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Institute
- Institut für Biochemie und Biologie (212) (remove)
Retinol-binding protein 4 (RBP4) is the major transport protein for retinol in blood. Recent evidence from genetic mouse models shows that circulating RBP4 derives exclusively from hepatocytes. Because RBP4 is elevated in obesity and associates with the development of glucose intolerance and insulin resistance, we tested whether a liver-specific overexpression of RBP4 in mice impairs glucose homeostasis. We used adeno-associated viruses (AAV) that contain a highly liver-specific promoter to drive expression of murine RBP4 in livers of adult mice. The resulting increase in serum RBP4 levels in these mice was comparable with elevated levels that were reported in obesity. Surprisingly, we found that increasing circulating RBP4 had no effect on glucose homeostasis. Also during a high-fat diet challenge, elevated levels of RBP4 in the circulation failed to aggravate the worsening of systemic parameters of glucose and energy homeostasis. These findings show that liver-secreted RBP4 does not impair glucose homeostasis. We conclude that a modest increase of its circulating levels in mice, as observed in the obese, insulin-resistant state, is unlikely to be a causative factor for impaired glucose homeostasis.
Mammalian aldehyde oxidases (AOX) are molybdo-flavoenzymes of pharmacological and pathophysiologic relevance that are involved in phase I drug metabolism and, as a product of their enzymatic activity, are also involved in the generation of reactive oxygen species. So far, the physiologic role of aldehyde oxidase 1 in the human body remains unknown. The human enzyme hAOX1 is characterized by a broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into their corresponding carboxylic acids, and hydroxylating various heteroaromatic rings. The enzyme uses oxygen as terminal electron acceptor to produce hydrogen peroxide and superoxide during turnover. Since hAOX1 and, in particular, some natural variants produce not only H2O2 but also high amounts of superoxide, we investigated the effect of both ROS molecules on the enzymatic activity of hAOX1 in more detail. We compared hAOX1 to the high-O-2(.-)-producing natural variant L438V for their time-dependent inactivation with H2O2/O-2(.-) during substrate turnover. We show that the inactivation of the hAOX1 wild-type enzyme is mainly based on the production of hydrogen peroxide, whereas for the variant L438V, both hydrogen peroxide and superoxide contribute to the time-dependent inactivation of the enzyme during turnover. Further, the level of inactivation was revealed to be substrate-dependent: using substrates with higher turnover numbers resulted in a faster inactivation of the enzymes. Analysis of the inactivation site of the enzyme identified a loss of the terminal sulfido ligand at the molybdenum active site by the produced ROS during turnover.
It is well known that functional diversity strongly affects ecosystem functioning. However, even in rather simple model communities consisting of only two or, at best, three trophic levels, the relationship between multitrophic functional diversity and ecosystem functioning appears difficult to generalize, because of its high contextuality. In this study, we considered several differently structured tritrophic food webs, in which the amount of functional diversity was varied independently on each trophic level. To achieve generalizable results, largely independent of parametrization, we examined the outcomes of 128,000 parameter combinations sampled from ecologically plausible intervals, with each tested for 200 randomly sampled initial conditions. Analysis of our data was done by training a random forest model. This method enables the identification of complex patterns in the data through partial dependence graphs, and the comparison of the relative influence of model parameters, including the degree of diversity, on food-web properties. We found that bottom-up and top-down effects cascade simultaneously throughout the food web, intimately linking the effects of functional diversity of any trophic level to the amount of diversity of other trophic levels, which may explain the difficulty in unifying results from previous studies. Strikingly, only with high diversity throughout the whole food web, different interactions synergize to ensure efficient exploitation of the available nutrients and efficient biomass transfer to higher trophic levels, ultimately leading to a high biomass and production on the top level. The temporal variation of biomass showed a more complex pattern with increasing multitrophic diversity: while the system initially became less variable, eventually the temporal variation rose again because of the increasingly complex dynamical patterns. Importantly, top predator diversity and food-web parameters affecting the top trophic level were of highest importance to determine the biomass and temporal variability of any trophic level. Overall, our study reveals that the mechanisms by which diversity influences ecosystem functioning are affected by every part of the food web, hampering the extrapolation of insights from simple monotrophic or bitrophic systems to complex natural food webs.
Plant cell biology
(2021)
PIN-FORMED (PIN) polar protein localization directs transport of the growth and developmental regulator auxin in plants. Once established after cytokinesis, PIN polarity requires maintenance. Now, direct interactions between PIN, MAB4/MEL and PID proteins suggest self-reinforced maintenance of PIN polarity through limiting lateral diffusion.
