of Srebf1 expression was paralleled by reduced local methylation of a specific CpG site within the Srebf1 gene. Thus, we provided evidence that Se-dependent effects on lipogenesis involve epigenetic mechanisms. (C) 2017 The Authors. Published by Elsevier Inc.
G(0)W(0) calculations for predicting vertical ionization potentials (IPs) and electron affinities of molecules and clusters are known to show a significant dependence on the density functional theory (DFT) starting point. A number of nonempirical procedures to find an optimal starting point have been proposed, typically based on tuning the amount of HF exchange in the underlying hybrid functional specifically for the system at hand. For the case of pi-conjugated molecular chains, these approaches lead to a significantly different amount of HF exchange for different oligomer sizes. In this study, we analyze if and how strongly this size dependence affects the ability of nonempirical tuning approaches to predict accurate IPs for pi-conjugated molecular chains of increasing chain length. To this end, we employ three different nonempirical tuning procedures for the G(0)W(0) starting point to calculate the IP of polyene oligomers up to 22 repeat units and compare the results to highly accurate coupled-cluster calculations. We find that, despite its size dependence, using an IP-tuned hybrid functional as a starting point for G(0)W(0) yields excellent agreement with the reference data for all chain lengths.
Chytridiomycota, often referred to as chytrids, can be virulent parasites with the potential to inflict mass mortalities on hosts, causing e.g. changes in phytoplankton size distributions and succession, and the delay or suppression of bloom events. Molecular environmental surveys have revealed an unexpectedly large diversity of chytrids across a wide range of aquatic ecosystems worldwide. As a result, scientific interest towards fungal parasites of phytoplankton has been gaining momentum in the past few years. Yet, we still know little about the ecology of chytrids, their life cycles, phylogeny, host specificity and range. Information on the contribution of chytrids to trophic interactions, as well as co-evolutionary feedbacks of fungal parasitism on host populations is also limited. This paper synthesizes ideas stressing the multifaceted biological relevance of phytoplankton chytridiomycosis, resulting from discussions among an international team of chytrid researchers. It presents our view on the most pressing research needs for promoting the integration of chytrid fungi into aquatic ecology.
Identifying behavioral mechanisms that underlie observed movement patterns is difficult when animals employ sophisticated cognitive‐based strategies. Such strategies may arise when timing of return visits is important, for instance to allow for resource renewal or territorial patrolling. We fitted spatially explicit random‐walk models to GPS movement data of six wolves (Canis lupus; Linnaeus, 1758) from Alberta, Canada to investigate the importance of the following: (1) territorial surveillance likely related to renewal of scent marks along territorial edges, to reduce intraspecific risk among packs, and (2) delay in return to recently hunted areas, which may be related to anti‐predator responses of prey under varying prey densities. The movement models incorporated the spatiotemporal variable “time since last visit,” which acts as a wolf's memory index of its travel history and is integrated into the movement decision along with its position in relation to territory boundaries and information on local prey densities. We used a model selection framework to test hypotheses about the combined importance of these variables in wolf movement strategies. Time‐dependent movement for territory surveillance was supported by all wolf movement tracks. Wolves generally avoided territory edges, but this avoidance was reduced as time since last visit increased. Time‐dependent prey management was weak except in one wolf. This wolf selected locations with longer time since last visit and lower prey density, which led to a longer delay in revisiting high prey density sites. Our study shows that we can use spatially explicit random walks to identify behavioral strategies that merge environmental information and explicit spatiotemporal information on past movements (i.e., “when” and “where”) to make movement decisions. The approach allows us to better understand cognition‐based movement in relation to dynamic environments and resources.
Recent reports of increasing iron (Fe) concentrations in freshwaters are of concern, given the fundamental role of Fe in biogeochemical processes. Still, little is known about the frequency and geographical distribution of Fe trends or about the underlying drivers. We analyzed temporal trends of Fe concentrations across 340 water bodies distributed over 10 countries in northern Europe and North America in order to gain a clearer understanding of where, to what extent, and why Fe concentrations are on the rise. We found that Fe concentrations have significantly increased in 28% of sites, and decreased in 4%, with most positive trends located in northern Europe. Regions with rising Fe concentrations tend to coincide with those with organic carbon (OC) increases. Fe and OC increases may not be directly mechanistically linked, but may nevertheless be responding to common regional-scale drivers such as declining sulfur deposition or hydrological changes. A role of hydrological factors was supported by covarying trends in Fe and dissolved silica, as these elements tend to stem from similar soil depths. A positive relationship between Fe increases and conifer cover suggests that changing land use and expanded forestry could have contributed to enhanced Fe export, although increases were also observed in nonforested areas. We conclude that the phenomenon of increasing Fe concentrations is widespread, especially in northern Europe, with potentially significant implications for wider ecosystem biogeochemistry, and for the current browning of freshwaters.
Following escape into the cytoplasm of host cells, Burkholderia pseudomallei and the related species Burkholderia thailandensis employ the type VI secretion system 5 ( T6SS-5) to induce plasma membrane fusion with an adjacent host cell. This process leads to the formation of multinucleated giant cells and facilitates bacterial access to an uninfected host cell in a direct manner. Despite its importance in virulence, the mechanism of the T6SS-5 and the role of host cell factors in cell-cell fusion remain elusive. To date, the T6SS-5 is the only system of bacterial origin known to induce host-cell fusion. To gain insight into the nature of T6SS-5-stimulated membrane fusion, we investigated the contribution of cholesterol and proteins exposed on the host cell surface, which were shown to be critically involved in virus-mediated giant cell formation. In particular, we analyzed the effect of host cell surface protein and cholesterol depletion on the formation of multinucleated giant cells induced by B. thailandensis. Acute protease treatment of RAW264.7 macrophages during infection with B. thailandensis followed by agarose overlay assays revealed a strong reduction in the number of cell-cell fusions compared with EDTA treated cells. Similarly, proteolytic treatment of specifically infected donor cells or uninfected recipient cells significantly decreased multinucleated giant cell formation. Furthermore, modulating host cell cholesterol content by acute cholesterol depletion from cellular membranes by methyl-beta-cyclodextrin treatment or exogenous addition of cholesterol impaired the ability of B. thailandensis to induce cell-cell fusions. The requirement of physiological cholesterol levels suggests that the membrane organization or mechanical properties of the lipid bilayer influence the fusion process. Altogether, our data suggest that membrane fusion induced by B. pseudomallei and B. thailandensis involves a complex interplay between the T6SS-5 and the host cell.
Protected areas are arguably the most important instrument of biodiversity conservation. To keep them fit under climate change, their management needs to be adapted to address related direct and indirect changes. In our study we focus on the adaptation of conservation management planning, evaluating management plans of 60 protected areas throughout Germany with regard to their climate change-robustness. First, climate change-robust conservation management was defined using 11 principles and 44 criteria, which followed an approach similar to sustainability standards. We then evaluated the performance of individual management plans concerning the climate change-robustness framework. We found that climate change-robustness of protected areas hardly exceeded 50 percent of the potential performance, with most plans ranking in the lower quarter. Most Natura 2000 protected areas, established under conservation legislation of the European Union, belong to the sites with especially poor performance, with lower values in smaller areas. In general, the individual principles showed very different rates of accordance with our principles, but similarly low intensity. Principles with generally higher performance values included holistic knowledge management, public accountability and acceptance as well as systemic and strategic coherence. Deficiencies were connected to dealing with the future and uncertainty. Lastly, we recommended the presented principles and criteria as essential guideposts that can be used as a checklist for working towards more climate change-robust planning.
Pollination syndromes and their predictive power regarding actual plant-animal interactions have been controversially discussed in the past. We investigate pollination syndromes in Balsaminaceae, utilizing quantitative respectively categorical data sets of flower morphometry, signal and reward traits for 86 species to test for the effect of different types of data on the test patterns retrieved. Cluster Analyses of the floral traits are used in combination with independent pollinator observations. Based on quantitative data we retrieve seven clusters, six of them corresponding to plausible pollination syndromes and one additional, well-supported cluster comprising highly divergent floral architectures. This latter cluster represents a non-syndrome of flowers not segregated by the specific data set here used. Conversely, using categorical data we obtained only a rudimentary resolution of pollination syndromes, in line with several earlier studies. The results underscore that the use of functional, exactly quanitified trait data has the power to retrieve pollination syndromes circumscribed by the specific data used. Data quality can, however, not be replaced by sheer data volume. With this caveat, it is possible to identify pollination syndromes from large datasets and to reliably extrapolate them for taxa for which direct observations are unavailable.
