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We studied the short- (12 h) and long-term (144 h) response of Daphnia pulex lipases to quality shifts in diets consisting of different mixtures of the green alga Scenedesmus with the cyanobacterium Synechococcus, two species with contrasting lipid compositions. The lipase/esterase activity in both the gut and the body tissues had fast responses to the diet shift and increased with higher dietary contributions of Synechococcus. When screening the Daphnia genome for TAG lipases, we discovered a large gene-family expansion of these enzymes. We used a subset of eight genes for mRNA expression analyses and distinguished between influences of time and diet on the observed gene expression patterns. We identified five diet-responsive lipases of which three showed a sophisticated short- and long-term pattern of expression in response to small changes in food-quality. Furthermore, the gene expression of one of the lipases was strongly correlated to lipase/esterase activity in the gut suggesting its potentially major role in digestion. These findings demonstrate that the lipid-related enzymatic machinery of D. pulex is finely tuned to diet and might constitute an important mechanism of physiological adaptation in nutritionally complex environments.
Understanding how variance in environmental factors affects physiological performance, population growth, and persistence is central in ecology. Despite recent interest in the effects of variance in single biological drivers, such as temperature, we have lacked a comprehensive framework for predicting how the variances and covariances between multiple environmental factors will affect physiological rates. Here, we integrate current theory on variance effects with co-limitation theory into a single unified conceptual framework that has general applicability. We show how the framework can be applied (1) to generate mathematically tractable predictions of the physiological effects of multiple fluctuating co-limiting factors, (2) to understand how each co-limiting factor contributes to these effects, and (3) to detect mechanisms such as acclimation or physiological stress when they are at play. We show that the statistical covariance of co-limiting factors, which has not been considered before, can be a strong driver of physiological performance in various ecological contexts. Our framework can provide powerful insights on how the global change-induced shifts in multiple environmental factors affect the physiological performance of organisms.
Massive historical land cover changes in the Central European lowlands have resulted in a forest distribution that now comprises small remnants of ancient forests and more recently established post-agricultural forests. Here, land-use history is considered a key driver of recent herb-layer community changes, where an extinction debt in ancient forest remnants and/or a colonization credit in post-agricultural forests are being paid over time. On a regional scale, these payments should in theory lead toward a convergence in species richness between ancient and post-agricultural forests over time. In this study, we tested this assumption with a resurvey of 117 semi-permanent plots in the well-studied deciduous forests of the Prignitz region (Brandenburg, NE Germany), where we knew that the plant communities of post-agricultural stands exhibit a colonization credit while the extinction debt in ancient stands has largely been paid. We compared changes in the species richness of all herb layer species, forest specialists and ancient forest indicator species between ancient and post-agricultural stands with linear mixed effect models and determined the influence of patch connectivity on the magnitude of species richness changes. Species richness increased overall, but the richness of forest specialists increased significantly more in post-agricultural stands and was positively influenced by higher patch connectivity, indicating a convergence in species richness between the ancient and postagricultural stands. Furthermore, the richness of ancient forest indicator species only increased significantly in post-agricultural stands. For the first time, we were able to verify a gradual payment of the colonization credit in post-agricultural forest stands using a comparison of actual changes in temporal species richness.
Recently, Kocyan & Wiland-Szymańska (2016) have published a thorough research article on one of the outstanding members of the family Hypoxidaceae on the Seychelles, which resulted in the raise of a new genus (Friedmannia Kocyan & Wiland-Szymańska 2016: 60) to accommodate the former Curculigo seychellensis Bojer ex Baker (1877: 368). However, it has turned out that the name Friedmannia Chantanachat & Bold (1962: 45) already exists in literature for a green alga, which renders the new hypoxid genus illegitimate (Melbourne Code; McNeill et al. 2012). Therefore, we assign a new generic epithet to Curculigo seychellensis.
This study was conducted in order to examine the differences between visualizers and verbalizers in the way they gaze at pictures and texts while learning. Using a collection of questionnaires, college students were classified according to their visual or verbal cognitive style and were asked to learn about two different, in terms of subject and type of knowledge, topics by means of text-picture combinations. Eye-tracking was used to investigate their gaze behavior. The results show that visualizers spent significantly more time inspecting pictures than verbalizers, while verbalizers spent more time inspecting texts. Results also suggest that both visualizers' and verbalizers' way of learning is active but mostly within areas providing the source of information in line with their cognitive style (pictures or text). Verbalizers tended to enter non-informative, irrelevant areas of pictures sooner than visualizers. The comparison of learning outcomes showed that the group of visualizers achieved better results than the group of verbalizers on a comprehension test.
Background
The hypopharyngeal gland of worker bees contributes to the production of the royal jelly fed to queens and larvae. The gland consists of thousands of two-cell units that are composed of a secretory cell and a duct cell and that are arranged in sets of about 12 around a long collecting duct.
Results
By fluorescent staining, we have examined the morphogenesis of the hypopharyngeal gland during pupal life, from a saccule lined by a pseudostratified epithelium to the elaborate organ of adult worker bees. The hypopharyngeal gland develops as follows. (1) Cell proliferation occurs during the first day of pupal life in the hypopharyngeal gland primordium. (2) Subsequently, the epithelium becomes organized into rosette-like units of three cells. Two of these will become the secretory cell and the duct cell of the adult secretory units; the third cell contributes only temporarily to the development of the secretory units and is eliminated by apoptosis in the second half of pupal life. (3) The three-cell units of flask-shaped cells undergo complex changes in cell morphology. Thus, by mid-pupal stage, the gland is structurally similar to the adult hypopharyngeal gland. (4) Concomitantly, the prospective secretory cell attains its characteristic subcellular organization by the invagination of a small patch of apical membrane domain, its extension to a tube of about 100 μm in length (termed a canaliculus), and the expansion of the tube to a diameter of about 3 μm. (6) Finally, the canaliculus-associated F-actin system becomes reorganized into rings of bundled actin filaments that are positioned at regular distances along the membrane tube.
Conclusions
The morphogenesis of the secretory units in the hypopharyngeal gland of the worker bee seems to be based on a developmental program that is conserved, with slight modification, among insects for the production of dermal glands. Elaboration of the secretory cell as a unicellular seamless epithelial tube occurs by invagination of the apical membrane, its extension likely by targeted exocytosis and its expansion, and finally the reorganisation of the membrane-associated F-actin system. Our work is fundamental for future studies of environmental effects on hypopharyngeal gland morphology and development.
Background
The hypopharyngeal gland of worker bees contributes to the production of the royal jelly fed to queens and larvae. The gland consists of thousands of two-cell units that are composed of a secretory cell and a duct cell and that are arranged in sets of about 12 around a long collecting duct.
Results
By fluorescent staining, we have examined the morphogenesis of the hypopharyngeal gland during pupal life, from a saccule lined by a pseudostratified epithelium to the elaborate organ of adult worker bees. The hypopharyngeal gland develops as follows. (1) Cell proliferation occurs during the first day of pupal life in the hypopharyngeal gland primordium. (2) Subsequently, the epithelium becomes organized into rosette-like units of three cells. Two of these will become the secretory cell and the duct cell of the adult secretory units; the third cell contributes only temporarily to the development of the secretory units and is eliminated by apoptosis in the second half of pupal life. (3) The three-cell units of flask-shaped cells undergo complex changes in cell morphology. Thus, by mid-pupal stage, the gland is structurally similar to the adult hypopharyngeal gland. (4) Concomitantly, the prospective secretory cell attains its characteristic subcellular organization by the invagination of a small patch of apical membrane domain, its extension to a tube of about 100 μm in length (termed a canaliculus), and the expansion of the tube to a diameter of about 3 μm. (6) Finally, the canaliculus-associated F-actin system becomes reorganized into rings of bundled actin filaments that are positioned at regular distances along the membrane tube.
Conclusions
The morphogenesis of the secretory units in the hypopharyngeal gland of the worker bee seems to be based on a developmental program that is conserved, with slight modification, among insects for the production of dermal glands. Elaboration of the secretory cell as a unicellular seamless epithelial tube occurs by invagination of the apical membrane, its extension likely by targeted exocytosis and its expansion, and finally the reorganisation of the membrane-associated F-actin system. Our work is fundamental for future studies of environmental effects on hypopharyngeal gland morphology and development.
Background: The hypopharyngeal gland of worker bees contributes to the production of the royal jelly fed to queens and larvae. The gland consists of thousands of two-cell units that are composed of a secretory cell and a duct cell and that are arranged in sets of about 12 around a long collecting duct. Results: By fluorescent staining, we have examined the morphogenesis of the hypopharyngeal gland during pupal life, from a saccule lined by a pseudostratified epithelium to the elaborate organ of adult worker bees. The hypopharyngeal gland develops as follows. (1) Cell proliferation occurs during the first day of pupal life in the hypopharyngeal gland primordium. (2) Subsequently, the epithelium becomes organized into rosette-like units of three cells. Two of these will become the secretory cell and the duct cell of the adult secretory units; the third cell contributes only temporarily to the development of the secretory units and is eliminated by apoptosis in the second half of pupal life. (3) The three-cell units of flask-shaped cells undergo complex changes in cell morphology. Thus, by mid-pupal stage, the gland is structurally similar to the adult hypopharyngeal gland. (4) Concomitantly, the prospective secretory cell attains its characteristic subcellular organization by the invagination of a small patch of apical membrane domain, its extension to a tube of about 100 mu m in length (termed a canaliculus), and the expansion of the tube to a diameter of about 3 mu m. (6) Finally, the canaliculus-associated F-actin system becomes reorganized into rings of bundled actin filaments that are positioned at regular distances along the membrane tube. Conclusions: The morphogenesis of the secretory units in the hypopharyngeal gland of the worker bee seems to be based on a developmental program that is conserved, with slight modification, among insects for the production of dermal glands. Elaboration of the secretory cell as a unicellular seamless epithelial tube occurs by invagination of the apical membrane, its extension likely by targeted exocytosis and its expansion, and finally the reorganisation of the membrane-associated F-actin system. Our work is fundamental for future studies of environmental effects on hypopharyngeal gland morphology and development.
Preface
(2017)
Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner.
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.