While habitat loss is a known key driver of biodiversity decline, the impact of other landscape properties, such as patch isolation, is far less clear. When patch isolation is low, species may benefit from a broader range of foraging opportunities, but are at the same time adversely affected by higher predation pressure from mobile predators. Although previous approaches have successfully linked such effects to biodiversity, their impact on local and metapopulation dynamics has largely been ignored. Since population dynamics may also be affected by environmental disturbances that temporally change the degree of patch isolation, such as periodic changes in habitat availability, accurate assessment of its link with isolation is highly challenging. To analyze the effect of patch isolation on the population dynamics on different spatial scales, we simulate a three-species meta-food chain on complex networks of habitat patches and assess the average variability of local populations and metapopulations, as well as the level of synchronization among patches. To evaluate the impact of periodic environmental disturbances, we contrast simulations of static landscapes with simulations of dynamic landscapes in which 30 percent of the patches periodically become unavailable as habitat. We find that increasing mean patch isolation often leads to more asynchronous population dynamics, depending on the parameterization of the food chain. However, local population variability also increases due to indirect effects of increased dispersal mortality at high mean patch isolation, consequently destabilizing metapopulation dynamics and increasing extinction risk. In dynamic landscapes, periodic changes of patch availability on a timescale much slower than ecological interactions often fully synchronize the dynamics. Further, these changes not only increase the variability of local populations and metapopulations, but also mostly overrule the effects of mean patch isolation. This may explain the often small and inconclusive impact of mean patch isolation in natural ecosystems.
Self-organised formation of spatial patterns is known from a variety of different ecosystems, yet little is known about how these patterns affect the diversity of communities. Here, we use a food chain model in which autotroph diversity is described by a continuous distribution of a trait that affects both growth and defence against heterotrophs. On isolated patches, diversity is always lost over time due to stabilising selection, and the local communities settle on one of two alternative stable community states that are characterised by a dominance of either defended or undefended species. In a metacommunity context, dispersal can destabilise these states and complex spatio-temporal patterns in the species' abundances emerge. The resulting biomass-trait feedback increases local diversity by an order of magnitude compared to scenarios without self-organised pattern formation, thereby maintaining the ability of communities to adapt to potential future changes in biotic or abiotic environmental conditions.
Eastern Africa has been a prime target for scientific drilling because it is rich in key paleoanthropological sites as well as in paleolakes, containing valuable paleoclimatic information on evolutionary time scales. The Hominin Sites and Paleolakes Drilling Project (HSPDP) explores these paleolakes with the aim of reconstructing environmental conditions around critical episodes of hominin evolution. Identification of biological taxa based on their sedimentary ancient DNA (sedaDNA) traces can contribute to understand past ecological and climatological conditions of the living environment of our ancestors. However, sedaDNA recovery from tropical environments is challenging because high temperatures, UV irradiation, and desiccation result in highly degraded DNA. Consequently, most of the DNA fragments in tropical sediments are too short for PCR amplification. We analyzed sedaDNA in the upper 70 m of the composite sediment core of the HSPDP drill site at Chew Bahir for eukaryotic remnants. We first tested shotgun high throughput sequencing which leads to metagenomes dominated by bacterial DNA of the deep biosphere, while only a small fraction was derived from eukaryotic, and thus probably ancient, DNA. Subsequently, we performed cross-species hybridization capture of sedaDNA to enrich ancient DNA (aDNA) from eukaryotic remnants for paleoenvironmental analysis, using established barcoding genes (cox1 and rbcL for animals and plants, respectively) from 199 species that may have had relatives in the past biosphere at Chew Bahir. Metagenomes yielded after hybridization capture are richer in reads with similarity to cox1 and rbcL in comparison to metagenomes without prior hybridization capture. Taxonomic assignments of the reads from these hybridization capture metagenomes also yielded larger fractions of the eukaryotic domain. For reads assigned to cox1, inferred wet periods were associated with high inferred relative abundances of putative limnic organisms (gastropods, green algae), while inferred dry periods showed increased relative abundances for insects. These findings indicate that cross-species hybridization capture can be an effective approach to enhance the information content of sedaDNA in order to explore biosphere changes associated with past environmental conditions, enabling such analyses even under tropical conditions.
License to flower
(2021)
The dictyostelium centrosome
(2021)
The centrosome of Dictyostelium amoebae contains no centrioles and consists of a cylindrical layered core structure surrounded by a corona harboring microtubule-nucleating gamma-tubulin complexes. It is the major centrosomal model beyond animals and yeasts. Proteomics, protein interaction studies by BioID and superresolution microscopy methods led to considerable progress in our understanding of the composition, structure and function of this centrosome type. We discuss all currently known components of the Dictyostelium centrosome in comparison to other centrosomes of animals and yeasts.
A drop of immunity
(2021)
AAA+ proteins (ATPases associated with various cellular activities) catalyze the energy-dependent movement or rearrangement of macromolecules. A new study addresses the important question of how to design a selective chemical inhibitor for specific proteins in this diverse superfamily. The powerful chemical genetics approach adds to a growing toolbox of applications that allow dissection of the functions of distinct AAA+ proteins in vivo, facilitating the first steps toward effective drug development.