Functional interactions of plastidial phosphorylase (Phol) and starch branching enzymes (BEs) from the developing rice endosperm are the focus of this study. In the presence of both Phol and BE, the same branched primer molecule is elongated and further branched almost simultaneously even at very low glucan concentrations present in the purified enzyme preparations. By contrast, in the absence of any BE, glucans are not, to any significant extent, elongated by Phol. Based on our in vitro data, in the developing rice endosperm, Phol appears to be weakly associated with any of the BE isozymes. By using fluorophore-labeled malto-oligosaccharides, we identified maltose as the smallest possible primer for elongation by Phol. Linear dextrins act as carbohydrate substrates for BEs. By functionally interacting with a BE, Phol performs two essential functions during the initiation of starch biosynthesis in the rice endosperm: First, it elongates maltodextrins up to a degree of polymerization of at least 60. Second, by closely interacting with BEs, Phol is able to elongate branched glucans efficiently and thereby synthesizes branched carbohydrates essential for the initiation of amylopectin biosynthesis.
Since its discovery over two decades ago as an important cell death regulator in Arabidopsis thaliana, the role of LESION SIMULATING DISEASE 1 (LSD1) has been studied intensively within both biotic and abiotic stress responses as well as with respect to plant fitness regulation. However, its molecular mode of action remains enigmatic. Here, we demonstrate that nucleo-cytoplasmic LSD1 interacts with a broad range of other proteins that are engaged in various molecular pathways such as ubiquitination, methylation, cell cycle control, gametogenesis, embryo development and cell wall formation. The interaction of LSD1 with these partners is dependent on redox status, as oxidative stress significantly changes the quantity and types of LSD1-formed complexes. Furthermore, we show that LSD1 regulates the number and size of leaf mesophyll cells and affects plant vegetative growth. Importantly, we also reveal that in addition to its function as a scaffold protein, LSD1 acts as a transcriptional regulator. Taken together, our results demonstrate that LSD1 plays a dual role within the cell by acting as a condition-dependent scaffold protein and as a transcription regulator.
1. During the last couple of decades, invasive species have become a worldwide problem in many freshwater systems. Besides higher plants and animals, microbes, in particular the potentially toxic cyanobacterium Cylindrospermopsis raciborskii, has attracted increasing attention, due to its spread towards temperate zones of the northern and southern hemisphere. A number of advantageous functional traits and a high intraspecific plasticity have been suggested to explain its invasion success. 2. The aim of this study was to examine intraspecific functional trait variability in 12 different isolates of C.raciborskii originating from different lakes in an invaded region in Northeast Germany. We measured growth rate, C:N:P ratios, chlorophyll-a content and the abundance of heterocysts under nutrient-replete and phosphorus-limited conditions. Moreover, the isolate-specific morphology and grazing losses by an herbivorous rotifer, as a top-down force, were studied. 3. DNA fingerprinting revealed that all isolates were genetically different. C.raciborskii exhibited a large variability in all measured traits among isolates. The C:P, N:P and Chl-a:C ratios differed by a factor of two or more. The trait variability among isolates was higher under nutrient-replete conditions, except for the C:P ratio, which varied most during phosphorus limitation. The susceptibility to grazing, calculated as maximum ingestion rates of the rotifer Brachionus calyciflorus on C.raciborskii, varied most among isolates, but was not related to any of the measured physiological or morphological traits, i.e. no trade-off was found. 4. Ecological and genetic clustering did not match, indicating that the genetic relationship based on DNA fingerprinting did not cover ecological differences. 5. Our results show a high trait variability within locally occurring and partly co-occurring C.raciborskii isolates. No overall trade-offs between the measured functional traits were found. This demonstrates the ecological relevance of linking multiple traits, e.g. competitive and consumptive. Furthermore, this study emphasises the importance of analysing more than one strain of a species, as different strains show different trait values potentially relevant for their invasibility and the field of general trait-based ecology.
Climatic change is expected to affect individual life histories and population dynamics, potentially increasing vulnerability to extinction. The importance of genetic diversity has been highlighted for adaptation and population persistence. However, whether responses of life-history traits to a given environmental condition depend on the genetic characteristics of a population remains elusive. Here we tested this hypothesis in the lizard Zootoca vivipara by simultaneously manipulating habitat humidity, a major climatic predictor of Zootoca’s distribution, and adult male color morph frequency, a trait with genome-wide linkage. Interactive effects of humidity and morph frequency had immediate effects on growth and body condition of juveniles and yearlings, as well as on adult survival, and delayed effects on offspring size. In yearlings, higher humidity led to larger female body size and lower humidity led to higher male compared to female survival. In juveniles and yearlings, some treatment effects were compensated over time. The results show that individual responses to environmental conditions depend on the population’s color morph frequency, age class, and sex and that these affect intra– and inter–age class competition. Moreover, humidity affected the competitive environment rather than imposing trait-based selection on specific color morphs. This indicates that species’ responses to changing environments (e.g., to climate change) are highly complex and difficult to accurately reconstruct and predict without information on the genetic characteristics and demographic structure of populations.
The outermost cell layer of plant roots (epidermis) constantly encounters environmental challenges. The epidermal outer plasma membrane domain harbours the PENETRATION3 (PEN3)/ABCG36/PDR8 ATP-binding cassette transporter that confers non-host resistance to several pathogens. Here, we show that the Arabidopsis ENDOPLASMIC RETICULUM-ARRESTED PEN3 (EAP3) BTB/POZ-domain protein specifically mediates PEN3 exit from the endoplasmic reticulum and confers resistance to a root-penetrating fungus, providing prime evidence for BTB/POZ-domain protein-dependent membrane trafficking underlying disease resistance.
Reciprocal selection between aphids, their protective endosymbionts, and the parasitoid wasps that prey upon them offers an opportunity to study the basis of their coevolution. We investigated adaptation to symbiont‐conferred defense by rearing the parasitoid wasp Lysiphlebus fabarum on aphids (Aphis fabae) possessing different defensive symbiont strains (Hamiltonella defensa). After ten generations of experimental evolution, wasps showed increased abilities to parasitize aphids possessing the H. defensa strain they evolved with, but not aphids possessing the other strain. We show that the two symbiont strains encode different toxins, potentially creating different targets for counter‐adaptation. Phenotypic and behavioral comparisons suggest that neither life‐history traits nor oviposition behavior differed among evolved parasitoid lineages. In contrast, comparative transcriptomics of adult female wasps identified a suite of differentially expressed genes among lineages, even when reared in a common, symbiont‐free, aphid host. In concurrence with the specificity of each parasitoid lineages’ infectivity, most differentially expressed parasitoid transcripts were also lineage‐specific. These transcripts are enriched with putative venom toxins and contain highly expressed, potentially defensive viral particles. Together, these results suggest that wild populations of L. fabarum employ a complicated offensive arsenal with sufficient genetic variation for wasps to adapt rapidly and specifically to their hosts’ microbial defenses.
Reliable information on past and present vegetation is important to project future changes, especially for rapidly transitioning areas such as the boreal treeline. To study past vegetation, pollen analysis is common, while current vegetation is usually assessed by field surveys. Application of detailed sedimentary DNA (sedDNA) records has the potential to enhance our understanding of vegetation changes, but studies systematically investigating the power of this proxy are rare to date. This study compares sedDNA metabarcoding and pollen records from surface sediments of 31 lakes along a north-south gradient of increasing forest cover in northern Siberia (Taymyr peninsula) with data from field surveys in the surroundings of the lakes. sedDNA metabarcoding recorded 114 plant taxa, about half of them to species level, while pollen analyses identified 43 taxa, both exceeding the 31 taxa found by vegetation field surveys. Increasing Larix percentages from north to south were consistently recorded by all three methods and principal component analyses based on percentage data of vegetation surveys and DNA sequences separated tundra from forested sites. Comparisons of the ordinations using procrustes and protest analyses show a significant fit among all compared pairs of records. Despite similarities of sedDNA and pollen records, certain idiosyncrasies, such as high percentages of Alnus and Betula in all pollen and high percentages of Salix in all sedDNA spectra, are observable. Our results from the tundra to single-tree tundra transition zone show that sedDNA analyses perform better than pollen in recording site-specific richness (i.e., presence/absence of taxa in the vicinity of the lake) and perform as well as pollen in tracing vegetation composition.