Ancient DNA of extinct species from the Pleistocene and Holocene has provided valuable evolutionary insights. However, these are largely restricted to mammals and high latitudes because DNA preservation in warm climates is typically poor. In the tropics and subtropics, non-avian reptiles constitute a significant part of the fauna and little is known about the genetics of the many extinct reptiles from tropical islands. We have reconstructed the near-complete mitochondrial genome of an extinct giant tortoise from the Bahamas (Chelonoidis alburyorum) using an approximately 1000-year-old humerus from a water-filled sinkhole (blue hole) on Great Abaco Island. Phylogenetic and molecular clock analyses place this extinct species as closely related to Galapagos (C. niger complex) and Chaco tortoises (C. chilensis), and provide evidence for repeated overseas dispersal in this tortoise group. The ancestors of extant Chelonoidis species arrived in South America from Africa only after the opening of the Atlantic Ocean and dispersed from there to the Caribbean and the Galapagos Islands. Our results also suggest that the anoxic, thermally buffered environment of blue holes may enhance DNA preservation, and thus are opening a window for better understanding evolution and population history of extinct tropical species, which would likely still exist without human impact.
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.
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.
Plastid protein biosynthesis occurs on bacterial-type 70S ribosomes consisting of a large (50S) and a small (30S) subunit. However, since many steps of ribosome biogenesis are not thermodynamically favorable at biological conditions, it requires many assembly factors. One group of assembly factors, circularly permuted GTPases, was implicated in 30S subunit maturation in E. coli, by a protein RsgA. RsgA orthologues are present in bacteria and plastid-containing species and in silico analysis revealed presence of a RsgA-like protein in Arabidopsis thaliana. To functionally characterize the Arabidopsis orthologue, two AtRsgA T-DNA insertion lines were analyzed in this study. The exon line (rsgA-e) led to embryo lethality, while the intron line (rsgA-i) caused severe dwarf, pale green phenotype. Further investigation of rsgA-i mutant line revealed defects in chloroplast biogenesis which led to increased number of chloroplasts, decreased chloroplast size, decreased air space between mesophyll cells and smaller shoot apical meristems, which showed unusual proplastid accumulation. Moreover, rsgA-i plants showed reduction in chlorophyll A and B content, decreased electron transport rate and photosynthetic efficiency. Further analyses revealed that the protein is involved in chloroplast 30S subunit maturation. Interestingly, we observed that while chloroplast-targeted and chloroplast-encoded proteins are generally downregulated in the mutant, a contrasting upregulation of the corresponding transcripts is observed, indicating an elaborate compensatory mechanism. To conclude, the study presented here reveals a ribosome assembly factor and a compensatory mechanism activated during impaired chloroplast function.
Aquaporins occupy an essential role in sustaining the salt/water balance in various cells types and tissues. Here, we present new insights into aqp-8 expression and regulation in Caenorhabditis elegans. We show, that upon exposure to osmotic stress, aqp-8 exhibits a distinct expression pattern within the excretory cell compared to other C. elegans aquaporins expressed. This expression is correlated to the osmolarity of the surrounding medium and can be activated physiologically by osmotic stress or genetically in mutants with constitutively active osmotic stress response. In addition, we found aqp-8 expression to be constitutively active in the TRPV channel mutant osm-9(ok1677). In a genome-wide RNAi screen we identified additional regulators of aqp-8. Many of these regulators are connected to chemosensation by the amphid neurons, e.g., odr-10 and gpa-6, and act as suppressors of aqp-8 expression. We postulate from our results, that aqp-8 plays an important role in sustaining the salt/water balance during a secondary response to hyper-osmotic stress. Upon its activation aqp-8 promotes vesicle docking to the lumen of the excretory cell and thereby enhances the ability to secrete water and transport osmotic active substances or waste products caused by protein damage. In summary, aqp-8 expression and function is tightly regulated by a network consisting of the osmotic stress response, neuronal chemosensation as well as the response to protein damage. These new insights in maintaining the salt/water balance in C. elegans will help to reveal the complex homeostasis network preserved throughout species.
Single-cell quantification of transcription kinetics and variability promotes a mechanistic understanding of gene regulation. Here, using single-molecule RNA fluorescence in situ hybridization and mathematical modeling, we dissect cellular RNA dynamics for Arabidopsis FLOWERING LOCUS C (FLC). FLC expression quantitatively determines flowering time and is regulated by antisense (COOLAIR) transcription. In cells without observable COOLAIR expression, we quantify FLC transcription initiation, elongation, intron processing, and lariat degradation, as well as mRNA release from the locus and degradation. In these heterogeneously sized cells, FLC mRNA number increases linearly with cell size, resulting in a large cell-to-cell variability in transcript level. This variation is accounted for by cell-sizedependent, Poissonian FLC mRNA production, but not by large transcriptional bursts. In COOLAIRexpressing cells, however, antisense transcription increases with cell size and contributes to FLC transcription decreasing with cell size. Our analysis therefore reveals an unexpected role for antisense transcription in modulating the scaling of transcription with cell size.
The oceans absorb about a quarter of the annually produced anthropogenic atmospheric carbon dioxide (CO2), resulting in a decrease in surface water pH, a process termed ocean acidification (OA). Surprisingly little is known about how OA affects the physiology of heterotrophic bacteria or the coupling of heterotrophic bacteria to phytoplankton when nutrients are limited. Previous experiments were, for the most part, undertaken during productive phases or following nutrient additions designed to stimulate algal blooms. Therefore, we performed an in situ large-volume mesocosm (similar to 55 m(3)) experiment in the Baltic Sea by simulating different fugacities of CO2 (fCO(2)) extending from present to future conditions. The study was conducted in July-August after the nominal spring bloom, in order to maintain low-nutrient conditions throughout the experiment. This resulted in phytoplankton communities dominated by small-sized functional groups (picophytoplankton). There was no consistent fCO(2)-induced effect on bacterial protein production (BPP), cell-specific BPP (csBPP) or biovolumes (BVs) of either free-living (FL) or particle-associated (PA) heterotrophic bacteria, when considered as individual components (univariate analyses). Permutational Multivariate Analysis of Variance (PERMANOVA) revealed a significant effect of the fCO(2) treatment on entire assemblages of dissolved and particulate nutrients, metabolic parameters and the bacteria-phytoplankton community. However, distance-based linear modelling only identified fCO(2) as a factor explaining the variability observed amongst the microbial community composition, but not for explaining variability within the metabolic parameters. This suggests that fCO(2) impacts on microbial metabolic parameters occurred indirectly through varying physicochemical parameters and microbial species composition. Cluster analyses examining the co-occurrence of different functional groups of bacteria and phytoplankton further revealed a separation of the four fCO(2)-treated mesocosms from both control mesocosms, indicating that complex trophic interactions might be altered in a future acidified ocean. Possible consequences for nutrient cycling and carbon export are still largely unknown, in particular in a nutrient-limited ocean.
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.
Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.
For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness.
For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness.
For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness.
The assembly of large DNA constructs coding for entire pathways poses a major challenge in the field of synthetic biology. Here, we present AssemblX, a novel, user-friendly and highly efficient multi-gene assembly strategy. The software-assisted AssemblX process allows even unexperienced users to rapidly design, build and test DNA constructs with currently up to 25 functional units, from 75 or more subunits. At the gene level, AssemblX uses scar-free, overlap-based and sequence-independent methods, allowing the unrestricted design of transcriptional units without laborious parts domestication. The assembly into multi-gene modules is enabled via a standardized, highly efficient, polymerase chain reaction-free and virtually sequence-independent scheme, which relies on rare cutting restriction enzymes and optimized adapter sequences. Selection and marker switching strategies render the whole process reliable, rapid and very effective. The assembly product can be easily transferred to any desired expression host, making AssemblX useful for researchers from various fields.
With Saccharomyces cerevisiae being a commonly used host organism for synthetic biology and biotechnology approaches, the work presented here aims at the development of novel tools to improve and facilitate pathway engineering and heterologous protein production in yeast. Initially, the multi-part assembly strategy AssemblX was established, which allows the fast, user-friendly and highly efficient construction of up to 25 units, e.g. genes, into a single DNA construct. To speed up complex assembly projects, starting from sub-gene fragments and resulting in mini-chromosome sized constructs, AssemblX follows a level-based approach: Level 0 stands for the assembly of genes from multiple sub-gene fragments; Level 1 for the combination of up to five Level 0 units into one Level 1 module; Level 2 for linkages of up to five Level 1 modules into one Level 2 module. This way, all Level 0 and subsequently all Level 1 assemblies can be carried out simultaneously. Individually planned, overlap-based Level 0 assemblies enable scar-free and sequence-independent assemblies of transcriptional units, without limitations in fragment number, size or content. Level 1 and Level 2 assemblies, which are carried out via predefined, computationally optimized homology regions, follow a standardized, highly efficient and PCR-free scheme. AssemblX follows a virtually sequence-independent scheme with no need for time-consuming domestication of assembly parts. To minimize the risk of human error and to facilitate the planning of assembly projects, especially for individually designed Level 0 constructs, the whole AssemblX process is accompanied by a user-friendly webtool. This webtool provides the user with an easy-to-use operating surface and returns a bench-protocol including all cloning steps. The efficiency of the assembly process is further boosted through the implementation of different features, e.g. ccdB counter selection and marker switching/reconstitution. Due to the design of homology regions and vector backbones the user can flexibly choose between various overlap-based cloning methods, enabling cost-efficient assemblies which can be carried out either in E. coli or yeast. Protein production in yeast is additionally supported by a characterized library of 40 constitutive promoters, fully integrated into the AssemblX toolbox. This provides the user with a starting point for protein balancing and pathway engineering. Furthermore, the final assembly cassette can be subcloned into any vector, giving the user the flexibility to transfer the individual construct into any host organism different from yeast.