The prevalence of diseases associated with misfolded proteins increases with age. When cellular defense mechanisms become limited, misfolded proteins form aggregates and may also develop more stable cross-β structures ultimately forming amyloid aggregates. Amyloid aggregates are associated with neurodegenerative diseases such as Alzheimer’s disease and Huntington’s disease. The formation of amyloid deposits, their toxicity and cellular defense mechanisms have been intensively studied. However, surprisingly little is known about the effects of protein aggregates on cellular signal transduction. It is also not understood whether the presence of aggregation-prone, but still soluble proteins affect signal transduction.
In this study, the still soluble aggregation-prone HttExon1Q74 and its amyloid aggregates were used to analyze the effect of amyloid aggregates on internalization and receptor activation of G protein-coupled receptors (GPCRs), the largest protein family of mammalian cell surface receptors involved in signal transduction. The aggregated HttExon1Q74, but not its soluble form, could inhibit ligand-induced clathrin-mediated endocytosis (CME) of various GPCRs. Most likely this inhibitory effect is based on a terminal sequestration of the HSC70 chaperone to the aggregates which is necessary for CME. Using the vasopressinV1a receptor (V1aR) and the corticotropin-releasing factor receptor 1 (CRF1R) as a model, it could be shown that the presence of HttExon1Q74 aggregates and the inhibition of ligand-induced CME leads to an accumulation of desensitized receptors at the plasma membrane. In turn, this disrupts Gq-mediated Ca2+ signaling and Gs-mediated cAMP signaling of the V1aR and the CRF1R respectively. In contrast to HttExon1Q74 amyloid aggregates, soluble HttExon1Q74 as well as amorphous aggregates did not inhibit GPCR internalization and signaling demonstrating that cellular signal transduction mechanisms are specifically impaired in response to the formation of amyloid aggregates.
In addition, preliminary experiments could show that HttExon1Q74 aggregates provoke an increase in membrane expression of a protein from a structurally and functionally unrelated membrane protein family, namely the serotonin transporter SERT. As SERT is the main pharmacological target to treat depression this could shed light on this commonly occurring comorbidity in neurodegenerative diseases, in particular in early disease states.
Due to global climate change providing food security for an increasing world population is a big challenge. Especially abiotic stressors have a strong negative effect on crop yield. To develop climate-adapted crops a comprehensive understanding of molecular alterations in the response of varying levels of environmental stresses is required. High throughput or ‘omics’ technologies can help to identify key-regulators and pathways of abiotic stress responses. In addition to obtain omics data also tools and statistical analyses need to be designed and evaluated to get reliable biological results.
To address these issues, I have conducted three different studies covering two omics technologies. In the first study, I used transcriptomic data from the two polymorphic Arabidopsis thaliana accessions, namely Col-0 and N14, to evaluate seven computational tools for their ability to map and quantify Illumina single-end reads. Between 92% and 99% of the reads were mapped against the reference sequence. The raw count distributions obtained from the different tools were highly correlated. Performing a differential gene expression analysis between plants exposed to 20 °C or 4°C (cold acclimation), a large pairwise overlap between the mappers was obtained. In the second study, I obtained transcript data from ten different Oryza sativa (rice) cultivars by PacBio Isoform sequencing that can capture full-length transcripts. De novo reference transcriptomes were reconstructed resulting in 38,900 to 54,500 high-quality isoforms per cultivar. Isoforms were collapsed to reduce sequence redundancy and evaluated, e.g. for protein completeness level (BUSCO), transcript length, and number of unique transcripts per gene loci. For the heat and drought tolerant aus cultivar N22, I identified around 650 unique and novel transcripts of which 56 were significantly differentially expressed in developing seeds during combined drought and heat stress. In the last study, I measured and analyzed the changes in metabolite profiles of eight rice cultivars exposed to high night temperature (HNT) stress and grown during the dry and wet season on the field in the Philippines. Season-specific changes in metabolite levels, as well as for agronomic parameters, were identified and metabolic pathways causing a yield decline at HNT conditions suggested.
In conclusion, the comparison of mapper performances can help plant scientists to decide on the right tool for their data. The de novo reconstruction of rice cultivars without a genome sequence provides a targeted, cost-efficient approach to identify novel genes responding to stress conditions for any organism. With the metabolomics approach for HNT stress in rice, I identified stress and season-specific metabolites which might be used as molecular markers for crop improvement in the future.
Bottom-up synthetic biology is used for the understanding of how a cell works. It is achieved through developing techniques to produce lipid-based vesicular structures as cellular mimics. The most common techniques used to produce cellular mimics or synthetic cells is through electroformation and swelling method. However, the abovementioned techniques cannot efficiently encapsulate macromolecules such as proteins, enzymes, DNA and even liposomes as synthetic organelles. This urges the need to develop new techniques that can circumvent this issue and make the artificial cell a reality where it is possible to imitate a eukaryotic cell through encapsulating macromolecules. In this thesis, the aim to construct a cell system using giant unilamellar vesicles (GUVs) to reconstitute the mitochondrial molybdenum cofactor biosynthetic pathway. This pathway is highly conserved among all life forms, and therefore is known for its biological significance in disorders induced through its malfunctioning. Furthermore, the pathway itself is a multi-step enzymatic reaction that takes place in different compartments. Initially, GTP in the mitochondrial matrix is converted to cPMP in the presence of cPMP synthase. Further, produced cPMP is transported across the membrane to the cytosol, to be converted by MPT synthase into MPT. This pathway provides a possibility to address the general challenges faced in the development of a synthetic cell, to encapsulate large biomolecules with good efficiency and greater control and to evaluate the enzymatic reactions involved in the process.