There is the tendency to assume that endangered species have been both genetically and demographically healthier in the past, so that any genetic erosion observed today was caused by their recent decline. The Iberian lynx (Lynx pardinus) suffered a dramatic and continuous decline during the 20th century, and now shows extremely low genome- and species-wide genetic diversity among other signs of genomic erosion. We analyze ancient (N = 10), historical (N = 245), and contemporary (N = 172) samples with microsatellite and mitogenome data to reconstruct the species' demography and investigate patterns of genetic variation across space and time. Iberian lynx populations transitioned from low but significantly higher genetic diversity than today and shallow geographical differentiation millennia ago, through a structured metapopulation with varying levels of diversity during the last centuries, to two extremely genetically depauperate and differentiated remnant populations by 2002. The historical subpopulations show varying extents of genetic drift in relation to their recent size and time in isolation, but these do not predict whether the populations persisted or went finally extinct. In conclusion, current genetic patterns were mainly shaped by genetic drift, supporting the current admixture of the two genetic pools and calling for a comprehensive genetic management of the ongoing conservation program. This study illustrates how a retrospective analysis of demographic and genetic patterns of endangered species can shed light onto their evolutionary history and this, in turn, can inform conservation actions.
We investigated changes in quality and quantity of extracellular and biomass-derived organic matter (OM) from three axenic algae (genera Rhodomonas, Chlamydomonas, Coelastrum) during growth of Limnohabitans parvus, Limnohabitans planktonicus and Polynucleobacter acidiphobus representing important clusters of freshwater planktonic Betaproteobacteria. Total extracellular and biomass-derived OM concentrations from each alga were approximately 20 mg l(-1) and 1 mg l(-1) respectively, from which up to 9% could be identified as free carbohydrates, polyamines, or free and combined amino acids. Carbohydrates represented 54%-61% of identified compounds of the extracellular OM from each alga. In biomass-derived OM of Rhodomonas and Chlamydomonas 71%-77% were amino acids and polyamines, while in that of Coelastrum 85% were carbohydrates. All bacteria grew on alga-derived OM of Coelastrum, whereas only Limnohabitans strains grew on OM from Rhodomonas and Chlamydomonas. Bacteria consumed 24%-76% and 38%-82% of all identified extracellular and biomass-derived OM compounds respectively, and their consumption was proportional to the concentration of each OM compound in the different treatments. The bacterial biomass yield was higher than the total identifiable OM consumption indicating that bacteria also utilized other unidentified alga-derived OM compounds. Bacteria, however, also produced specific OM compounds suggesting enzymatic polymer degradation or de novo exudation.
Despite increasing concerns about microplastic (MP) pollution in aquatic ecosystems, there is insufficient knowledge on how MP affect fungal communities. In this study, we explored the diversity and community composition of fungi attached to polyethylene (PE) and polystyrene (PS) particles incubated in different aquatic systems in north-east Germany: the Baltic Sea, the River Warnow and a wastewater treatment plant. Based on next generation 18S rRNA gene sequencing, 347 different operational taxonomic units assigned to 81 fungal taxa were identified on PE and PS. The MP-associated communities were distinct from fungal communities in the surrounding water and on the natural substrate wood. They also differed significantly among sampling locations, pointing towards a substrate and location specific fungal colonization. Members of Chytridiomycota, Cryptomycota and Ascomycota dominated the fungal assemblages, suggesting that both parasitic and saprophytic fungi thrive in MP biofilms. Thus, considering the worldwide increasing accumulation of plastic particles as well as the substantial vector potential of MP, especially these fungal taxa might benefit from MP pollution in the aquatic environment with yet unknown impacts on their worldwide distribution, as well as biodiversity and food web dynamics at large.
Butterflies rank among the most threatened animal groups throughout Europe. However, current population trends differ among species. The nettle-feeding butterflies Aglais io and Aglais urticae cope successfully with the anthropogenic land-use change. Both species are assumed to be pre-adapted to higher nitrogen contents in their host plant, stinging nettle (Urtica dioica). However, it is currently unknown, whether this pre-adaptation enables both Aglais species to cope successfully or even to benefit from the excessive nitrogen availabilities in nettles growing in modern farmlands. For this reason, this study focused on the response of both Aglais species to unfertilized nettles compared to nettles receiving 150 or 300 kg N ha(-1) yr(-1) (i.e., common fertilizer quantities of modern-day agriculture). Fertilized nettles were characterized by higher nitrogen concentrations and lower C:N ratios compared to the control group. In both Aglais species, the individuals feeding on fertilized nettles had higher survival rates, shorter larval periods and heavier pupae and, in A. urticae also longer forewings. All these trait shifts are beneficial for the individuals, lowering their risk to die before reproduction and increasing their reproductive potential. These responses agree with the well-accepted nitrogen-limitation hypothesis predicting a positive relationship between the nitrogen content of the diet and the performance of herbivorous insects. Furthermore, our findings suggest that the increasing abundance of both Aglais species may result not only from the increasing spread of nettles into the farmland but also from changes in their quality due to the eutrophication of the landscape during recent decades.
The epidermis of aerial plant organs is thought to be limiting for growth, because it acts as a continuous load-bearing layer, resisting tension. Leaf epidermis contains jigsaw puzzle piece-shaped pavement cells whose shape has been proposed to be a result of subcellular variations in expansion rate that induce local buckling events. Paradoxically, such local compressive buckling should not occur given the tensile stresses across the epidermis. Using computational modeling, we show that the simplest scenario to explain pavement cell shapes within an epidermis under tension must involve mechanical wall heterogeneities across and along the anticlinal pavement cell walls between adjacent cells. Combining genetics, atomic force microscopy, and immunolabeling, we demonstrate that contiguous cell walls indeed exhibit hybrid mechanochemical properties. Such biochemical wall heterogeneities precede wall bending. Altogether, this provides a possible mechanism for the generation of complex plant cell shapes.
Saber-toothed cats (Machairodontinae) are among the most widely recognized representatives of the now largely extinct Pleistocene megafauna. However, many aspects of their ecology, evolution, and extinction remain uncertain. Although ancient-DNA studies have led to huge advances in our knowledge of these aspects of many other megafauna species (e.g., mammoths and cave bears), relatively few ancient-DNA studies have focused on saber-toothed cats [1-3], and they have been restricted to short fragments of mitochondrial DNA. Here we investigate the evolutionary history of two lineages of saber-toothed cats (Smilodon and Homotherium) in relation to living carnivores and find that the Machairodontinae form a well-supported clade that is distinct from all living felids. We present partial mitochondrial genomes from one S. populator sample and three Homotherium sp. samples, including the only Late Pleistocene Homotherium sample from Eurasia [4]. We confirm the identification of the unique Late Pleistocene European fossil through ancient-DNA analyses, thus strengthening the evidence that Homotherium occurred in Europe over 200,000 years later than previously believed. This in turn forces a re-evaluation of its demography and extinction dynamics. Within the Machairodontinae, we find a deep divergence between Smilodon and Homotherium (similar to 18 million years) but limited diversity between the American and European Homotherium specimens. The genetic data support the hypothesis that all Late Pleistocene (or post-Villafrancian) Homotherium should be considered a single species, H. latidens, which was previously proposed based on morphological data [5, 6].
The homodinuclear ruthenium(II) complex [{Ru(l-N4Me2)}(2)(-tape)](PF6)(4) {[1](PF6)(4)} (l-N4Me2=N,N-dimethyl-2,11-diaza[3.3](2,6)-pyridinophane, tape=1,6,7,12-tetraazaperylene) can store one or two electrons in the energetically low-lying * orbital of the bridging ligand tape. The corresponding singly and doubly reduced complexes [{Ru(l-N4Me2)}(2)(-tape(.-))](PF6)(3) {[2](PF6)(3)} and [{Ru(l-N4Me2)}(2)(-tape(2-))](PF6)(2) {[3](PF6)(2)}, respectively, were electrochemically generated, successfully isolated and fully characterized by single-crystal X-ray crystallography, spectroscopic methods and magnetic susceptibility measurements. The singly reduced complex [2](PF6)(3) contains the -radical tape(.-) and the doubly reduced [3](PF6)(2) the diamagnetic dianion tape(2-) as bridging ligand, respectively. Nucleophilic aromatic substitution at the bridging tape in [1](4+) by two sulfite units gave the complex [{Ru(l-N4Me2)}(2){-tape-(SO3)(2)}](2+) ([4](2+)). Complex dication [4](2+) was exploited as a redox mediator between an anaerobic homogenous reaction solution of an enzyme system (sulfite/sulfite oxidase) and the electrode via participation of the low-energy *-orbital of the disulfonato-substituted bridging ligand tape-(SO3)(2)(2-) (E-red1=-0.1V versus Ag/AgCl/1m KCl in water).