As successful production of heterologous compounds generally requires a precise adjustment of protein levels or even manipulation of the host genome to e.g. inhibit unwanted feedback regulations, the optogenetic transcriptional regulation tool PhiReX was designed. In recent years, light induction was reported to enable easy, reversible, fast, non-toxic and nearly gratuitous regulation, thereby providing manifold advantages compared to conventional chemical inducers. The optogenetic interface established in this study is based on the photoreceptor PhyB and its interacting protein PIF3. Both proteins, derived from Arabidopsis thaliana, dimerize in a red/far-red light-responsive manner. This interaction depends on a chromophore, naturally not available in yeast. By fusing split proteins to both components of the optical dimerizer, active enzymes can be reconstituted in a light-dependent manner. For the construction of the red/far-red light sensing gene expression system PhiReX, a customizable synTALE-DNA binding domain was fused to PhyB, and a VP64 activation domain to PIF3. The synTALE-based transcription factor allows programmable targeting of any desired promoter region. The first, plasmid-based PhiReX version mediates chromophore- and light-dependent expression of the reporter gene, but required further optimization regarding its robustness, basal expression and maximum output. This was achieved by genome-integration of the optical regulator pair, by cloning the reporter cassette on a high-copy plasmid and by additional molecular modifications of the fusion proteins regarding their cellular localization. In combination, this results in a robust and efficient activation of cells over an incubation time of at least 48 h. Finally, to boost the potential of PhiReX for biotechnological applications, yeast was engineered to produce the chromophore. This overcomes the need to supply the expensive and photo-labile compound exogenously. The expression output mediated through PhiReX is comparable to the strong constitutive yeast TDH3 promoter and - in the experiments described here - clearly exceeds the commonly used galactose inducible GAL1 promoter.
The fast-developing field of synthetic biology enables the construction of complete synthetic genomes. The upcoming Synthetic Yeast Sc2.0 Project is currently underway to redesign and synthesize the S. cerevisiae genome. As a prerequisite for the so-called “SCRaMbLE” system, all Sc2.0 chromosomes incorporate symmetrical target sites for Cre recombinase (loxPsym sites), enabling rearrangement of the yeast genome after induction of Cre with the toxic hormonal substance beta-estradiol. To overcome the safety concern linked to the use of beta-estradiol, a red light-inducible Cre recombinase, dubbed L-SCRaMbLE, was established in this study. L-SCRaMbLE was demonstrated to allow a time- and chromophore-dependent recombination with reliable off-states when applied to a plasmid containing four genes of the beta-carotene pathway, each flanked with loxPsym sites. When directly compared to the original induction system, L-SCRaMbLE generates a larger variety of recombination events and lower basal activity. In conclusion, L-SCRaMbLE provides a promising and powerful tool for genome rearrangement.
The three tools developed in this study provide so far unmatched possibilities to tackle complex synthetic biology projects in yeast by addressing three different stages: fast and reliable biosynthetic pathway assembly; highly specific, orthogonal gene regulation; and tightly controlled synthetic evolution of loxPsym-containing DNA constructs.
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.
Mathematical models of bacterial growth have been successfully applied to study the relationship between antibiotic drug exposure and the antibacterial effect. Since these models typically lack a representation of cellular processes and cell physiology, the mechanistic integration of drug action is not possible on the cellular level. The cellular mechanisms of drug action, however, are particularly relevant for the prediction, analysis and understanding of interactions between antibiotics. Interactions are also studied experimentally, however, a lacking consent on the experimental protocol hinders direct comparison of results. As a consequence, contradictory classifications as additive, synergistic or antagonistic are reported in literature.
In the present thesis we developed a novel mathematical model for bacterial growth that integrates cell-level processes into the population growth level. The scope of the model is to predict bacterial growth under antimicrobial perturbation by multiple antibiotics in vitro.
To this end, we combined cell-level data from literature with population growth data for Bacillus subtilis, Escherichia coli and Staphylococcus aureus. The cell-level data described growth-determining characteristics of a reference cell, including the ribosomal concentration and efficiency. The population growth data comprised extensive time-kill curves for clinically relevant antibiotics (tetracycline, chloramphenicol, vancomycin, meropenem, linezolid, including dual combinations).
The new cell-level approach allowed for the first time to simultaneously describe single and combined effects of the aforementioned antibiotics for different experimental protocols, in particular different growth phases (lag and exponential phase). Consideration of ribosomal dynamics and persisting sub-populations explained the decreased potency of linezolid on cultures in the lag phase compared to exponential phase cultures. The model captured growth rate dependent killing and auto-inhibition of meropenem and - also for vancomycin exposure - regrowth of the bacterial cultures due to adaptive resistance development. Stochastic interaction surface analysis demonstrated the pronounced antagonism between meropenem and linezolid to be robust against variation in the growth phase and pharmacodynamic endpoint definition, but sensitive to a change in the experimental duration.
Furthermore, the developed approach included a detailed representation of the bacterial cell-cycle. We used this representation to describe septation dynamics during the transition of a bacterial culture from the exponential to stationary growth phase. Resulting from a new mechanistic understanding of transition processes, we explained the lag time between the increase in cell number and bacterial biomass during the transition from the lag to exponential growth phase. Furthermore, our model reproduces the increased intracellular RNA mass fraction during long term exposure of bacteria to chloramphenicol.
In summary, we contribute a new approach to disentangle the impact of drug effects, assay readout and experimental protocol on antibiotic interactions. In the absence of a consensus on the corresponding experimental protocols, this disentanglement is key to translate information between heterogeneous experiments and also ultimately to the clinical setting.
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.
Background: Animals show consistent individual behavioural patterns over time and over situations. This phenomenon has been referred to as animal personality or behavioural syndromes. Little is known about consistency of animal personalities over entire life times. We investigated the repeatability of behaviour in common voles (Microtus arvalis) at different life stages, with different time intervals, and in different situations. Animals were tested using four behavioural tests in three experimental groups: 1. before and after maturation over three months, 2. twice as adults during one week, and 3. twice as adult animals over three months, which resembles a substantial part of their entire adult life span of several months.
Results: Different behaviours were correlated within and between tests and a cluster analysis showed three possible behavioural syndrome-axes, which we name boldness, exploration and activity. Activity and exploration behaviour in all tests was highly repeatable in adult animals tested over one week. In animals tested over maturation, exploration behaviour was consistent whereas activity was not. Voles that were tested as adults with a three-month interval showed the opposite pattern with stable activity but unstable exploration behaviour.
Conclusions: The consistency in behaviour over time suggests that common voles do express stable personality over short time. Over longer periods however, behaviour is more flexible and depending on life stage (i.e. tested before/after maturation or as adults) of the tested individual. Level of boldness or activity does not differ between tested groups and maintenance of variation in behavioural traits can therefore not be explained by expected future assets as reported in other studies.
EDTA and NTA effectively tune the mineralization of calcium phosphate from bulk aqueous solution
(2017)
This study describes the effects of nitrilotriacetic acid (NTA) and ethylenediaminotetraacetic acid (EDTA) on themineralization of calciumphosphate from bulk aqueous solution. Mineralization was performed between pH 6 and 9 and with NTA or EDTA concentrations of 0, 5, 10, and 15 mM. X-ray diffraction and infrared spectroscopy show that at low pH, mainly brushite precipitates and at higher pH, mostly hydroxyapatite forms. Both additives alter the morphology of the precipitates. Without additive, brushite precipitates as large plates. With NTA, the morphology changes to an unusual rod-like shape. With EDTA, the edges of the particles are rounded and disk-like particles form. Conductivity and pH measurements suggest that the final products form through several intermediate steps.
EDTA and NTA effectively tune the mineralization of calcium phosphate from bulk aqueous solution
(2017)
This study describes the effects of nitrilotriacetic acid (NTA) and ethylenediaminotetraacetic acid (EDTA) on themineralization of calciumphosphate from bulk aqueous solution. Mineralization was performed between pH 6 and 9 and with NTA or EDTA concentrations of 0, 5, 10, and 15 mM. X-ray diffraction and infrared spectroscopy show that at low pH, mainly brushite precipitates and at higher pH, mostly hydroxyapatite forms. Both additives alter the morphology of the precipitates. Without additive, brushite precipitates as large plates. With NTA, the morphology changes to an unusual rod-like shape. With EDTA, the edges of the particles are rounded and disk-like particles form. Conductivity and pH measurements suggest that the final products form through several intermediate steps.
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.
Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g., high-fat diets) or overweight and insulin resistance (e.g., methionine-choline-deficient diets), or they are based on monogenetic defects (e.g., ob/ob mice). In the current study, a Western-type diet containing soybean oil with high n-6-PUFA and 0.75% cholesterol (SOD + Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice, which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast, a soybean oil-containing Western-type diet without cholesterol (SOD) induced only mild steatosis but not hepatic inflammation, fibrosis, weight gain or insulin resistance. Another high-fat diet, mainly consisting of lard and supplemented with fructose in drinking water (LAD + Fru), resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD + Cho, but livers were devoid of inflammation and fibrosis. Although both LAD + Fru-and SOD + Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD + Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. In summary, dietary cholesterol in the SOD + Cho diet may trigger hepatic inflammation and fibrosis. SOD + Cho-fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH.
The existence of diverse and active microbial ecosystems in the deep subsurface – a biosphere that was originally considered devoid of life – was discovered in multiple microbiological studies. However, most of the studies are restricted to marine ecosystems, while our knowledge about the microbial communities in the deep subsurface of lake systems and their potentials to adapt to changing environmental conditions is still fragmentary. This doctoral thesis aims to build up a unique data basis for providing the first detailed high-throughput characterization of the deep biosphere of lacustrine sediments and to emphasize how important it is to differentiate between the living and the dead microbial community in deep biosphere studies.
In this thesis, up to 3.6 Ma old sediments (up to 317 m deep) of the El’gygytgyn Crater Lake were examined, which represents the oldest terrestrial climate record of the Arctic. Combining next generation sequencing with detailed geochemical characteristics and other environmental parameters, the microbial community composition was analyzed in regard to changing climatic conditions within the last 3.6 Ma to 1.0 Ma (Pliocene and Pleistocene). DNA from all investigated sediments was successfully extracted and a surprisingly diverse (6,910 OTUs) and abundant microbial community in the El’gygytgyn deep sediments were revealed. The bacterial abundance (10³-10⁶ 16S rRNA copies g⁻¹ sediment) was up to two orders of magnitudes higher than the archaeal abundance (10¹-10⁵) and fluctuates with the Pleistocene glacial/interglacial cyclicality. Interestingly, a strong increase in the microbial diversity with depth was observed (approximately 2.5 times higher diversity in Pliocene sediments compared to Pleistocene sediments). The increase in diversity with depth in the Lake El’gygytgyn is most probably caused by higher sedimentary temperatures towards the deep sediment layers as well as an enhanced temperature-induced intra-lake bioproductivity and higher input of allochthonous organic-rich material during Pliocene climatic conditions. Moreover, the microbial richness parameters follow the general trends of the paleoclimatic parameters, such as the paleo-temperature and paleo-precipitation. The most abundant bacterial representatives in the El’gygytgyn deep biosphere are affiliated with the phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria, which are also commonly distributed in the surrounding permafrost habitats. The predominated taxon was the halotolerant genus Halomonas (in average 60% of the total reads per sample).