For this purpose, the emulsion-based technique was developed and optimised to allow rapid production of GUVs (~18 min) with high encapsulation efficiency (80%). This was made possible by optimizing various parameters such as density, type of oil, the impact of centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume. Furthermore, the method was optimised in microtiter plates for direct experimentation and visualization after the GUV formation. Using this technique, the two steps - formation of cPMP from GTP and the formation of MPT from cPMP were encapsulated in different sets of GUVs to mimic the two compartments. Two independent fluorescence-based detection systems were established to confirm the successful encapsulation and conversion of the reactants. Alternatively, the enzymes produced using bacterial expression and measured. Following the successful encapsulation and evaluation of enzymatic reactions, cPMP transport across mitochondrial membrane has been mimicked using GUVs using a complex mitochondrial lipid composition. It was found that the cPMP interaction with the lipid bilayer results in transient pore-formation and leakage of internal contents.
Overall, it can be concluded that in this thesis a novel technique has been optimised for fast production of functional synthetic cells. The individual enzymatic steps of the Moco biosynthetic pathway have successfully implemented and quantified within these cellular mimics.
There is a general consensus that diverse ecological communities are better equipped to adapt to changes in their environment, but our understanding of the mechanisms by which they do so remains incomplete. Accurately predicting how the global biodiversity crisis affects the functioning of ecosystems, and the services they provide, requires extensive knowledge about these mechanisms.
Mathematical models of food webs have been successful in uncovering many aspects of the link between diversity and ecosystem functioning in small food web modules, containing at most two adaptive trophic levels. Meaningful extrapolation of this understanding to the functioning of natural food webs remains difficult, due to the presence of complex interactions that are not always accurately captured by bitrophic descriptions of food webs. In this dissertation, we expand this approach to tritrophic food web models by including the third trophic level. Using a functional trait approach, coexistence of all species is ensured using fitness-balancing trade-offs. For example, the defense-growth trade-off implies that species may be defended against predation, but this defense comes at the cost of a lower maximal growth rate. In these food webs, the functional diversity on a given trophic level can be varied by modifying the trait differences between the species on that level.
In the first project, we find that functional diversity promotes high biomass on the top level, which, in turn, leads to a reduction in the temporal variability due to compensatory dynamical patterns governed by the top level. Next, these results are generalized by investigating the average behavior of tritrophic food webs, for wide intervals of all parameters describing species interactions in the food web. We find that the diversity on the top level is most important for determining the biomass and temporal variability of all other trophic levels, and show how biomass is only transferred efficiently to the top level when diversity is high everywhere in the food web. In the third project, we compare the response of a simple food chain against a nutrient pulse perturbation, to that of a food web with diversity on every trophic level. By joint consideration of the resistance, resilience, and elasticity, we uncover that the response is efficiently buffered when biomass on the top level is high, which is facilitated by functional diversity on every trophic level in the food web. Finally, in the fourth project, we show that even in a simple consumer-resource model without any diversity, top-down control on the intermediate level frequently causes the phase difference between the intermediate and basal level to deviate from the quarter-cycle lag rule. By adding a top predator, we show that these deviations become even more likely, and anti-phase cycles are often observed.
The combined results of these projects show how the properties of the top trophic level, including its functional diversity, have a decisive influence on the functioning of tritrophic food webs from a mechanistic perspective. Because top species are often among the most vulnerable to extinction, our results emphasize the importance of their conservation in ecosystem management and restoration strategies.
As the ongoing trend of developing smart materials that can reversibly switch geometry stimulated by environmental control addressed increasing attention in many research fields, especially for biomedical or soft robotic applications. Shape-memory polymers (SMPs), which can change shape, stiffness, size, and structure when exposed to an external stimulus, are intensively explored as encouraging material candidates for achieving multifunctionality, and for miniaturizing into micro-components to expand the applications. Besides, the geometrical design has gained growing attention for creating engineering applications, such as bi-stable mechanisms, and has the potential to be explored by implementing SMP for new functions. In this context, this thesis aimed to develop smart micro-/nano-objects based on SMP and explore new functions by geometrical design using SMP. Here, two types of stimuli-responsive objects capable of one-way temperature-memory effect (TME) or free-standing reversible actuation e.g., micro/nanofibers (i) and microcuboids (ii) at different aspects were explored. At first, it was hypothesized that the advanced atomic force microscopy (AFM) platform can be established to study individual polymeric micro-/nanofibers (i) in terms of incorporation and characterization of a reversible shape-memory actuation capability. Crystallizable material was chosen for preparing the fibers and the molecular alignment within the fibers among different diameters will influence the crystallization-induced elongation during cooling that determined the reversible effect. For the second type, microcuboids (ii), it was hypothesized that a programming and quantification approach can be developed to enable the realization and characterization of a one-way micro-TME and micro-shape-memory polymer actuation (SMPA) in microcuboids. The responsive temperature of one-way shape transformation can be tuned by programming temperature (Tp) and the separation temperature (Tsep) for post-programming can influence the actuation. Finally, a geometrical design with bi-stability was combined with SME to create new functions of shape actuation. It was hypothesized that the predicted bi-stable or mono-stable structures can be achieved with the aid of digital fabrication methods. Using shape-memory effect (SME), the alteration of bi-stable and mono-stable can initiate shape transformation with a larger magnitude and higher energy output.