Ectoine plays an important role in protecting biomolecules and entire cells against environmental stressors such as salinity, freezing, drying and high temperatures. Recent studies revealed that ectoine also provides effective protection for human skin cells from damage caused by UV-A radiation. These protective properties make ectoine a valuable compound and it is applied as an active ingredient in numerous pharmaceutical devices and cosmetics. Interestingly, the underlying mechanism resulting in protecting cells from radiation is not yet fully understood. Here we present a study on ectoine and its protective influence on DNA during electron irradiation. Applying gel electrophoresis and atomic force microscopy, we demonstrate for the first time that ectoine prevents DNA strand breaks caused by ionizing electron radiation. The results presented here point to future applications of ectoine for instance in cancer radiation therapy.
Antarctic ice-covered lakes are exceptional sites for studying the ecology of aquatic fungi under conditions of minimal human disturbance. In this study, we explored the diversity and community composition of fungi in five permanently covered lake basins located in the Taylor and Miers Valleys of Antarctica. Based on analysis of the 18S rRNA sequences, we showed that fungal taxa represented between 0.93% and 60.32% of the eukaryotic sequences. Cryptomycota and Chytridiomycota dominated the fungal communities in all lakes; however, members of Ascomycota, Basidiomycota, Zygomycota, and Blastocladiomycota were also present. Of the 1313 fungal OTUs identified, the two most abundant, belonging to LKM11 and Chytridiaceae, comprised 74% of the sequences. Significant differences in the community structure were determined among lakes, water depths, habitat features (i.e., brackish vs. freshwaters), and nucleic acids (DNA vs. RNA), suggesting niche differentiation. Network analysis suggested the existence of strong relationships among specific fungal phylotypes as well as between fungi and other eukaryotes. This study sheds light on the biology and ecology of basal fungi in aquatic systems. To our knowledge, this is the first report showing the predominance of early diverging lineages of fungi in pristine limnetic ecosystems, particularly of the enigmatic phylum Cryptomycota.
Animal reproductive proteins, especially those in the seminal fluid, have been shown to have higher levels of divergence than non-reproductive proteins and are often evolving adaptively. Seminal fluid proteins have been implicated in the formation of reproductive barriers between diverging lineages, and hence represent interesting candidates underlying speciation. RNA-seq was used to generate the first male reproductive transcriptome for the New Zealand tree weta species Hemideina thoracica and H. crassidens. We identified 865 putative reproductive associated proteins across both species, encompassing a diverse range of functional classes. Candidate gene sequencing of nine genes across three Hemideina, and two Deinacrida species suggests that H. thoracica has the highest levels of intra-specific genetic diversity. Non-monophyly was observed in the majority of sequenced genes indicating that either gene flow may be occurring between the species, or that reciprocal monophyly at these loci has yet to be attained. Evidence for positive selection was found for one lectin-related reproductive protein, with an overall omega of 7.65 and one site in particular being under strong positive selection. This candidate gene represents the first step in the identification of proteins underlying the evolutionary basis of weta reproduction and speciation.
An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.
It is unclear whether Indo-European languages in Europe spread from the Pontic steppes in the late Neolithic, or from Anatolia in the Early Neolithic. Under the former hypothesis, people of the Globular Amphorae culture (GAC) would be descended from Eastern ancestors, likely representing the Yamnaya culture. However, nuclear (six individuals typed for 597 573 SNPs) and mitochondrial (11 complete sequences) DNA from the GAC appear closer to those of earlier Neolithic groups than to the DNA of all other populations related to the Pontic steppe migration. Explicit comparisons of alternative demographic models via approximate Bayesian computation confirmed this pattern. These results are not in contrast to Late Neolithic gene flow from the Pontic steppes into Central Europe. However, they add nuance to this model, showing that the eastern affinities of the GAC in the archaeological record reflect cultural influences from other groups from the East, rather than the movement of people.
Seasonal and spatial functional shifts in phytoplankton communities of five tropical reservoirs
(2017)
Trait-based approaches have become increasingly important and valuable in understanding phytoplankton community assembly and composition. These approaches allow for comparisons between water bodies with different species composition. We hypothesize that similar changes in environmental conditions lead to similar responses with regard to functional traits of phytoplankton communities, regardless of trophic state or species composition. We studied the phytoplankton (species composition, community trait mean and diversity) of five reservoirs in Brazil along a trophic gradient from ultra-oligotrophic to meso-eutrophic. Samples at two seasons (summer/rainy and winter/dry) with a horizontal and vertical resolution were taken. Using multivariate analysis, the five reservoirs separated, despite some overlap, according to their environmental variables (mainly total phosphorus, conductivity, pH, chlorophyll a). However, between the seasonal periods, the reservoirs shifted in a similar direction in the multi-dimensional space. The seasonal response of the overall phytoplankton community trait mean differed between the ultra-oligotrophic and the other reservoirs, with three reservoirs exhibiting a very similar community trait mean despite considerable differences in species composition. Within-season differences between different water layers were low. The functional diversity was also unrelated to the trophic state of the reservoirs. Thus, seasonal environmental changes had strong influence on the functional characteristics of the phytoplankton community in reservoirs with distinct trophic condition and species composition. These results demonstrate that an ataxonomic trait-based approach is a relevant tool for comparative studies in phytoplankton ecology.
The amyloid precursor-like protein 1 (APLP1) is a type I transmembrane protein that plays a role in synaptic adhesion and synaptogenesis. Past investigations indicated that APLP1 is involved in the formation of protein-protein complexes that bridge the junctions between neighboring cells. Nevertheless, APLP1-APLP1 trans interactions have never been directly observed in higher eukaryotic cells. Here, we investigated APLP1 interactions and dynamics directly in living human embryonic kidney cells using fluorescence fluctuation spectroscopy techniques, namely cross-correlation scanning fluorescence correlation spectroscopy and number and brightness analysis. Our results show that APLP1 forms homotypic trans complexes at cell-cell contacts. In the presence of zinc ions, the protein forms macroscopic clusters, exhibiting an even higher degree of trans binding and strongly reduced dynamics. Further evidence from giant plasma membrane vesicles suggests that the presence of an intact cortical cytoskeleton is required for zinc-induced cis multimerization. Subsequently, large adhesion platforms bridging interacting cells are formed through APLP1-APLP1 trans interactions. Taken together, our results provide direct evidence that APLP1 functions as a neuronal zinc-dependent adhesion protein and allow a more detailed understanding of the molecular mechanisms driving the formation of APLP1 adhesion platforms.
Bacterial biofilm formation is a widespread phenomenon and a complex process requiring a set of genes facilitating the initial adhesion, maturation, and production of the extracellular polymeric matrix and subsequent dispersal of bacteria. Most studies on Escherichia coli biofilm formation have investigated nonpathogenic E. coli K-12 strains. Due to the extensive focus on laboratory strains in most studies, there is poor information regarding biofilm formation by pathogenic E. coli isolates. In this study, we genotypically and phenotypically characterized 187 human clinical E. coli isolates representing various pathotypes (e.g., uropathogenic, enteropathogenic, and enteroaggregative E. coli). We investigated the presence of biofilm-associated genes ("genotype") and phenotypically analyzed the isolates for motility and curli and cellulose production ("phenotype"). We developed a new screening method to examine the in vitro biofilm formation ability. In summary, we found a high prevalence of biofilm-associated genes. However, we could not detect a biofilm-associated gene or specific phenotype correlating with the biofilm formation ability. In contrast, we did identify an association of increased biofilm formation with a specific E. coli pathotype. Enteroaggregative E. coli (EAEC) was found to exhibit the highest capacity for biofilm formation. Using our image-based technology for the screening of biofilm formation, we demonstrated the characteristic biofilm formation pattern of EAEC, consisting of thick bacterial aggregates. In summary, our results highlight the fact that biofilm-promoting factors shown to be critical for biofilm formation in nonpathogenic strains do not reflect their impact in clinical isolates and that the ability of biofilm formation is a defined characteristic of EAEC. IMPORTANCE Bacterial biofilms are ubiquitous and consist of sessile bacterial cells surrounded by a self-produced extracellular polymeric matrix. They cause chronic and device-related infections due to their high resistance to antibiotics and the host immune system. In nonpathogenic Escherichia coli, cell surface components playing a pivotal role in biofilm formation are well known. In contrast, there is poor information for their role in biofilm formation of pathogenic isolates. Our study provides insights into the correlation of biofilm-associated genes or specific phenotypes with the biofilm formation ability of commensal and pathogenic E. coli. Additionally, we describe a newly developed method enabling qualitative biofilm analysis by automated image analysis, which is beneficial for high-throughput screenings. Our results help to establish a better understanding of E. coli biofilm formation.