Additionally, this doctoral thesis focuses on the live/dead differentiation of microbes in cultures and environmental samples. While established methods (e.g., fluorescence in situ hybridization, RNA analyses) are not applicable to the challenging El’gygytgyn sediments, two newer methods were adapted to distinguish between DNA from live cells and free (extracellular, dead) DNA: the propidium monoazide (PMA) treatment and the cell separation adapted for low amounts of DNA. The applicability of the DNA-intercalating dye PMA was successfully evaluated to mask free DNA of different cultures of methanogenic archaea, which play a major role in the global carbon cycle. Moreover, an optimal procedure to simultaneously treat bacteria and archaea was developed using 130 µM PMA and 5 min of photo-activation with blue LED light, which is also applicable on sandy environmental samples with a particle load of ≤ 200 mg mL⁻¹. It was demonstrated that the soil texture has a strong influence on the PMA treatment in particle-rich samples and that in particular silt and clay-rich samples (e.g., El’gygytgyn sediments) lead to an insufficient shielding of free DNA by PMA. Therefore, a cell separation protocol was used to distinguish between DNA from live cells (intracellular DNA) and extracellular DNA in the El’gygytgyn sediments. While comparing these two DNA pools with a total DNA pool extracted with a commercial kit, significant differences in the microbial composition of all three pools (mean distance of relative abundance: 24.1%, mean distance of OTUs: 84.0%) was discovered. In particular, the total DNA pool covers significantly fewer taxa than the cell-separated DNA pools and only inadequately represents the living community. Moreover, individual redundancy analyses revealed that the microbial community of the intra- and extracellular DNA pool are driven by different environmental factors. The living community is mainly influenced by life-dependent parameters (e.g., sedimentary matrix, water availability), while the extracellular DNA is dependent on the biogenic silica content. The different community-shaping parameters and the fact, that a redundancy analysis of the total DNA pool explains significantly less variance of the microbial community, indicate that the total DNA represents a mixture of signals of the live and dead microbial community.
This work provides the first fundamental data basis of the diversity and distribution of microbial deep biosphere communities of a lake system over several million years. Moreover, it demonstrates the substantial importance of extracellular DNA in old sediments. These findings may strongly influence future environmental community analyses, where applications of live/dead differentiation avoid incorrect interpretations due to a failed extraction of the living microbial community or an overestimation of the past community diversity in the course of total DNA extraction approaches.
Plant-soil feedback effects can be masked by aboveground herbivory under natural field conditions
(2017)
For plants, herbivory and interactions with their surrounding soil ecosystem are crucial factors influencing individual performance and plant-community composition. Until now, research has mostly focused on individual effects of herbivory or plant-soil feedbacks (PSFs) on plant growth and community composition, but few studies have explicitly investigated herbivory in the context of PSFs. These few studies, however, were performed under greenhouse conditions even though PSFs and herbivory may differ between greenhouse and field conditions. Therefore, we performed a field experiment in a grassland, testing the growth responses of three grass species that consistently differ in local abundance, on soils previously conditioned by these species. We tested these PSF effects for the three species both in the presence and in the absence of aboveground herbivores. Without herbivores, the two subdominant species suffered from negative PSF effects. However, in the presence of herbivores and on heterospecific soils, the same two species experienced a significant loss of shoot biomass, whereas, in contrast, enhanced root growth was observed on conspecific soils, resulting in overall neutral PSF effects. The dominant species was not damaged by herbivores and showed overall neutral PSF effects in the field with and without herbivores. Our study provides empirical evidence that negative PSF effects that exist under natural field conditions in grasslands can be overwhelmed by aboveground herbivory. Hence, potential PSF effects might not be detected in the field, because other abiotic and biotic interactions such as aboveground herbivory have stronger effects on plant performance and might therefore mask or override these PSF effects.
Dam construction on the Zeya River, which is an important tributary of the Amur River in Far East Russia, has caused significant declines in water levels and frequency of floods in the adjacent floodplains since 1980. However, an extreme flood event occurred in 2013. Populations of six crane species were monitored before and after these drastic water level changes at Muraviovka Park in Far East Russia, an important breeding and stop-over site. Individuals were counted by territory mapping during the breeding season (2000-2015) and by roosting site counts during autumn migration (2006-2015). The objective of this study was to evaluate whether changes in water levels had a significant impact on local and migratory crane populations. We found a positive effect of flooding on numbers of breeding Red-crowned Cranes (Grus japonensis) and White-naped Cranes (Antigone vipio), as well as on numbers of roosting Hooded Cranes (Grus monacha) in autumn. Siberian Cranes (Leucogeranus leucogeranus) were only observed after the wetlands were flooded. The results of this study highlight the importance of elevated Amur River water levels for crane populations of global importance.
The relevance of vegetation structures and small water bodies for bats foraging above farmland
(2017)
Bats are known to forage and commute close to vegetation structures when moving across the agricultural matrix, but the role of isolated landscape elements in arable fields for bat activity is unknown. Therefore, we investigated the influence of small isolated ponds which lie within arable fields close to vegetation structures on the flight and foraging activity of bats. Additionally, we compared species-specific activity measures between forest edges and linear structures such as hedgerows. We repeatedly recorded bat activity using passive acoustic monitoring along 20 transects extending from the vegetation edge up to 200 m into the arable field (hereafter: edge-field interface) with a small pond present at five transects per edge type (linear vs. forest). Using generalized linear mixed effect models, we analyzed the effects of edge type, pond presence and the season on species-specific flight and foraging activity within the edge-field interface. We found a higher flight activity of Nyctalus noctula and Pipistrellus pygmaeus above the arable field when a pond was present. Furthermore, Pipistrellus nathusii and Pipistrellus pipistrellus foraged more frequently at forest edges than at linear structures (e.g. hedgerows). Additionally, we found three major patterns of seasonal variation in the activity of bats along the edge-field interface. This study highlights the species-specific and dynamic use of forest and hedgerow or tree line edges by bats and their importance for different bat species in the agricultural landscape. Further, additional landscape elements such as small isolated ponds within arable fields might support the activity of bats above the open agricultural landscape, thereby facilitating agroecosystem functioning. Therefore, additional landscape elements within managed areas should be restored and protected against the conversion into arable land and better linked to surrounding landscape elements in order to efficiently support bats within the agroecosystem.
Biodiversity and intact ecological interactions form the basis for functional and resilient ecosystems that maintain optimal conditions for life on earth. During the second half of the 20th century, especially land-use changes and an intensification of agricultural management caused an unprecedented loss of biodiversity in agroecosystems worldwide. Concerns have been raised that the ongoing loss of biodiversity would ultimately lead to impaired ecological interactions and ecosystem functioning in agricultural landscapes. In order to stop biodiversity loss while producing enough food for a growing world population, we need to gain detailed knowledge on ecological interactions and the functioning of agroecosystems as a whole.
Bats (Chiroptera) represent an important component of global biodiversity, occupy a variety of ecological niches and fulfill numerous ecosystem services. Especially in temperate zone agroecosystems, bats were repeatedly reported to contribute to the reduction of pest insects above intensively managed arable fields. However, bat populations have been decimated by the consequence of land-use intensification which led to their legal protection status in the European Union (Council of Europe, 1979). The increasing number of wind turbines on arable fields poses an additional threat to bats as they might get injured or killed when flying too close to wind turbine blades. Although a large amount of land area is covered by arable fields, not much is known about how bats use the intensively managed agricultural landscape.
In the present thesis, my general aim was to identify the relevance of factors at different spatiotemporal scales for shaping species-specific bat activity above intensively managed arable fields. Therefore, I repeatedly monitored bat activity above open arable fields in a landscape dominated by agriculture which is located in Northeast Brandenburg, Germany. From 2012 to 2014, I recorded echolocation calls of bats on a total of 113 sites using a passive acoustic approach. I obtained a total of 27,779 recordings, identified the recorded echolocation calls manually to species level and calculated species-specific bat activity measures. Depending on the focus of research, I modeled the obtained species-specific activity measures using generalized linear and additive mixed effect models. In Chapter I, I focused on identifying seasonal patterns in several species-specific activity measures of different functional bat groups. In Chapter II, I investigated small-scale effects of landscape elements, such as hedgerows and forest edges, on the flight and foraging activity of different bat species along the edge-field interface. Additionally, I aimed at identifying whether these effects are influenced by small ponds located within arable field and whether these effects change across seasons. In Chapter III, my aim was to investigate the interaction between factors from different spatiotemporal levels on the flight and foraging activity of bats above arable fields. At the small spatial scale, I focused on prey availability, at a large spatial scale on selected parameters which describe landscape characteristics and at the temporal scale on seasonal effects.
The major findings obtained in each chapter can be summarized in the following three points. The first major finding is that not only landscape elements on a small spatial scale, e.g. a hedgerow at the edge of an arable field, but also landscape characteristics on a large spatial scale, e.g. landscape composition, shaped species-specific bat activity above open arable fields. This activity was also strongly influenced by interactions between landscape characteristics and local prey availability. Second, the influence of landscape elements and characteristics on bat activity above arable fields was not constant over time but changed across seasons with the strongest impact during summer as compared to spring and autumn. Third, I found indications of ecosystem service provided by N. noctula and P. nathusii in all three chapters, as especially these bat species were repeatedly found to forage above arable fields. This foraging activity was positively influenced by the proximity to landscape elements at the edge of the arable field but also by the presence of small ponds within the arable field.