In the first part, the method to quantify the reversible SMPA of a single micro/nano crystallizable fiber with geometry change during the actuation was explored. Electrospinning was used to prepare poly (ε-caprolactone) (PCL) micro/nanofiber with different diameters, which were fixed by UV glue and crosslinked on the structured silicon wafer. Using AFM, the programming, as well as the observation of recovery and reversible displacement of the fiber, were performed by vertical three-point bending at the free suspended part. A plateau tip was chosen to achieve stable contact and longer working distance for performing larger deformation, enabling intensified reversible SMPA of single fibers. In this way, programming strains of 39 ± 1% or 46 ± 1% were realized for fiber with a diameter of 1 ± 0.2 µm and 300 ± 50 nm, which were bent at 80 °C and fixed at 10 °C. Values for the reversible elongation of εrev = 3.4 ± 0.1% and 10.5 ± 0.1% were obtained for a single micro and nanofiber respectively between 10 and 60 °C. The higher actuation effect observed for nanofiber demonstrated that the highly compact and oriented crystallites in nanofibers, which determined the pronounced εrev compared to the thick microfibers. Besides, a stable reversible actuation of a nanofiber can be tracked by AFM tip up to 10 cycles, indicating a sustainable application can be achieved on the fiber actuators. The findings obtained for cPCL micro-/nano-fibers will help design and evaluate the next generation polymeric microactuators or micromanipulators.
The second part of the thesis studies the shape-memory effect (SME) of a single individual SMP micro-object by controlling deformation temperatures during programming and actuation temperatures during reversible change. In this work, microcuboids of crosslinked poly[ethylene-co-(vinyl acetate)] (cPEVA) elastomers with 18 wt% vinyl acetate (VA) contents were successfully prepared by template-based replication from polydimethylsiloxane (PDMS) mold. The micro-TME and micro-SMPA were observed and studied based on micro-geometry change using optical microscopy (OM) and AFM. Different switching temperatures of shape recovery were achieved from 55 °C to 86 °C by tuning Tp from 55 °C to 100 °C, indicating a successful implementation of micro-TME on individual microcuboid. For micro-SMPA functionalization, microcuboids were deformed by compression at 100 °C and the change in single particle height was monitored during cyclic heating and cooling between various Tseps from 60 °C to 85 °C and 20 °C. The micro-SMPA on a single microcuboid was achieved with a reversible strain in the range of 2 to 7%, whereby higher compression ratio CR and Tsep induced prominent reversible strain. The results achieved in this work demonstrated the successful functionalization of microcuboids with different SMEs by controlling temperatures during programming and actuation processes. Based on these achievements, such micro-objects can be further designed as on demand switchable microactuators or release systems with adjustable working temperatures.
In the last part of the work, a new function of shape-memory polymeric bi-stable 3D structured film was designed and fabricated. The SME and geometrical design of compliant mechanics were merged to enable switching between bi-stable and mono-stable states, which generate snap movement that mimics the Venus flytrap. A truncated tetrahedron structure with a slope angle as a tunable parameter to alter the bi-stability was chosen for the study to combine with
SME. It was anticipated that the structured film designed with a slope angle of 30° exhibited mono-stable behavior, and such a structure with a slope angle of 45° exhibited bi-stable behavior. Then the structured SMP film of designed mono-stable shape was successfully fabricated using soft lithography based on 3D printed master molds supported from digital manufacturing. The structured mold was also used in programming the SMP film into the structure with a higher slope angle to attain bi-stability. Finally, the switching between bi-stable and mono-stable states was successfully realized using SME, which introduces snapping movement triggered by heat. The implementation of compliant mechanisms by the SME increased the magnitude of thermally induced reconfiguration without additional external force.
To sum up, the results of the thesis support the development of smart objects capable of one-way micro-TME, free-standing reversible actuation, or bi-stability mediated shape-memory reconfiguration. Electrospinning and template-based method were used for fabrication with good control of geometry and low size dispersity. Microscopy methods especially the AFM platform with decent sensitivity was developed for implementation as well as characterization of SME on individual micro-/nanoobjects. Implementation of bi-stability improves the shape transformation amplitude of thermally triggered SMP. These findings can give novel insights for designing polymer-based actuators or soft robotics.