Trade-offs between functional traits are ubiquitous in nature and can promote species coexistence depending on their shape. Classic theory predicts that convex trade-offs facilitate coexistence of specialized species with extreme trait values (extreme species) while concave trade-offs promote species with intermediate trait values (intermediate species). We show here that this prediction becomes insufficient when the traits translate non-linearly into fitness which frequently occurs in nature, e.g., an increasing length of spines reduces grazing losses only up to a certain threshold resulting in a saturating or sigmoid trait-fitness function. We present a novel, general approach to evaluate the effect of different trade-off shapes on species coexistence. We compare the trade-off curve to the invasion boundary of an intermediate species invading the two extreme species. At this boundary, the invasion fitness is zero. Thus, it separates trait combinations where invasion is or is not possible. The invasion boundary is calculated based on measurable trait-fitness relationships. If at least one of these relationships is not linear, the invasion boundary becomes non-linear, implying that convex and concave trade-offs not necessarily lead to different coexistence patterns. Therefore, we suggest a new ecological classification of trade-offs into extreme-favoring and intermediate-favoring which differs from a purely mathematical description of their shape. We apply our approach to a well-established model of an empirical predator-prey system with competing prey types facing a trade-off between edibility and half-saturation constant for nutrient uptake. We show that the survival of the intermediate prey depends on the convexity of the trade-off. Overall, our approach provides a general tool to make a priori predictions on the outcome of competition among species facing a common trade-off in dependence of the shape of the trade-off and the shape of the trait-fitness relationships.
This study tested systematically at two spatial scales for key traits shaping within-species ecotypic differentiation under increasing aridity. It assessed different plant strategy theories and considered potential implications for climate change. We studied the widespread Mediterranean grass Brachypodium hybridum. At large scale, we tested 14 populations along a steep natural aridity gradient (114-954 mm annual rainfall). At small scale, we tested the microclimatic contrast between plants originating from corresponding north (more mesic) and south (more arid) exposed hillslopes. Fifteen traits were measured in the greenhouse, including the popular traits of the LeafHeight- Seed scheme (SLA, plant height, seed mass), several traits on phenology, architecture, growth, fitness, and rarely measured root traits. Clear trait shifts indicated ecotypic differentiation along the large-scale gradient. Earlier phenology, higher reproductive allocation and reduced root investment characterized arid ecotypes. Surprisingly, no trait of the Leaf-Height-Seed scheme shifted with aridity and root responses were opposite to the theory of optimal resource partitioning. Trait differences between north and south exposures were small, often inconsistent between sites, and poorly matched the trends across the large-scale gradient. South exposures thus appeared unlikely to harbour distinct ecotypes better adapted to aridity. Our findings highlight ecotypes as a crucial way how species span environmental gradients, yet underpinning their restriction at small spatial scales. In combination, this possibly renders populations more vulnerable to climate change. We draw attention to specific, partly unexpected traits and pose the question whether the LeafHeight- Seed scheme has limited applicability for intraspecific investigations in drylands.
Arabidopsis EARLY FLOWERING3 increases salt tolerance by suppressing salt stress response pathways
(2017)
Intraspecific trait variability plays an important role in species adaptation to climate change. However, it still remains unclear how plants in semi-arid environments respond to increasing aridity. We investigated the intraspecific trait variability of two common Mediterranean annuals (Geropogon hybridus and Crupina crupinastrum) with similar habitat preferences. They were studied along a steep precipitation gradient in Israel similar to the maximum predicted precipitation changes in the eastern Mediterranean basin (i.e. -30% until 2100). We expected a shift from competitive ability to stress tolerance with decreasing precipitation and tested this expectation by measuring key functional traits (canopy and seed release height, specific leaf area, N-and P-leaf content, seed mass). Further, we evaluated generative bet-hedging strategies by different seed traits. Both species showed different responses along the precipitation gradient. C. crupinastrum exhibited only decreased plant height toward saridity, while G. hybridus showed strong trends of generative adaptation to aridity. Different seed trait indices suggest increased bet-hedging of G. hybridus in arid environments. However, no clear trends along the precipitation gradient were observed in leaf traits (specific leaf area and leaf N-/P-content) in both species. Moreover, variance decomposition revealed that most of the observed trait variation (>> 50%) is found within populations. The findings of our study suggest that responses to increased aridity are highly species-specific and local environmental factors may have a stronger effect on intraspecific trait variation than shifts in annual precipitation. We therefore argue that trait-based analyses should focus on precipitation gradients that are comparable to predicted precipitation changes and compare precipitation effects to effects of local environmental factors. (C) 2017 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.
Biotic and abiotic particles shape the microspatial architecture that defines the microbial aquatic habitat, being particles highly variable in size and quality along oceanic horizontal and vertical gradients. We analysed the prokaryotic (bacterial and archaeal) diversity and community composition present in six distinct particle size classes ranging from the pico-to the microscale (0.2 to 200 lm). Further, we studied their variations along oceanographic horizontal (from the coast to open oceanic waters) and vertical (from the ocean surface into the meso-and bathypelagic ocean) gradients. In general, prokaryotic community composition was more variable with depth than in the transition from the coast to the open ocean. Comparing the six size-fractions, distinct prokaryotic communities were detected in each size-fraction, and whereas bacteria were more diverse in the larger size-fractions, archaea were more diverse in the smaller size-fractions. Comparison of prokaryotic community composition among particle size-fractions showed that most, but not all, taxonomic groups have a preference for a certain size-fraction sustained with depth. Species sorting, or the presence of diverse ecotypes with distinct size-fraction preferences, may explain why this trend is not conserved in all taxa.
Hybrid lead halide perovskites are introduced as charge generation layers (CGLs) for the accurate determination of electron mobilities in thin organic semiconductors. Such hybrid perovskites have become a widely studied photovoltaic material in their own right, for their high efficiencies, ease of processing from solution, strong absorption, and efficient photogeneration of charge. Time-of-flight (ToF) measurements on bilayer samples consisting of the perovskite CGL and an organic semiconductor layer of different thickness are shown to be determined by the carrier motion through the organic material, consistent with the much higher charge carrier mobility in the perovskite. Together with the efficient photon-to-electron conversion in the perovskite, this high mobility imbalance enables electron-only mobility measurement on relatively thin application-relevant organic films, which would not be possible with traditional ToF measurements. This architecture enables electron-selective mobility measurements in single components as well as bulk-heterojunction films as demonstrated in the prototypical polymer/fullerene blends. To further demonstrate the potential of this approach, electron mobilities were measured as a function of electric field and temperature in an only 127 nm thick layer of a prototypical electron-transporting perylene diimide-based polymer, and found to be consistent with an exponential trap distribution of ca. 60 meV. Our study furthermore highlights the importance of high mobility charge transporting layers when designing perovskite solar cells.
Disentangling eco-evolutionary dynamics of predator-prey coevolution: the case of antiphase cycles
(2017)
The impact of rapid predator-prey coevolution on predator-prey dynamics remains poorly understood, as previous modelling studies have given rise to contradictory conclusions and predictions. Interpreting and reconciling these contradictions has been challenging due to the inherent complexity of model dynamics, defying mathematical analysis and mechanistic understanding. We develop a new approach here, based on the Geber method for deconstructing eco-evolutionary dynamics, for gaining such understanding. We apply this approach to a co-evolutionary predator-prey model to disentangle the processes leading to either antiphase or 1/4-lag cycles. Our analysis reveals how the predator-prey phase relationship is driven by the temporal synchronization between prey biomass and defense dynamics. We further show when and how prey biomass and trait dynamics become synchronized, resulting in antiphase cycles, allowing us to explain and reconcile previous modelling and empirical predictions. The successful application of our proposed approach provides an important step towards a comprehensive theory on eco-evolutionary feedbacks in predator-prey systems.