In light of the obtained findings, I strongly recommend protecting and most importantly recreating semi-natural landscape elements in the agricultural landscape. Furthermore, I strongly recommend against the construction of wind turbines close to these linear woody vegetation edges as bats were found to be active close to these landscape elements. Additionally, the operation times for wind turbines should be down-regulated during the mating and migration period in autumn due to high bat activity above arable fields. Since bats are considered being good bioindicators, effective conservation measures for bats might contribute to the protection of species from other taxa leading to an overall support of biodiversity in agricultural landscapes. In their entirety, the findings in this thesis contribute to the knowledge of different aspects of bat ecology and shed light on the complex interplay between factors from different spatiotemporal levels that shape bat activity above arable fields. Additionally, they can serve as a basis for the improvement and development of conservation measures for bats in agricultural landscapes.
Diabetic nephropathy is one of the most frequent, devastating and costly complications of diabetes. The available therapeutic approaches are limited. Dipeptidyl peptidase type 4 (DPP-4) inhibitors represent a new class of glucose-lowering drugs that might also have reno-protective properties. DPP-4 exists in two forms: a plasma membranebound form and a soluble form, and can exert many biological actions mainly through its peptidase activity and interaction with extracellular matrix components. The kidneys have the highest DPP-4 expression level in mammalians. DPP-4 expression and urinary activity are up-regulated in diabetic nephropathy, highlighting its role as a potential target to manage diabetic nephropathy. Preclinical animal studies and some clinical data suggest that DPP-4 inhibitors decrease the progression of diabetic nephropathy in a blood pressure-and glucose-independent manner. Many studies reported that these reno-protective effects could be due to increased half-life of DPP-4 substrates such as glucagon-like peptide-1 (GLP-1) and stromal derived factor-1 alpha (SDF-1a). However, the underlying mechanisms are far from being completely understood and clearly need further investigations.
Recent advances in gene function prediction rely on ensemble approaches that integrate results from multiple inference methods to produce superior predictions. Yet, these developments remain largely unexplored in plants. We have explored and compared two methods to integrate 10 gene co-function networks for Arabidopsis thaliana and demonstrate how the integration of these networks produces more accurate gene function predictions for a larger fraction of genes with unknown function. These predictions were used to identify genes involved in mitochondrial complex I formation, and for five of them, we confirmed the predictions experimentally. The ensemble predictions are provided as a user-friendly online database, EnsembleNet. The methods presented here demonstrate that ensemble gene function prediction is a powerful method to boost prediction performance, whereas the EnsembleNet database provides a cutting-edge community tool to guide experimentalists.
We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D-1/2 = 1.7 +/- 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods.
The determination of the microscopic dose-damage relationship for DNA in an aqueous environment is of a fundamental interest for dosimetry and applications in radiation therapy and protection. We combine GEANT4 particle-scattering simulations in water with calculations concerning the movement of biomolecules to obtain the energy deposit in the biologically relevant nanoscopic volume. We juxtaposition these results to the experimentally determined damage to obtain the dose-damage relationship at a molecular level. This approach is tested for an experimentally challenging system concerning the direct irradiation of plasmid DNA (pUC19) in water with electrons as primary particles. Here a microscopic target model for the plasmid DNA based on the relation of lineal energy and radiation quality is used to calculate the effective target volume. It was found that on average fewer than two ionizations within a 7.5-nm radius around the sugar-phosphate backbone are sufficient to cause a single strand break, with a corresponding median lethal energy deposit being E-1/2 = 6 +/- 4 eV. The presented method is applicable for ionizing radiation (e.g.,.gamma rays, x rays, and electrons) and a variety of targets, such as DNA, proteins, or cells.
Poly(ether imide) (PEI), which can be chemically functionalized with biologically active ligands, has emerged as a potential biomaterial for medical implants. Electrospun PEI scaffolds have shown advantageous properties, such as enhanced endothelial cell adherence, proliferation and low platelet adhesion in in vitro experiments. In this study, the in vivo behaviour of electrospun PEI scaffolds and PEI films was examined in a murine subcutaneous implantation model. Electrospun PEI scaffolds and films were surgically implanted subcutaneously in the dorsae of mice. The surrounding subcutaneous tissue response was examined via histopathological examination at 7 and 28days after implantation. No serious adverse events were observed for both types of PEI implants. The presence of macrophages or foreign body giant cells in the vicinity of the implants and the formation of a fibrous capsule indicated a normal foreign body reaction towards PEI films and scaffolds. Capsule thickness and inflammatory infiltration cells significantly decreased for PEI scaffolds during days 7-28 while remaining unchanged for PEI films. The infiltration of cells into the implant was observed for PEI scaffolds 7days after implantation and remained stable until 28days of implantation. Additionally some, but not all, PEI scaffold implants induced the formation of functional blood vessels in the vicinity of the implants. Conclusively, this study demonstrates the in vivo biocompatibility of PEI implants, with favourable properties of electrospun PEI scaffolds regarding tissue integration and wound healing.
The effects of biodiversity on ecosystem functioning generally increase over time, but the underlying processes remain unclear. Using 26 long-term grassland and forest experimental ecosystems, we demonstrate that biodiversity-ecosystem functioning relationships strengthen mainly by greater increases in functioning in high-diversity communities in grasslands and forests. In grasslands, biodiversity effects also strengthen due to decreases in functioning in low-diversity communities. Contrasting trends across grasslands are associated with differences in soil characteristics.
Modeling a secular trend by Monte Carlo simulation of height biased migration in a spatial network
(2017)
Background: In a recent Monte Carlo simulation, the clustering of body height of Swiss military conscripts within a spatial network with characteristic features of the natural Swiss geography was investigated. In this study I examined the effect of migration of tall individuals into network hubs on the dynamics of body height within the whole spatial network. The aim of this study was to simulate height trends. Material and methods: Three networks were used for modeling, a regular rectangular fishing net like network, a real world example based on the geographic map of Switzerland, and a random network. All networks contained between 144 and 148 districts and between 265-307 road connections. Around 100,000 agents were initially released with average height of 170 cm, and height standard deviation of 6.5 cm. The simulation was started with the a priori assumption that height variation within a district is limited and also depends on height of neighboring districts (community effect on height). In addition to a neighborhood influence factor, which simulates a community effect, body height dependent migration of conscripts between adjacent districts in each Monte Carlo simulation was used to re-calculate next generation body heights. In order to determine the direction of migration for taller individuals, various centrality measures for the evaluation of district importance within the spatial network were applied. Taller individuals were favored to migrate more into network hubs, backward migration using the same number of individuals was random, not biased towards body height. Network hubs were defined by the importance of a district within the spatial network. The importance of a district was evaluated by various centrality measures. In the null model there were no road connections, height information could not be delivered between the districts. Results: Due to the favored migration of tall individuals into network hubs, average body height of the hubs, and later, of the whole network increased by up to 0.1 cm per iteration depending on the network model. The general increase in height within the network depended on connectedness and on the amount of height information that was exchanged between neighboring districts. If higher amounts of neighborhood height information were exchanged, the general increase in height within the network was large (strong secular trend). The trend in the homogeneous fishnet like network was lowest, the trend in the random network was highest. Yet, some network properties, such as the heteroscedasticity and autocorrelations of the migration simulation models differed greatly from the natural features observed in Swiss military conscript networks. Autocorrelations of district heights for instance, were much higher in the migration models. Conclusion: This study confirmed that secular height trends can be modeled by preferred migration of tall individuals into network hubs. However, basic network properties of the migration simulation models differed greatly from the natural features observed in Swiss military conscripts. Similar network-based data from other countries should be explored to better investigate height trends with Monte Carlo migration approach.
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.
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.
The transition from hunting and gathering to farming involved profound cultural and technological changes. In Western and Central Europe, these changes occurred rapidly and synchronously after the arrival of early farmers of Anatolian origin [1-3], who largely replaced the local Mesolithic hunter-gatherers [1, 4-6]. Further east, in the Baltic region, the transition was gradual, with little or no genetic input from incoming farmers [7]. Here we use ancient DNA to investigate the relationship between hunter-gatherers and farmers in the Lower Danube basin, a geographically intermediate area that is characterized by a rapid Neolithic transition but also by the presence of archaeological evidence that points to cultural exchange, and thus possible admixture, between hunter-gatherers and farmers. We recovered four human paleogenomes (1.13 to 4.13 coverage) from Romania spanning a time transect between 8.8 thousand years ago (kya) and 5.4 kya and supplemented them with two Mesolithic genomes (1.73- and 5.33) from Spain to provide further context on the genetic background of Mesolithic Europe. Our results show major Western hunter-gatherer (WHG) ancestry in a Romanian Eneolithic sample with a minor, but sizeable, contribution from Anatolian farmers, suggesting multiple admixture events between hunter-gatherers and farmers. Dietary stableisotope analysis of this sample suggests a mixed terrestrial/ aquatic diet. Our results provide support for complex interactions among hunter-gatherers and farmers in the Danube basin, demonstrating that in some regions, demic and cultural diffusion were not mutually exclusive, but merely the ends of a continuum for the process of Neolithization.
The aim of the study was to investigate the correlation of heights of conscripts living in neighboring districts in Poland. The study used 10% of a nationally representative sample of 26,178 males 18.5-19.5 years old examined during the National survey of Polish conscripts conducted in 2001. The sample represented all regions and social strata of the country and included 354 different districts within 16 voivodships (provinces). Analyses were performed with the R statistical software. A small but significant correlation (0.24, p < 0.0001) was observed for height between 1st order neighboring districts. Correlations decreased with increased distances between neighboring districts, but remained significant for 7th node neighbors (0.18, p < 0.0001). Regarding voivodships (provinces), average height showed a geographical trend from the northwest (relatively tall) to the southeast (relatively short), and the correlation was stronger for first order neighboring provinces (0.796, p < 0.001). This study revealed clusters of tall people and short people, providing a support for hypothesis of the community effect in height. Small correlations between 1st order neighbors than in another country (Switzerland) may be associated with differences in geography, since in Poland there are no natural barriers (e.g., mountains) and road infrastructure is well-developed.