Rapid screening of infected people plays a crucial role in interrupting infection chains. However, the current methods for identification of bacteria are very tedious and labor intense. Fast on-site screening for pathogens based on volatile organic compounds (VOCs) by ion mobility spectrometry (IMS) could help to differentiate between healthy and potentially infected subjects. As a first step towards this, the feasibility of differentiating between seven different bacteria including resistant strains was assessed using IMS coupled to multicapillary columns (MCC-IMS). The headspace above bacterial cultures was directly drawn and analyzed by MCC-IMS after 90 min of incubation. A cluster analysis software and statistical methods were applied to select discriminative VOC clusters. As a result, 63 VOC clusters were identified, enabling the differentiation between all investigated bacterial strains using canonical discriminant analysis. These 63 clusters were reduced to 7 discriminative VOC clusters by constructing a hierarchical classification tree. Using this tree, all bacteria including resistant strains could be classified with an AUC of 1.0 by receiver-operating characteristic analysis. In conclusion, MCC-IMS is able to differentiate the tested bacterial species, even the non-resistant and their corresponding resistant strains, based on VOC patterns after 90 min of cultivation. Although this result is very promising, in vivo studies need to be performed to investigate if this technology is able to also classify clinical samples. With a short analysis time of 5 min, MCC-IMS is quite attractive for a rapid screening for possible infections in various locations from hospitals to airports. Key Points center dot Differentiation of bacteria by MCC-IMS is shown after 90-min cultivation. center dot Non-resistant and resistant strains can be distinguished. center dot Classification of bacteria is possible based on metabolic features.
Deconstructing the Gestalt
(2021)
Snakes-a subset of lizards-have traditionally been divided into two major groups based on feeding mechanics: "macrostomy," involving the ingestion of proportionally large prey items; and "microstomy," the lack of this ability. "Microstomy"-considered present in scolecophidian and early-diverging alethinophidian snakes-is generally viewed as a symplesiomorphy shared with non-snake lizards. However, this perspective of "microstomy" as plesiomorphic and morphologically homogenous fails to recognize the complexity of this condition and its evolution across "microstomatan" squamates. To challenge this problematic paradigm, we formalize a new framework for conceptualizing and testing the homology of overall character complexes, or "morphotypes," which underlies our re-assessment of "microstomy." Using micro-computed tomography (micro-CT) scans, we analyze the morphology of the jaws and suspensorium across purported "microstomatan" squamates (scolecophidians, early-diverging alethinophidians, and non-snake lizards) and demonstrate that key components of the jaw complex are not homologous at the level of primary character state identity across these taxa. Therefore, rather than treating "microstomy" as a uniform condition, we instead propose that non-snake lizards, early-diverging alethinophidians, anomalepidids, leptotyphlopids, and typhlopoids each exhibit a unique and nonhomologous jaw morphotype: "minimal-kinesis microstomy," "snout-shifting," "axle-brace maxillary raking," "mandibular raking," and "single-axle maxillary raking," respectively. The lack of synapomorphy among scolecophidians is inconsistent with the notion of scolecophidians representing an ancestral snake condition, and instead reflects a hypothesis of the independent evolution of fossoriality, miniaturization, and "microstomy" in each scolecophidian lineage. We ultimately emphasize that a rigorous approach to comparative anatomy is necessary in constructing evolutionary hypotheses that accurately reflect biological reality.
The ubiquitous freshwater cyanobacterium Microcystis is remarkably successful, showing a high tolerance against fluctuations in environmental conditions. It frequently forms dense blooms which can accumulate significant amounts of the hepatotoxin microcystin, which plays an extracellular role as an infochemical but also acts intracellularly by interacting with proteins of the carbon metabolism, notably with the CO2 fixing enzyme RubisCO. Here we demonstrate a direct link between external microcystin and its intracellular targets. Monitoring liquid cultures of Microcystis in a diel experiment revealed fluctuations in the extracellular microcystin content that correlate with an increase in the binding of microcystin to intracellular proteins. Concomitantly, reversible relocation of RubisCO from the cytoplasm to the cell’s periphery was observed. These variations in RubisCO localization were especially pronounced with cultures grown at higher cell densities. We replicated these effects by adding microcystin externally to cultures grown under continuous light. Thus, we propose that microcystin may be part of a fast response to conditions of high light and low carbon that contribute to the metabolic flexibility and the success of Microcystis in the field.