The Cheb Basin (NW Bohemia, Czech Republic) is a shallow, neogene intracontinental basin. It is a non-volcanic region which features frequent earthquake swarms and large-scale diffuse degassing of mantle-derived CO2 at the surface that occurs in the form of CO2-rich mineral springs and wet and dry mofettes. So far, the influence of CO2 degassing onto the microbial communities has been studied for soil environments, but not for aquatic systems. We hypothesized, that deep-trenching CO2 conduits interconnect the subsurface with the surface. This admixture of deep thermal fluids should be reflected in geochemical parameters and in the microbial community compositions. In the present study four mineral water springs and two wet mofettes were investigated through an interdisciplinary survey. The waters were acidic and differed in terms of organic carbon and anion/cation concentrations. Element geochemical and isotope analyses of fluid components were used to verify the origin of the fluids. Prokaryotic communities were characterized through quantitative PCR and Illumina 16S rRNA gene sequencing. Putative chemolithotrophic, anaerobic and microaerophilic organisms connected to sulfur (e.g., Sulfuricurvum, Sulfurimonas) and iron (e.g., Gallionella, Sideroxydans) cycling shaped the core community. Additionally, CO2-influenced waters form an ecosystem containing many taxa that are usually found in marine or terrestrial subsurface ecosystems. Multivariate statistics highlighted the influence of environmental parameters such as pH, Fe2+ concentration and conductivity on species distribution. The hydrochemical and microbiological survey introduces a new perspective on mofettes. Our results support that mofettes are either analogs or rather windows into the deep biosphere and furthermore enable access to deeply buried paleo-sediments.
JUNGBRUNNEN1 Confers Drought Tolerance Downstream of the HD-Zip I Transcription Factor AtHB13
(2017)
Low water availability is the major environmental factor limiting growth and productivity of plants and crops and is therefore considered of high importance for agriculture affected by climate change. Identifying regulatory components controlling the response and tolerance to drought stress is thus of major importance. The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) from Arabidopsis thaliana extends leaf longevity under non-stress growth conditions, lowers cellular hydrogen peroxide (H2O2) level, and enhances tolerance against heat stress and salinity. Here, we additionally find that JUB1 strongly increases tolerance to drought stress in Arabidopsis when expressed from both, a constitutive (CaMV 35S) and an abiotic stress-induced (RD29A) promoter. Employing a yeast one-hybrid screen we identified HD-Zip class I TF AtHB13 as an upstream regulator of JUB1. AtHB13 has previously been reported to act as a positive regulator of drought tolerance. AtHB13 and JUB1 thereby establish a joint drought stress control module.
Pollen-based quantitative reconstructions of past climate variables is a standard palaeoclimatic approach. Despite knowing that the spatial extent of the calibration-set affects the reconstruction result, guidance is lacking as to how to determine a suitable spatial extent of the pollen-climate calibration-set. In this study, past mean annual precipitation (P-ann) during the Holocene (since 11.5 cal ka BP) is reconstructed repeatedly for pollen records from Qinghai Lake (36.7 degrees N, 100.5 degrees E; north-east Tibetan Plateau), Gonghai Lake (38.9 degrees N, 112.2 degrees E; north China) and Sihailongwan Lake (42.3 degrees N, 126.6 degrees E; north-east China) using calibration-sets of varying spatial extents extracted from the modern pollen dataset of China and Mongolia (2559 sampling sites and 168 pollen taxa in total). Results indicate that the spatial extent of the calibration-set has a strong impact on model performance, analogue quality and reconstruction diagnostics (absolute value, range, trend, optimum). Generally, these effects are stronger with the modern analogue technique (MAT) than with weighted averaging partial least squares (WA-PLS). With respect to fossil spectra from northern China, the spatial extent of calibration-sets should be restricted to radii between ca. 1000 and 1500 km because small-scale calibration-sets (<800 km radius) will likely fail to include enough spatial variation in the modern pollen assemblages to reflect the temporal range shifts during the Holocene, while too broad a scale calibration-set (>1500 km radius) will include taxa with very different pollen-climate relationships. (C) 2017 Elsevier Ltd. All rights reserved.
Heterostyly is a fascinating adaptation to promote outbreeding and a classical paradigm of botany. In the most common type of heterostyly, plants either form flowers with long styles and short stamens, or short styles and long stamens. This reciprocal organ positioning reduces pollen wastage and promotes cross-pollination, thus increasing male fitness. In addition, in many heterostylous species selfing and the generation of unfit progeny due to inbreeding depression is limited by a self-incompatibility system, thus promoting female fitness. The two floral forms are genetically determined by the S locus as a complex supergene, namely a chromosomal region containing several individual genes that control the different traits, such as style or stamen length, and are held together by very tight linkage due to suppressed recombination. Recent molecular-genetic studies in several systems, including Turnera, Fagopyrum, Linum, and Primula have begun to identify and characterize the causal heterostyly genes residing at the S locus. An emerging theme from several families is that the dominant S haplotype represents a hemizygous region not present on the recessive s haplotype. This provides an explanation for the suppressed recombination and suggests a scenario for the chromosomal evolution of the S locus. In this review, we discuss the results from recent molecular-genetic analyses in light of the classical models on the genetics and evolution of heterostyly.
Stoichiometric Correlation Analysis: Principles of Metabolic Functionality from Metabolomics Data
(2017)
Recent advances in metabolomics technologies have resulted in high-quality (time-resolved) metabolic profiles with an increasing coverage of metabolic pathways. These data profiles represent read-outs from often non-linear dynamics of metabolic networks. Yet, metabolic profiles have largely been explored with regression-based approaches that only capture linear relationships, rendering it difficult to determine the extent to which the data reflect the underlying reaction rates and their couplings. Here we propose an approach termed Stoichiometric Correlation Analysis (SCA) based on correlation between positive linear combinations of log-transformed metabolic profiles. The log-transformation is due to the evidence that metabolic networks can be modeled by mass action law and kinetics derived from it. Unlike the existing approaches which establish a relation between pairs of metabolites, SCA facilitates the discovery of higherorder dependence between more than two metabolites. By using a paradigmatic model of the tricarboxylic acid cycle we show that the higher-order dependence reflects the coupling of concentration of reactant complexes, capturing the subtle difference between the employed enzyme kinetics. Using time-resolved metabolic profiles from Arabidopsis thaliana and Escherichia coli, we show that SCA can be used to quantify the difference in coupling of reactant complexes, and hence, reaction rates, underlying the stringent response in these model organisms. By using SCA with data from natural variation of wild and domesticated wheat and tomato accession, we demonstrate that the domestication is accompanied by loss of such couplings, in these species. Therefore, application of SCA to metabolomics data from natural variation in wild and domesticated populations provides a mechanistic way to understanding domestication and its relation to metabolic networks.
This is a cumulative dissertation comprising three original studies (one published, one in revision, one submitted; Effective December 2017) investigating how reptile species in arid Australia respond to various climatic parameters at different spatial scales and analysing the two potential main underlying mechanisms: thermoregulatory behaviour and species interactions. This dissertation combines extensive individual-based field data across trophic levels, selected field experiments, statistical analyses, and predictive modelling techniques. Mechanisms and processes detected in this dissertation can now be used to predict potential future changes in the community of arid-zone lizards. This knowledge will help improving our fundamental understanding of the consequences of global change and thereby prevent biodiversity loss in a vulnerable ecosystem.
In this work the human AOX1 was characterized and detailed aspects regarding the expression, the enzyme kinetics and the production of reactive oxygen species (ROS) were investigated. The hAOX1 is a cytosolic enzyme belonging to the molybdenum hydroxylase family. Its catalytically active form is a homodimer with a molecular weight of 300 kDa. Each monomer (150 kDa) consists of three domains: a N-terminal domain (20 kDa) containing two [2Fe-2S] clusters, a 40 kDa intermediate domain containing a flavin adenine dinucleotide (FAD), and a C-terminal domain (85 kDa) containing the substrate binding pocket and the molybdenum cofactor (Moco). The hAOX1 has an emerging role in the metabolism and pharmacokinetics of many drugs, especially aldehydes and N- heterocyclic compounds.
In this study, the hAOX1 was hetereogously expressed in E. coli TP1000 cells, using a new codon optimized gene sequence which improved the expressed protein yield of around 10-fold compared to the previous expression systems for this enzyme. To increase the catalytic activity of hAOX1, an in vitro chemical sulfuration was performed to favor the insertion of the equatorial sulfido ligand at the Moco with consequent increased enzymatic activity of around 10-fold. Steady-state kinetics and inhibition studies were performed using several substrates, electron acceptors and inhibitors. The recombinant hAOX1 showed higher catalytic activity when molecular oxygen was used as electron acceptor. The highest turn over values were obtained with phenanthridine as substrate. Inhibition studies using thioridazine (phenothiazine family), in combination with structural studies performed in the group of Prof. M.J. Romão, Nova Universidade de Lisboa, showed a new inhibition site located in proximity of the dimerization site of hAOX1. The inhibition mode of thioridazine resulted in a noncompetitive inhibition type. Further inhibition studies with loxapine, a thioridazine-related molecule, showed the same type of inhibition. Additional inhibition studies using DCPIP and raloxifene were carried out.