The oomycete Plasmopara obducens was first described on wild Impatiens noli-tangere in Germany in 1877. About 125 years later the first occurrence of P. obducens on cultivated I. walleriana in the United Kingdom was reported, and a worldwide epidemic followed. Although this pathogen is a major threat for ornamental busy lizzy, the identity of the pathogen remained unconfirmed and the high host specificity observed for the genus Plasmopara cast doubts regarding its determination as P. obducens. In this study, using multigene phylogenies and morphological investigation, it is revealed that P. obducens on I. noli-tangere is not the conspecific with the pathogen affecting I. walleriana and another ornamental balsam, I. balsamina. As a consequence, the new names P. destructor and P. velutina are introduced for the pathogens of I. walleriana and I. balsamina, respectively.
The Western honey bee (Apis mellifera) is widely used as commercial pollinator in worldwide agriculture and, therefore, plays an important role in global food security. Among the parasites and pathogens threatening health and survival of honey bees are two species of microsporidia, Nosema apis and Nosema ceranae. Nosema ceranae is considered an emerging pathogen of the Western honey bee. Reports on the spread of N. ceranae suggested that this presumably highly virulent species is replacing its more benign congener N. apis in the global A. mellifera population. We here present a 12 year longitudinal cohort study on the prevalence of N. apis and N. ceranae in Northeast Germany. Between 2005 and 2016, a cohort of about 230 honey bee colonies originating from 23 apiaries was sampled twice a year (spring and autumn) resulting in a total of 5,600 bee samples which were subjected to microscopic and molecular analysis for determining the presence of infections with N. apis or/and N. ceranae. Throughout the entire study period, both N. apis- and N. ceranae-infections could be diagnosed within the cohort. Logistic regression analysis of the prevalence data demonstrated a significant increase of N. ceranae-infections over the last 12 years, both in autumn (reflecting the development during the summer) and in spring (reflecting the development over winter) samples. Cell culture experiments confirmed that N. ceranae has a higher proliferative potential than N. apis at 27. and 33 degrees C potentially explaining the increase in N. ceranae prevalence during summer. In autumn, characterized by generally low infection prevalence, this increase was accompanied by a significant decrease in N. apis- infection prevalence. In contrast, in spring, the season with a higher prevalence of infection, no significant decrease of N. apis infections despite a significant increase in N. ceranae infections could be observed. Therefore, our data do not support a general advantage of N. ceranae over N. apis and an overall replacement of N. apis by N. ceranae in the studied honey bee population.
Many lakes exhibit seasonal stratification, during which they develop strong thermal and chemical gradients. An expansion of depth-integrated monitoring programs has provided insight into the importance of organic carbon processing that occurs below the upper mixed layer. However, the chemical and physical drivers of metabolism and metabolic coupling remain unresolved, especially in the metalimnion. In this depth zone, sharp gradients in key resources such as light and temperature co-occur with dynamic physical conditions that influence metabolic processes directly and simultaneously hamper the accurate tracing of biological activity. We evaluated the drivers of metalimnetic metabolism and its associated uncertainty across 10 stratified lakes in Europe and North America. We hypothesized that the metalimnion would contribute highly to whole-lake functioning in clear oligotrophic lakes, and that metabolic rates would be highly variable in unstable polymictic lakes. Depth-integrated rates of gross primary production (GPP) and ecosystem respiration (ER) were modelled from diel dissolved oxygen curves using a Bayesian approach. Metabolic estimates were more uncertain below the epilimnion, but uncertainty was not consistently related to lake morphology or mixing regime. Metalimnetic rates exhibited high day-to-day variability in all trophic states, with the metalimnetic contribution to daily whole-lake GPP and ER ranging from 0% to 87% and < 1% to 92%, respectively. Nonetheless, the metalimnion of low-nutrient lakes contributed strongly to whole-lake metabolism on average, driven by a collinear combination of highlight, low surface-water phosphorous concentration and high metalimnetic volume. Consequently, a single-sensor approach does not necessarily reflect whole-ecosystem carbon dynamics in stratified lakes.
Extreme weather events can pervasively influence ecosystems. Observations in lakes indicate that severe storms in particular can have pronounced ecosystem-scale consequences, but the underlying mechanisms have not been rigorously assessed in experiments. One major effect of storms on lakes is the redistribution of mineral resources and plankton communities as a result of abrupt thermocline deepening. We aimed at elucidating the importance of this effect by mimicking in replicated large enclosures (each 9 m in diameter, ca. 20 m deep, ca. 1300 m 3 in volume) a mixing event caused by a severe natural storm that was previously observed in a deep clear-water lake. Metabolic rates were derived from diel changes in vertical profiles of dissolved oxygen concentrations using a Bayesian modelling approach, based on high-frequency measurements. Experimental thermocline deepening stimulated daily gross primary production (GPP) in surface waters by an average of 63% for > 4 weeks even though thermal stratification re-established within 5 days. Ecosystem respiration (ER) was tightly coupled to GPP, exceeding that in control enclosures by 53% over the same period. As GPP responded more strongly than ER, net ecosystem productivity (NEP) of the entire water column was also increased. These protracted increases in ecosystem metabolism and autotrophy were driven by a proliferation of inedible filamentous cyanobacteria released from light and nutrient limitation after they were entrained from below the thermocline into the surface water. Thus, thermocline deepening by a single severe storm can induce prolonged responses of lake ecosystem metabolism independent of other storm-induced effects, such as inputs of terrestrial materials by increased catchment run-off. This highlights that future shifts in frequency, severity or timing of storms are an important component of climate change, whose impacts on lake thermal structure will superimpose upon climate trends to influence algal dynamics and organic matter cycling in clear-water lakes. Keywords: climate variability, ecosystem productivity, extreme events, gross primary production, mesocosm, respiration stratified lakes
The appearance of the first leaf from the coleoptile in wheat seedlings (Triticum aestivum L.) coincides with the development of seedling susceptibility to water deficiency on the fifth day following imbibition. In dehydrated wheat seedlings, an increase in the protein carbonyl group has been observed. The coincidence of higher protein carbonylation levels with development of dehydration intolerance drew our attention. To gain more insight into the molecular basis of wheat drought tolerance, the seedling profiles of carbonylated proteins were analysed and compared. Two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry (MALDI-TOF and LC-MS/MS) were used to indicate and identify differential carbonylated proteins. Among the protein spots with at least a two-fold change in protein abundance in dehydrated seedlings in relation to control (well-watered) plants during the tolerant phase of growth, 19 carbonylated proteins increased and 18 carbonylated proteins decreased in abundance. Among 26 differentially expressed carbonylated proteins in sensitive seedlings, the abundance of 10 protein spots increased while that of 16 proteins decreased upon dehydration. We have demonstrated a link between protein carbonylation and seedling sensitivity to dehydration. The analysis of carbonylated protein profiles clearly showed that proteins with a potential role in the maintenance of dehydration tolerance in wheat seedlings are mainly linked to energy production, anti-fungal and/or insecticidal activity, or to the regulation of both protein synthesis and degradation.
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.
Cellular membranes constantly experience remodeling, as exemplified by morphological changes during endo- and exocytosis. Regulation of membrane morphology is essential for these processes. In this work, we attempt to establish a regulation path based on the use of photoswitches exhibiting conformational changes in model membranes, namely, giant unilamellar vesicles (GUVs). The mechanism of the changes in the GUVs’ morphology caused by isomerization of the photosensitive molecules has been previously explored but still remains elusive. We examine the morphological reshaping of GUVs in the presence of the photoswitch o-tetrafluoroazobenzene (F-azo) and show that the mechanism behind the resulting morphological changes involves both an increase in the membrane area and generation of a positive spontaneous curvature. First, we characterize the partitioning of F-azo in a single-component membrane using both experimental and computational approaches. The partition coefficient calculated from molecular dynamic simulations agrees with experimental data obtained with size-exclusion chromatography. Then, we implement the approach of vesicle electrodeformation in order to assess the increase in the membrane area, which is observed as a result of the conformational change of F-azo. Finally, the local and the effective membrane spontaneous curvatures were estimated from the observed shapes of vesicles exhibiting outward budding. We then extend the application of the F-azo to multicomponent lipid membranes, which exhibit a coexistence of domains in different liquid phases due to a miscibility gap between the lipids. We perform initial experiments to investigate whether F-azo can be employed to modulate the lateral lipid packing and organization. We observe either complete mixing of the domains or the appearing of disordered domains within the domains of more ordered phase. The type of behavior observed in response to the photoisomerization of F-azo was dependent on the used lipid composition. We believe that the findings introduced here will have an impact in understanding and controlling both lipid phase modulation and regulation of the membrane morphology in membrane systems.
Inactivation of thermogenic UCP1 as a historical contingency in multiple placental mammal clades
(2017)
Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism. Glulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs. Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.
The phylogenetic structure of communities (PSC) reveals how evolutionary history affects community assembly processes. However, there are important knowledge gaps on PSC patterns for annual communities and there is a need for studies along environmental gradients in dry ecosystems where several processes shape PSC. Here, we investigated the PSC of annual plants along an aridity gradient in Israel, including eight years, two spatial scales, the effects of shrubs on understory, and the phylogenetic signal of important traits. Increasing drought stress led to overdispersed PSC at the drier end of the gradient, indicating that species were less related than expected by chance. This was supported at a smaller spatial scale, where within the drier sites, communities in open- more arid- habitats were more overdispersed than those under nurse shrubs. Interestingly, some key traits related to drought resistance were not conserved in the phylogeny. Together, our findings suggested that while habitat filtering selected for drought resistance strategies, these strategies evolved independently along multiple contrasting evolutionary lineages. Our comprehensive PSC study provides strong evidence for the interacting effects of habitat filtering and plant- plant interactions, particularly highlighting that the conservative evolution of traits should not be assumed in future interpretations of PSC patterns.