Strong as a Hippo’s Heart: Biomechanical Hippo Signaling During Zebrafish Cardiac Development
(2021)
The heart is comprised of multiple tissues that contribute to its physiological functions. During development, the growth of myocardium and endocardium is coupled and morphogenetic processes within these separate tissue layers are integrated. Here, we discuss the roles of mechanosensitive Hippo signaling in growth and morphogenesis of the zebrafish heart. Hippo signaling is involved in defining numbers of cardiac progenitor cells derived from the secondary heart field, in restricting the growth of the epicardium, and in guiding trabeculation and outflow tract formation. Recent work also shows that myocardial chamber dimensions serve as a blueprint for Hippo signaling-dependent growth of the endocardium. Evidently, Hippo pathway components act at the crossroads of various signaling pathways involved in embryonic zebrafish heart development. Elucidating how biomechanical Hippo signaling guides heart morphogenesis has direct implications for our understanding of cardiac physiology and pathophysiology.
Objective
The Caribbean is an important global biodiversity hotspot. Adaptive radiations there lead to many speciation events within a limited period and hence are particularly prominent biodiversity generators. A prime example are freshwater fish of the genus Limia, endemic to the Greater Antilles. Within Hispaniola, nine species have been described from a single isolated site, Lake Miragoâne, pointing towards extraordinary sympatric speciation. This study examines the evolutionary history of the Limia species in Lake Miragoâne, relative to their congeners throughout the Caribbean.
Results
For 12 Limia species, we obtained almost complete sequences of the mitochondrial cytochrome b gene, a well-established marker for lower-level taxonomic relationships. We included sequences of six further Limia species from GenBank (total N = 18 species). Our phylogenies are in concordance with other published phylogenies of Limia. There is strong support that the species found in Lake Miragoâne in Haiti are monophyletic, confirming a recent local radiation. Within Lake Miragoâne, speciation is likely extremely recent, leading to incomplete lineage sorting in the mtDNA. Future studies using multiple unlinked genetic markers are needed to disentangle the relationships within the Lake Miragoâne clade.
Eastern Africa has been a prime target for scientific drilling because it is rich in key paleoanthropological sites as well as in paleolakes, containing valuable paleoclimatic information on evolutionary time scales. The Hominin Sites and Paleolakes Drilling Project (HSPDP) explores these paleolakes with the aim of reconstructing environmental conditions around critical episodes of hominin evolution. Identification of biological taxa based on their sedimentary ancient DNA (sedaDNA) traces can contribute to understand past ecological and climatological conditions of the living environment of our ancestors. However, sedaDNA recovery from tropical environments is challenging because high temperatures, UV irradiation, and desiccation result in highly degraded DNA. Consequently, most of the DNA fragments in tropical sediments are too short for PCR amplification. We analyzed sedaDNA in the upper 70 m of the composite sediment core of the HSPDP drill site at Chew Bahir for eukaryotic remnants. We first tested shotgun high throughput sequencing which leads to metagenomes dominated by bacterial DNA of the deep biosphere, while only a small fraction was derived from eukaryotic, and thus probably ancient, DNA. Subsequently, we performed cross-species hybridization capture of sedaDNA to enrich ancient DNA (aDNA) from eukaryotic remnants for paleoenvironmental analysis, using established barcoding genes (cox1 and rbcL for animals and plants, respectively) from 199 species that may have had relatives in the past biosphere at Chew Bahir. Metagenomes yielded after hybridization capture are richer in reads with similarity to cox1 and rbcL in comparison to metagenomes without prior hybridization capture. Taxonomic assignments of the reads from these hybridization capture metagenomes also yielded larger fractions of the eukaryotic domain. For reads assigned to cox1, inferred wet periods were associated with high inferred relative abundances of putative limnic organisms (gastropods, green algae), while inferred dry periods showed increased relative abundances for insects. These findings indicate that cross-species hybridization capture can be an effective approach to enhance the information content of sedaDNA in order to explore biosphere changes associated with past environmental conditions, enabling such analyses even under tropical conditions.
Semi-natural habitats (SNHs) are becoming increasingly scarce in modern agricultural landscapes. This may reduce natural ecosystem services such as pest control with its putatively positive effect on crop production. In agreement with other studies, we recently reported wheat yield reductions at field borders which were linked to the type of SNH and the distance to the border. In this experimental landscape-wide study, we asked whether these yield losses have a biotic origin while analyzing fungal seed and fungal leaf pathogens, herbivory of cereal leaf beetles, and weed cover as hypothesized mediators between SNHs and yield. We established experimental winter wheat plots of a single variety within conventionally managed wheat fields at fixed distances either to a hedgerow or to an in-field kettle hole. For each plot, we recorded the fungal infection rate on seeds, fungal infection and herbivory rates on leaves, and weed cover. Using several generalized linear mixed-effects models as well as a structural equation model, we tested the effects of SNHs at a field scale (SNH type and distance to SNH) and at a landscape scale (percentage and diversity of SNHs within a 1000-m radius). In the dry year of 2016, we detected one putative biotic culprit: Weed cover was negatively associated with yield values at a 1-m and 5-m distance from the field border with a SNH. None of the fungal and insect pests, however, significantly affected yield, neither solely nor depending on type of or distance to a SNH. However, the pest groups themselves responded differently to SNH at the field scale and at the landscape scale. Our findings highlight that crop losses at field borders may be caused by biotic culprits; however, their negative impact seems weak and is putatively reduced by conventional farming practices.