Extensive studies on the FAD active site of the hAOX1 were performed. Twenty new hAOX1 variants were produced and characterized. The hAOX1 variants generated in this work were divided in three groups: I) hAOX1 single nucleotide polymorphisms (SNP) variants; II) XOR- FAD loop hAOX1 variants; III) additional single point hAOX1 variants. The hAOX1 SNP variants G46E, G50D, G346R, R433P, A439E, K1231N showed clear alterations in their catalytic activity, indicating a crucial role of these residues into the FAD active site and in relation to the overall reactivity of hAOX1.
Furthermore, residues of the bovine XOR FAD flexible loop (Q423ASRREDDIAK433) were introduced in the hAOX1. FAD loop hAOX1 variants were produced and characterized for their stability and catalytic activity. Especially the variants hAOX1 N436D/A437D/L438I, N436D/A437D/L438I/I440K and Q434R/N436D/A437D/L438I/I440K showed decreased catalytic activity and stability. hAOX1 wild type and variants were tested for reactivity toward NADH but no reaction was observed.
Additionally, the hAOX1 wild type and variants were tested for the generation of reactive oxygen species (ROS). Interestingly, one of the SNP variants, hAOX1 L438V, showed a high ratio of superoxide prodction. This result showed a critical role for the residue Leu438 in the mechanism of oxygen radicals formation by hAOX1. Subsequently, further hAOX1 variants having the mutated Leu438 residue were produced. The variants hAOX1 L438A, L438F and L438K showed superoxide overproduction of around 85%, 65% and 35% of the total reducing equivalent obtained from the substrate oxidation.
The results of this work show for the first time a characterization of the FAD active site of the hAOX1, revealing the importance of specific residues involved in the generation of ROS and effecting the overall enzymatic activity of hAOX1. The hAOX1 SNP variants presented here indicate that those allelic variations in humans might cause alterations ROS balancing and clearance of drugs in humans.
Analyse der Funktion der dualen Lokalisation der 3-Mercaptopyruvat Sulfurtransferase im Menschen
(2017)
In littoral zones of lakes, multiple processes determine lake ecology and water quality. Lacustrine groundwater discharge (LGD), most frequently taking place in littoral zones, can transport or mobilize nutrients from the sediments and thus contribute significantly to lake eutrophication. Furthermore, lake littoral zones are the habitat of benthic primary producers, namely submerged macrophytes and periphyton, which play a key role in lake food webs and influence lake water quality. Groundwater-mediated nutrient-influx can potentially affect the asymmetric competition between submerged macrophytes and periphyton for light and nutrients. While rooted macrophytes have superior access to sediment nutrients, periphyton can negatively affect macrophytes by shading. LGD may thus facilitate periphyton production at the expense of macrophyte production, although studies on this hypothesized effect are missing.
The research presented in this thesis is aimed at determining how LGD influences periphyton, macrophytes, and the interactions between these benthic producers. Laboratory experiments were combined with field experiments and measurements in an oligo-mesotrophic hard water lake.
In the first study, a general concept was developed based on a literature review of the existing knowledge regarding the potential effects of LGD on nutrients and inorganic and organic carbon loads to lakes, and the effect of these loads on periphyton and macrophytes. The second study includes a field survey and experiment examining the effects of LGD on periphyton in an oligotrophic, stratified hard water lake (Lake Stechlin). This study shows that LGD, by mobilizing phosphorus from the sediments, significantly promotes epiphyton growth, especially at the end of the summer season when epilimnetic phosphorus concentrations are low. The third study focuses on the potential effects of LGD on submerged macrophytes in Lake Stechlin. This study revealed that LGD may have contributed to an observed change in macrophyte community composition and abundance in the shallow littoral areas of the lake. Finally, a laboratory experiment was conducted which mimicked the conditions of a seepage lake. Groundwater circulation was shown to mobilize nutrients from the sediments, which significantly promoted periphyton growth. Macrophyte growth was negatively affected at high periphyton biomasses, confirming the initial hypothesis.
More generally, this thesis shows that groundwater flowing into nutrient-limited lakes may import or mobilize nutrients. These nutrients first promote periphyton, and subsequently provoke radical changes in macrophyte populations before finally having a possible influence on the lake’s trophic state. Hence, the eutrophying effect of groundwater is delayed and, at moderate nutrient loading rates, partly dampened by benthic primary producers. The present research emphasizes the importance and complexity of littoral processes, and the need to further investigate and monitor the benthic environment. As present and future global changes can significantly affect LGD, the understanding of these complex interactions is required for the sustainable management of lake water quality.
Natural products and their derivatives have always been a source of drug leads. In particular, bacterial compounds have played an important role in drug development, for example in the field of antibiotics. A decrease in the discovery of novel leads from natural sources and the hope of finding new leads through the generation of large libraries of drug-like compounds by combinatorial chemistry aimed at specific molecular targets drove the pharmaceutical companies away from research on natural products. However, recent technological advances in genetics, bioinformatics and analytical chemistry have revived the interest in natural products. The ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of natural products generated by the action of post-translationally modifying enzymes on precursor peptides translated from mRNA by ribosomes. The great substrate promiscuity exhibited by many of the enzymes from RiPP biosynthetic pathways have led to the generation of hundreds of novel synthetic and semisynthetic variants, including variants carrying non-canonical amino acids (ncAAs). The microviridins are a family of RiPPs characterized by their atypical tricyclic structure composed of lactone and lactam rings, and their activity as serine protease inhibitors. The generalities of their biosynthetic pathway have already been described, however, the lack of information on details such as the protease responsible for cleaving off the leader peptide from the cyclic core peptide has impeded the fast and cheap production of novel microviridin variants. In the present work, knowledge on leader peptide activation of enzymes from other RiPP families has been extrapolated to the microviridin family, making it possible to bypass the need of a leader peptide. This feature allowed for the exploitation of the microviridin biosynthetic machinery for the production of novel variants through the establishment of an efficient one-pot in vitro platform. The relevance of this chemoenzymatic approach has been exemplified by the synthesis of novel potent serine protease inhibitors from both rationally-designed peptide libraries and bioinformatically predicted microviridins. Additionally, new structure-activity relationships (SARs) could be inferred by screening microviridin intermediates. The significance of this technique was further demonstrated by the simple incorporation of ncAAs into the microviridin scaffold.
Die Hybridomtechnik zur Produktion von monoklonalen Antikörpern ermöglichte einen großen Schritt in der Entwicklung von Immunoassays für die biochemische Forschung und klinische Diagnostik. Auch die Produktion von Antikörpern gegen niedermolekulare Analyten, Haptene, typische Targets in der Lebensmittel- und Umweltanalytik, erlangte in den letzten Jahren eine immer größere Bedeutung. Im Zuge der Durchführung der Hybridomtechnik werden tausende Antikörper-sezernierende und nicht-sezernierende Zellen generiert. Die Selektion der wenigen antigenselektiven Hybridomzellen zählt dabei zu den herausforderndsten Schritten für die Antikörpergewinnung. Bisherige Selektionsverfahren, wie die Limiting-Dilution-Klonierung in Verbindung mit Enzyme-linked Immunosorbent Assays (ELISAs), garantieren keine Monoklonalität und erlauben nur das Screening von einigen wenigen Zellklonen. Hingegen ermöglichen Hochdurchsatz-Selektionsmethoden, wie die Fluoreszenz-aktivierte Zellsortierung (FACS), einen sehr hohen Probendurchsatz. Eine Einzelzellablage garantiert hierbei Monoklonalität. Jedoch sind die dafür erforderlichen Zellmarkierungen oftmals zellschädigend oder aufwendig zu generieren. Auch ist bisher noch keine Markierungsmethode bekannt, die es ermöglicht, Hapten-selektive Hybridomzellen durchflusszytometrisch zu analysieren und eine FACS-Selektion durchzuführen.
Aus diesem Grund wurden in dieser Arbeit zwei Zellmarkierungsmethoden entwickelt, die dies ermöglichen sollten. Die membranständigen Antikörper von Hybridomzellen sollten entweder direkt oder indirekt immunfluoreszenz-markiert und dadurch für die Durchflusszytometrie und FACS-Selektion zugänglich gemacht werden. Die direkte Markierung wurde mittels eines Hapten-Fluorophor-Konjugats durchgeführt. Sie ermöglichte erstmalig den Anteil an Haptenselektiven Hybridomzellen in einer Hybridomzelllinie zu überprüfen. Dies konnte für zwei Hapten-selektive Hybridomzelllinien, die Antikörper gegen das Hormon 17β-Estradiol und das Cardenolid Digoxigenin bilden, gezeigt werden. Durchflusszytometrie und ELISAs lieferten vergleichbare Ergebnisse. Zellen, die Hapten-selektiv markiert werden konnten, sezernierten ebenfalls Hapten-selektive Antikörper. Des Weiteren konnte die direkte Markierung dazu genutzt werden, zwei Mykotoxin-selektive Hybridomzelllinien, welche Antikörper gegen Aflatoxin und Zearalenon bilden, auf Monoklonalität zu testen. Dies ist mittels ELISA nicht möglich. Die Markierungsmethode eignete sich jedoch nur für fixierte Hybridomzellen. Eine Markierung von lebenden Zellen konnte weder durchflusszytometrisch noch mittels konfokaler Laser-Scanning-Mikroskopie gezeigt werden.