Planktotrons
(2017)
We established a new indoor mesocosm facility, 12 fully controlled Planktotrons, designed to conduct marine and freshwater experiments for biodiversity and food web approaches using natural or artificial, benthic or planktonic communities. The Planktotrons are a unique and custom-tailored facility allowing long-term experiments. Wall growth can be inhibited by a rotating gate paddle with silicone lips. Additionally, temperature and light intensity are individually controllable for each Planktotron and the large volume (600 L) enables high-frequency or volume-intense measurements. In a pilot freshwater experiment various trophic levels of a pelagic food web were maintained for up to 90 d. First, an artificially assembled phytoplankton community of 11 species was inoculated in all Planktotrons. After 22 d, two ciliates were added to all, and three Daphnia species were added to six Planktotrons. After 72 d, dissolved organic matter (DOM, an alkaline soil extract) was added as an external disturbance to six of the 12 Planktotrons, involving three Planktotrons stocked with Daphnia and three without, respectively. We demonstrate the suitability of the Planktotrons for food web and biodiversity research. Variation among replicated Planktotrons (n=3 minimum) did not differ from other laboratory systems and field experiments. We investigated population dynamics and interactions among the different trophic levels, and found them affected by the sequence of ciliate and Daphnia addition and the disturbance caused by addition of DOM.
Immunodeficient mice are crucial models to evaluate the efficacy of monoclonal antibodies (mAbs). When studying mAb pharmacokinetics (PK), protection from elimination by binding to the neonatal Fc receptor (FcRn) is known to be a major process influencing the unspecific clearance of endogenous and therapeutic IgG. The concentration of endogenous IgG in immunodeficient mice, however is reduced, and this effect on the FcRn protection mechanism and subsequently on unspecific mAb clearance is unknown, yet of great importance for the interpretation of mAb PK data. We used a PBPK modelling approach to elucidate the influence of altered endogenous IgG concentrations on unspecific mAb clearance. To this end, we used PK data in immunodeficient mice, i.e. nude and severe combined immunodeficiency mice. To avoid impact of target-mediated clearance processes, we focussed on mAbs without affinity to a target antigen in these mice. In addition, intravenous immunoglobulin (IVIG) data of immunocompetent mice was used to study the impact of increased total IgG concentrations on unspecific therapeutic antibody clearance. The unspecific clearance is linear, whenever therapeutic IgG concentrations, i.e. mAb and IVIG concentrations are lower than FcRn; it can be non-linear if therapeutic IgG concentrations are larger than FcRn and endogenous IgG concentrations (e.g., under IVIG therapy). Unspecific mAb clearance of immunodeficient mice is effectively linear (under mAb doses as typically used in human). Studying the impact of reduced endogenous IgG concentrations on unspecific mAb clearance is of great relevance for the extrapolation to clinical species, e.g., when predicting mAb PK in immunosuppressed cancer patients.
Assembly of iron sulfur (FeS) clusters is an important process in living cells. The initial sulfur mobilization step for FeS cluster biosynthesis is catalyzed by L-cysteine desulfurase NFS1, a reaction that is localized in mitochondria in humans. In humans, the function of NFS1 depends on the ISD11 protein, which is required to stabilize its structure. The NFS1/ISD11 complex further interacts with scaffold protein ISCU and regulator protein frataxin, thereby forming a quaternary complex for FeS cluster formation. It has been suggested that the role of ISD11 is not restricted to its role in stabilizing the structure of NFS1, because studies of single-amino acid variants of ISD11 additionally demonstrated its importance for the correct assembly of the quaternary complex. In this study, we are focusing on the N-terminal region of ISD11 to determine the role of N-terminal amino acids in the formation of the complex with NFS1 and to reveal the mitochondria) targeting sequence for subcellular localization. Our in vitro studies with the purified proteins and in vivo studies in a cellular system show that the first 10 N-terminal amino acids of ISD11 are indispensable for the activity of NFS1 and especially the conserved "LYR" motif is essential for the role of ISD11 in forming a stable and active complex with NFS1.
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.
Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. The physiological function of mammalian AOX isoenzymes is still unclear, however, human AOX (hAOX1) is an emerging enzyme in phase-I drug metabolism. Indeed, the number of xenobiotics acting as hAOX1 substrates is increasing. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified within the hAOX1 gene. SNPs are a major source of inter-individual variability in the human population, and SNP-based amino acid exchanges in hAOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. In this report we selected ten novel SNPs resulting in amino acid exchanges in proximity to the FAD site of hAOX1 and characterized the purified enzymes after heterologous expression in Escherichia coli. The hAOX1 variants were characterized carefully by quantitative differences in their ability to produce superoxide radical. ROS represent prominent key molecules in physiological and pathological conditions in the cell. Our data reveal significant alterations in superoxide anion production among the variants. In particular the SNP-based amino acid exchange L438V in proximity to the isoalloxanzine ring of the FAD cofactor resulted in increased rate of superoxide radical production of 75%. Considering the high toxicity of the superoxide in the cell, the hAOX1-L438V SNP variant is an eventual candidate for critical or pathological roles of this natural variant within the human population.
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.
We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate.
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.
Aerobic anoxygenic phototrophs (AAPs) have been shown to exist in numerous marine and brackish environments where they are hypothesized to play important ecological roles. Despite their potential significance, the study of freshwater AAPs is in its infancy and limited to local investigations. Here, we explore the occurrence, diversity and distribution of AAPs in lakes covering a wide latitudinal gradient: Mongolian and German lakes located in temperate regions of Eurasia, tropical Great East African lakes, and polar permanently ice-covered Antarctic lakes. Our results show a widespread distribution of AAPs in lakes with contrasting environmental conditions and confirm that this group is composed of different members of the Alpha- and Betaproteobacteria. While latitude does not seem to strongly influence AAP abundance, clear patterns of community structure and composition along geographic regions were observed as indicated by a strong macro-geographical signal in the taxonomical composition of AAPs. Overall, our results suggest that the distribution patterns of freshwater AAPs are likely driven by a combination of small-scale environmental conditions (specific of each lake and region) and large-scale geographic factors (climatic regions across a latitudinal gradient).
Engineering artificial networks from modular components is a major challenge in synthetic biology. In the past years, single units, such as switches and oscillators, were successfully constructed and implemented. The effective integration of these parts into functional artificial self-regulated networks is currently on the verge of breakthrough. Here, we describe the design of a modular higher-order synthetic genetic network assembled from two independent self-sustained synthetic units: repressilators coupled via a modified quorum-sensing circuit. The isolated communication circuit and the network of coupled oscillators were analysed in mathematical modelling and experimental approaches. We monitored clustering of cells in groups of various sizes. Within each cluster of cells, cells oscillate synchronously, whereas the theoretical modelling predicts complete synchronization of the whole cellular population to be obtained approximately after 30 days. Our data suggest that self-regulated synchronization in biological systems can occur through an intermediate, long term clustering phase. The proposed artificial multicellular network provides a system framework for exploring how a given network generates a specific behaviour.
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.
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.
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.
The profits of excludability and transferability in redistributive land reform in central Namibia
(2017)
Policies which redistribute property rights to land can improve the well-being of rural households and can have overall growth effects. In many cases, however, land reforms are driven mainly by politically justified objectives. Under such circumstances, little emphasis is placed on whether and, if so, how property rights can increase productivity. Following 18 years of land reform implementation in Namibia, we evaluated 65 beneficiaries in Namibia. We assess to which degree land rights affects their farm income. The study focuses on Namibia’s two main commercial land reform instruments, namely the Farm Unit Resettlement Scheme and the Affirmative Action Loan Scheme. We find evidence that the majority of land reform projects are not profitable. Further, our study confirms the importance of the right to restrict land access compared with the right to transfer. The long-term leasehold contract seemingly provides sufficient incentives to make productive use of the land.
The Influence of Land Use Intensity on the Plant-Associated Microbiome of Dactylis glomerata L.
(2017)
In this study, we investigated the impact of different land use intensities (LUI) on the root-associated microbiome of Dactylis glomerata (orchardgrass). For this purpose, eight sampling sites with different land use intensity levels but comparable soil properties were selected in the southwest of Germany. Experimental plots covered land use levels from natural grassland up to intensively managed meadows. We used 16S rRNA gene based barcoding to assess the plant-associated community structure in the endosphere, rhizosphere and bulk soil of D. glomerata. Samples were taken at the reproductive stage of the plant in early summer. Our data indicated that roots harbor a distinct bacterial community, which clearly differed from the microbiome of the rhizosphere and bulk soil. Our results revealed Pseudomonadaceae, Enterobacteriaceae and Comamonadaceae as the most abundant endophytes independently of land use intensity. Rhizosphere and bulk soil were dominated also by Proteobacteria, but the most abundant families differed from those obtained from root samples. In the soil, the effect of land use intensity was more pronounced compared to root endophytes leading to a clearly distinct pattern of bacterial communities under different LUI from rhizosphere and bulk soil vs. endophytes. Overall, a change of community structure on the plant-soil interface was observed, as the number of shared OTUs between all three compartments investigated increased with decreasing land use intensity. Thus, our findings suggest a stronger interaction of the plant with its surrounding soil under low land use intensity. Furthermore, the amount and quality of available nitrogen was identified as a major driver for shifts in the microbiome structure in all compartments.
Sequelae of prematurity triggered by oxidative stress and free radical-mediated tissue damage have coined the term “oxygen radical disease of prematurity”. Caffeine, a potent free radical scavenger and adenosine receptor antagonist, reduces rates of brain damage in preterm infants. In the present study, we investigated the effects of caffeine on oxidative stress markers, anti-oxidative response, inflammation, redox-sensitive transcription factors, apoptosis, and extracellular matrix following the induction of hyperoxia in neonatal rats. The brain of a rat pups at postnatal Day 6 (P6) corresponds to that of a human fetal brain at 28–32 weeks gestation and the neonatal rat is an ideal model in which to investigate effects of oxidative stress and neuroprotection of caffeine on the developing brain. Six-day-old Wistar rats were pre-treated with caffeine and exposed to 80% oxygen for 24 and 48 h. Caffeine reduced oxidative stress marker (heme oxygenase-1, lipid peroxidation, hydrogen peroxide, and glutamate-cysteine ligase catalytic subunit (GCLC)), promoted anti-oxidative response (superoxide dismutase, peroxiredoxin 1, and sulfiredoxin 1), down-regulated pro-inflammatory cytokines, modulated redox-sensitive transcription factor expression (Nrf2/Keap1, and NFκB), reduced pro-apoptotic effectors (poly (ADP-ribose) polymerase-1 (PARP-1), apoptosis inducing factor (AIF), and caspase-3), and diminished extracellular matrix degeneration (matrix metalloproteinases (MMP) 2, and inhibitor of metalloproteinase (TIMP) 1/2). Our study affirms that caffeine is a pleiotropic neuroprotective drug in the developing brain due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties.