Dies gelang erst mit einer neu entwickelten indirekten Immunfluoreszenzmarkierung. Dabei wurden die Zellen zunächst mit einem Hapten-Peroxidase-Konjugat inkubiert, gefolgt von einem Fluorophor-markierten anti-HRP-Antikörper-Konjugat. Dies wurde für zwei Analyten, das Hormon Estron und das Antiepileptikum Carbamazepin, gezeigt. Die indirekte Markierung wurde erfolgreich dazu verwendet, Carbamazepin-selektive Hybridomzellen aus einem Fusionsansatz für die monoklonale Antikörperproduktion auszusortieren. Damit wurde erstmalig eine Zellmarkierungsmethode entwickelt, die eine Hochdurchsatz-Selektion lebender Hybridomzellen aus einem Fusionsansatz ermöglicht. Sie ist nicht zellschädigend und kann zusätzlich zur Selektion Hapten-selektiver Plasmazellen verwendet werden.
Hintergrund: Etablierte Protein- und Nukleinsäure-basierte Methoden für den spezifischen Pathogennachweis sind nur unter standardisierten Laborbedingungen von geschultem Personal durchführbar und daher mit einem hohen Zeit- und Kostenaufwand verbunden. In der Nukleinsäure-basierten Diagnostik kann durch die Einführung der isothermalen Amplifikation eine schnelle und kostengünstige Alternative zur Polymerase-Kettenreaktion (PCR) verwendet werden. Die Loop-mediated isothermal amplification (LAMP) bietet aufgrund der hohen Amplifikationseffizienz vielfältige Detektionsmöglichkeiten, die sowohl für Schnelltest- als auch für Monitoring-Anwendungen geeignet sind.
Ein wesentliches Ziel dieser Arbeit war die Verbesserung der Anwendbarkeit der LAMP und die Entwicklung einer neuen Methode für den einfachen, schnellen und günstigen Nachweis von Pathogenen mittels alternativer DNA- oder Pyrophosphat-abhängiger Detektionsverfahren. Hier wurden zunächst direkte und indirekte Detektionsmethoden untersucht und darauf aufbauend ein Verfahren entwickelt, mit dem neue Metallionen-abhängige Fluoreszenzfarbstoffe für die selektive Detektion von Pyrophosphat in der LAMP und anderen enzymatischen Reaktionen identifiziert werden können. Als Alternative für die DNA-basierte Detektion in der digitalen LAMP sollten die zuvor etablierten Farbstoffe für den Pyrophosphatnachweis in einer Emulsion getestet werden. Abschließend wurde ein neuer Reaktionsmechanismus für die effiziente Generierung hochmolekularer DNA unter isothermalen Bedingungen als Alternative zur LAMP entwickelt.
Ergebnisse: Für den Nachweis RNA- und DNA-basierter Phythopathogene konnte die Echtzeit- und Endpunktdetektion mit verschiedenen Farbstoffen in einem geschlossenen System etabliert werden. Hier wurde Berberin als DNA-interkalierender Fluoreszenzfarbstoff mit vergleichbarer Sensitivität zu SYBR Green und EvaGreen erfolgreich in der LAMP mit Echtzeitdetektion eingesetzt. Ein Vorteil von Berberin gegenüber den anderen Farbstoffen ist die Toleranz der DNA-Polymerase auch bei hohen Farbstoffkonzentrationen. Berberin kann daher auch in der geschlossenen LAMP-Reaktion ohne zusätzliche Anpassung der Reaktionsbedingungen für die Endpunktdetektion verwendet werden. Darüber hinaus konnte Hydroxynaphtholblau (HNB), das für den kolorimetrischen Endpunktnachweis bekannt ist, erstmals auch für die fluorimetrische Detektion der LAMP in Echtzeit eingesetzt werden. Zusätzlich konnten in der Arbeit weitere Metallionen-abhängige Farbstoffe zur indirekten Detektion der LAMP über das Pyrophosphat identifiziert werden. Dafür wurde eine iterative Methode entwickelt, mit der potenzielle Farbstoffe hinsichtlich ihrer Enzymkompatibilität und ihrer spektralen Eigenschaften bei An- oder Abwesenheit von Manganionen selektiert werden können. Mithilfe eines kombinatorischen Screenings im Mikrotiterplattenformat konnte die komplexe Konzentrationsabhängigkeit zwischen den einzelnen Komponenten für einen fluorimetrischen Verdrängungsnachweis untersucht werden. Durch die Visualisierung des Signal-Rausch-Verhältnis’ als Intensitätsmatrix (heatmap) konnten zunächst Alizarinrot S und Tetrazyklin unter simulierten Reaktionsbedingungen selektiert werden. In der anschließenden enzymatischen LAMP-Reaktion konnte insbesondere Alizarinrot S als günstiger, nicht-toxischer und robuster Fluoreszenzfarbstoff identifiziert werden und zeigte eine Pyrophosphat-abhängige Zunahme der Fluoreszenzintensität. Die zuvor etablierten Farbstoffe (HNB, Calcein und Alizarinrot S) konnten anschließend erfolgreich für die indirekte, fluorimetrische Detektion von Pyrophosphat in einer LAMP-optimierten Emulsion eingesetzt werden. Die Stabilität und Homogenität der generierten Emulsion wurde durch den Zusatz des Emulgators Poloxamer 188 verbessert. Durch die fluoreszenzmikroskopische Analyse der Emulsion war eine eindeutige Diskriminierung der positiven und negativen Tröpfchen vor allem bei Einsatz von Calcein und Alizarinrot S möglich. Aufgrund des komplexen Primer-Designs und der hohen Wahrscheinlichkeit unspezifischer Amplifikation in der LAMP wurde eine neue Bst DNA-Polymerase-abhängige isothermale Amplifikationsreaktion entwickelt. Durch die Integration einer spezifischen Linkerstruktur (abasische Stelle oder Hexaethylenglykol) zwischen zwei Primersequenzen konnte ein bifunktioneller Primer die effiziente Regenerierung der Primerbindungsstellen gewährleisten. Der neue Primer induziert nach der spezifischen Hybridisierung auf dem Templat die Rückfaltung zu einer Haarnadelstruktur und blockiert gleichzeitig die Polymeraseaktivität am Gegenstrang, wodurch eine autozyklische Amplifikation trotz konstanter Reaktionstemperatur möglich ist. Die Effizienz der „Hinge-initiated Primer dependent Amplification“ (HIP) konnte abschließend durch die Verkürzung der Distanz zwischen einem modifizierten Hinge-Primer und einem PCR-ähnlichen Primer verbessert werden.
Schlussfolgerung: Die LAMP hat sich aufgrund der hohen Robustheit und Effizienz zu einer leistungsfähigen Alternative für die klassische PCR in der molekularbiologischen Diagnostik entwickelt. Unterschiedliche Detektionsverfahren verbessern die Leistungsfähigkeit der qualitativen und quantitativen LAMP für die Feldanwendungen und für die Diagnostik, da die neuen DNA- und Pyrophosphat-abhängigen Nachweismethoden in einer geschlossenen Reaktion eingesetzt werden können und so eine einfache Pathogendiagnostik ermöglichen. Die gezeigten Methoden können darüber hinaus zu einer Kostensenkung und Zeitersparnis gegenüber den herkömmlichen Methoden beitragen. Ein attraktives Ziel stellt die Weiterentwicklung der HIP für den Pathogennachweis als Alternative zur LAMP dar. Hierbei können die neuen LAMP-Detektionsverfahren ebenfalls Anwendung finden. Die Verwendung von Bst DNA-Polymerase-abhängigen Reaktionen ermöglicht darüber hinaus die Integration einer robusten isothermalen Amplifikation in mikrofluidische Systeme. Durch die Kombination der Probenvorbereitung, Amplifikation und Detektion sind zukünftige Anwendungen mit kurzer Analysezeit und geringem apparativen Aufwand insbesondere in der Pathogendiagnostik möglich.