Sequelae of prematurity triggered by oxidative stress and free radical-mediated tissue damage have coined the term "oxygen radical disease of prematurity". Caffeine, a potent free radical scavenger and adenosine receptor antagonist, reduces rates of brain damage in preterm infants. In the present study, we investigated the effects of caffeine on oxidative stress markers, anti-oxidative response, inflammation, redox-sensitive transcription factors, apoptosis, and extracellular matrix following the induction of hyperoxia in neonatal rats. The brain of a rat pups at postnatal Day 6 (P6) corresponds to that of a human fetal brain at 28-32 weeks gestation and the neonatal rat is an ideal model in which to investigate effects of oxidative stress and neuroprotection of caffeine on the developing brain. Six-day-old Wistar rats were pre-treated with caffeine and exposed to 80% oxygen for 24 and 48 h. Caffeine reduced oxidative stress marker (heme oxygenase-1, lipid peroxidation, hydrogen peroxide, and glutamate-cysteine ligase catalytic subunit (GCLC)), promoted anti-oxidative response (superoxide dismutase, peroxiredoxin 1, and sulfiredoxin 1), down-regulated pro-inflammatory cytokines, modulated redox-sensitive transcription factor expression (Nrf2/Keap1, and NF kappa B), reduced pro-apoptotic effectors (poly (ADP-ribose) polymerase-1 (PARP-1), apoptosis inducing factor (AIF), and caspase-3), and diminished extracellular matrix degeneration (matrix metalloproteinases (MMP) 2, and inhibitor of metalloproteinase (TIMP) 1/2). Our study affirms that caffeine is a pleiotropic neuroprotective drug in the developing brain due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties.
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.
Background: Severe bacterial infections remain a major challenge in intensive care units because of their high prevalence and mortality. Adequate antibiotic exposure has been associated with clinical success in critically ill patients. The objective of this study was to investigate the target attainment of standard meropenem dosing in a heterogeneous critically ill population, to quantify the impact of the full renal function spectrum on meropenem exposure and target attainment, and ultimately to translate the findings into a tool for practical application. Methods: A prospective observational single-centre study was performed with critically ill patients with severe infections receiving standard dosing of meropenem. Serial blood samples were drawn over 4 study days to determine meropenem serum concentrations. Renal function was assessed by creatinine clearance according to the Cockcroft and Gault equation (CLCRCG). Variability in meropenem serum concentrations was quantified at the middle and end of each monitored dosing interval. The attainment of two pharmacokinetic/pharmacodynamic targets (100% T->MIC, 50% T->4xMIC) was evaluated for minimum inhibitory concentration (MIC) values of 2 mg/L and 8 mg/L and standard meropenem dosing (1000 mg, 30-minute infusion, every 8 h). Furthermore, we assessed the impact of CLCRCG on meropenem concentrations and target attainment and developed a tool for risk assessment of target non-attainment. Results: Large inter-and intra-patient variability in meropenem concentrations was observed in the critically ill population (n = 48). Attainment of the target 100% T->MIC was merely 48.4% and 20.6%, given MIC values of 2 mg/L and 8 mg/L, respectively, and similar for the target 50% T->4xMIC. A hyperbolic relationship between CLCRCG (25-255 ml/minute) and meropenem serum concentrations at the end of the dosing interval (C-8h) was derived. For infections with pathogens of MIC 2 mg/L, mild renal impairment up to augmented renal function was identified as a risk factor for target non-attainment (for MIC 8 mg/L, additionally, moderate renal impairment). Conclusions: The investigated standard meropenem dosing regimen appeared to result in insufficient meropenem exposure in a considerable fraction of critically ill patients. An easy-and free-to-use tool (the MeroRisk Calculator) for assessing the risk of target non-attainment for a given renal function and MIC value was developed.
Populations of several long-distance migratory songbirds in Eurasia are in peril, drastically illustrated by the recent range-wide population collapse in the Yellow-breasted Bunting Emberiza aureola. There are signals of a strong decline also in the Rustic Bunting E. rustica, but no range-wide assessment of population trends in this superabundant and widespread bunting species has yet been undertaken. The conservation status of Rustic Bunting is ‘Least Concern’ on the global IUCN Red List, but it has recently been upgraded to ‘Vulnerable’ on the European Red List. To assess the Rustic Bunting’s global conservation status we compiled, for the first time, population data across its breeding and wintering ranges. The analysis reveals a 75–87% decline in overall population size over the last 30 years and a 32–91% decline over the last 10 years. The trend estimates indicate that the long-term (30-year) range-wide population decline in the Rustic Bunting is of similar magnitude to two well-known examples of declining species within the same genus, the Yellow-breasted Bunting and the Ortolan Bunting E. hortulana. The magnitude of the range-wide population decline over the last 10 years suggests that the Rustic Bunting could be upgraded from ‘Least Concern’ to ‘Vulnerable’ or ‘Endangered’ on the IUCN global Red List. Agricultural intensification in the wintering range and intensified levels of disturbance, including logging and fire, in the breeding range could be important drivers of the range-wide population decline, and persecution could also contribute. Untangling threat factors and their interactions on Rustic Bunting is necessary for conservation, but hampered by our currently limited understanding of the relationships between population dynamics and different threats.
Plastic pollution is a major global concern with several million microplastic particles entering every day freshwater ecosystems via wastewater discharge. Microplastic particles stimulate biofilm formation (plastisphere) throughout the water column and have the potential to affect microbial community structure if they accumulate in pelagic waters, especially enhancing the proliferation of biohazardous bacteria. To test this scenario, we simulated the inflow of treated wastewater into a temperate lake using a continuous culture system with a gradient of concentration of microplastic particles. We followed the effect of microplastics on the microbial community structure and on the occurrence of integrase 1 (intl), a marker associated with mobile genetic elements known as a proxy for anthropogenic effects on the spread of antimicrobial resistance genes. The abundance of intl increased in the plastisphere with increasing microplastic particle concentration, but not in the water surrounding the microplastic particles. Likewise, the microbial community on microplastic was more similar to the original wastewater community with increasing microplastic concentrations. Our results show that microplastic particles indeed promote persistence of typical indicators of microbial anthropogenic pollution in natural waters, and substantiate that their removal from treated wastewater should be prioritised. (C) 2017 Elsevier Ltd. All rights reserved.
Background
A territory as a prerequisite for breeding limits the maximum number of breeders in a given area, and thus lowers the proportion of breeders if population size increases. However, some territorially breeding animals can have dramatic density fluctuations and little is known about the change from density-dependent processes to density-independence of breeding during a population increase or an outbreak. We suggest that territoriality, breeding suppression and its break-down can be understood with an incomplete-control model, developed for social breeders and social suppression.
Results
We studied density dependence in an arvicoline species, the bank vole, known as a territorial breeder with cyclic and non-cyclic density fluctuations and periodically high densities in different parts of its range. Our long-term data base from 38 experimental populations in large enclosures in boreal grassland confirms that breeding rates are density-regulated at moderate densities, probably by social suppression of subordinate potential breeders. We conducted an experiment, were we doubled and tripled this moderate density under otherwise the same conditions and measured space use, mortality, reproduction and faecal stress hormone levels (FGM) of adult females. We found that mortality did not differ among the densities, but the regulation of the breeding rate broke down: at double and triple densities all females were breeding, while at the low density the breeding rate was regulated as observed before. Spatial overlap among females increased with density, while a minimum territory size was maintained. Mean stress hormone levels were higher in double and triple densities than at moderate density.
Conclusions
At low and moderate densities, breeding suppression by the dominant breeders, But above a density-threshold (similar to a competition point), the dominance of breeders could not be sustained (incomplete control). In our experiment, this point was reached after territories could not shrink any further, while the number of intruders continued to increase with increasing density. Probably suppression becomes too costly for the dominants, and increasing number of other breeders reduces the effectiveness of threats. In wild populations, crossing this threshold would allow for a rapid density increase or population outbreaks, enabling territorial species to escape density-dependency.
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.
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.
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.
Direct visualization of APLP1 cell-cell adhesion platforms via fluorescence fluctuation spectroscopy
(2017)
Single molecule RNA fluorescent in situ hybridization (smFISH) enables gene transcription to be assessed at the cellular level. In this point of view article, we describe our recent smFISH research in the model plant Arabidopsis thaliana and discuss how this technique could further knowledge of plant gene transcription in the future.
Background: Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor.
Results: IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 mu L - 100 mu L). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter.
Conclusions: Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources.
The pyranopterin dithiolene (PDT) ligand is an integral component of the molybdenum cofactor (Moco) found in all molybdoenzymes with the sole exception of nitrogenase. However, the roles of the PDT in catalysis are still unknown. The PDT is believed to be bound to the proteins by an extensive hydrogen bonding network, and it has been suggested that these interactions may function to fine-tune Moco for electron- and atom-transfer reactivity in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe Moco-protein interactions using heavy-atom congeners of lumazine, molecules that bind tightly to both wild-type xanthine dehydrogenase (wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation to the corresponding violapterin product molecules. The resulting enzyme-product complexes possess intense near-IR absorption, allowing high-quality rR spectra to be collected on wt-XDH and the Q102G and Q197A variants. Small negative frequency shifts relative to wt-XDH are observed for the low-frequency Moco vibrations. These results are interpreted in the context of weak hydrogen-bonding and/or electrostatic interactions between Q102 and the -NH2 terminus of the PDT, and between Q197 and the terminal oxo of the Mo equivalent to O group. The Q102A, Q102G, Q197A, and Q197E variants do not appreciably affect the kinetic parameters k(red) and k(red)/K-D, indicating that a primary role for these glutamine residues is to stabilize and coordinate Moco in the active site of XO family enzymes but to not directly affect the catalytic throughput. Raman frequency shifts between wt-XDH and its Q102G variant suggest that the changes in the electron density at the Mo ion that accompany Mo oxidation during electron-transfer regeneration of the catalytically competent active site are manifest in distortions at the distant PDT amino terminus. This implies a primary role for the PDT as a conduit for facilitating enzymatic electron-transfer reactivity in xanthine oxidase family enzymes.