The ubiquitous freshwater cyanobacterium Microcystis is remarkably successful, showing a high tolerance against fluctuations in environmental conditions. It frequently forms dense blooms which can accumulate significant amounts of the hepatotoxin microcystin, which plays an extracellular role as an infochemical but also acts intracellularly by interacting with proteins of the carbon metabolism, notably with the CO2 fixing enzyme RubisCO. Here we demonstrate a direct link between external microcystin and its intracellular targets. Monitoring liquid cultures of Microcystis in a diel experiment revealed fluctuations in the extracellular microcystin content that correlate with an increase in the binding of microcystin to intracellular proteins. Concomitantly, reversible relocation of RubisCO from the cytoplasm to the cell’s periphery was observed. These variations in RubisCO localization were especially pronounced with cultures grown at higher cell densities. We replicated these effects by adding microcystin externally to cultures grown under continuous light. Thus, we propose that microcystin may be part of a fast response to conditions of high light and low carbon that contribute to the metabolic flexibility and the success of Microcystis in the field.
Due to their isolated and often fragmented nature, range margin populations are especially vulnerable to rapid environmental change. To maintain genetic diversity and adaptive potential, gene flow from disjunct populations might therefore be crucial to their survival. Translocations are often proposed as a mitigation strategy to increase genetic diversity in threatened populations. However, this also includes the risk of losing locally adapted alleles through genetic swamping. Human-mediated translocations of southern lineage specimens into northern German populations of the endangered European fire-bellied toad (Bombina bombina) provide an unexpected experimental set-up to test the genetic consequences of an intraspecific introgression from central population individuals into populations at the species range margin. Here, we utilize complete mitochondrial genomes and transcriptome nuclear data to reveal the full genetic extent of this translocation and the consequences it may have for these populations. We uncover signs of introgression in four out of the five northern populations investigated, including a number of introgressed alleles ubiquitous in all recipient populations, suggesting a possible adaptive advantage. Introgressed alleles dominate at the MTCH2 locus, associated with obesity/fat tissue in humans, and the DSP locus, essential for the proper development of epidermal skin in amphibians. Furthermore, we found loci where local alleles were retained in the introgressed populations, suggesting their relevance for local adaptation. Finally, comparisons of genetic diversity between introgressed and nonintrogressed northern German populations revealed an increase in genetic diversity in all German individuals belonging to introgressed populations, supporting the idea of a beneficial transfer of genetic variation from Austria into North Germany.
In C3 plants, CO2 diffuses into the leaf and is assimilated by the Calvin-Benson cycle in the mesophyll cells. It leaves Rubisco open to its side reaction with O2, resulting in a wasteful cycle known as photorespiration. A sharp fall in atmospheric CO2 levels about 30 million years ago have further increased the side reaction with O2. The pressure to reduce photorespiration led, in over 60 plant genera, to the evolution of a CO2-concentrating mechanism called C4 photosynthesis; in this mode, CO2 is initially incorporated into 4-carbon organic acids, which diffuse to the bundle sheath and are decarboxylated to provide CO2 to Rubisco. Some genera, like Flaveria, contain several species that represent different steps in this complex evolutionary process. However, the majority of terrestrial plant species did not evolve a CO2-concentrating mechanism and perform C3 photosynthesis.
This thesis compares photosynthetic metabolism in several species with C3, C4 and intermediate modes of photosynthesis. Metabolite profiling and stable isotope labelling were performed to detect inter-specific differences changes in metabolite profile and, hence, how a pathway operates. The results obtained were subjected to integrative data analyses like hierarchical clustering and principal component analysis, and were deepened by correlation analyses to uncover specific metabolic features and reaction steps that were conserved or differed between species.
The main findings are that Calvin-Benson cycle metabolite profiles differ between C3 and C4 species and between different C3 species, including a very different response to rising irradiance in Arabidopsis and rice. These findings confirm Calvin-Benson cycle operation diverged between C3 and C4 species and, most unexpectedly, even between different C3 species. Moreover, primary metabolic profiles supported the current C4 evolutionary model in the genus Flaveria and also provided new insights and opened up new questions. Metabolite profiles also point toward a progressive adjustment of the Calvin-Benson cycle during the evolution of C4 photosynthesis. Overall, this thesis point out the importance of a metabolite-centric approach to uncover underlying differences in species apparently sharing the same photosynthetic routes and as a valid method to investigate evolutionary transition between C3 and C4 photosynthesis.
Identification of chemical mediators that regulate the specialized metabolism in Nostoc punctiforme
(2021)
Specialized metabolites, so-called natural products, are produced by a variety of different organisms, including bacteria and fungi. Due to their wide range of different biological activities, including pharmaceutical relevant properties, microbial natural products are an important source for drug development. They are encoded by biosynthetic gene clusters (BGCs), which are a group of locally clustered genes. By screening genomic data for genes encoding typical core biosynthetic enzymes, modern bioinformatical approaches are able to predict a wide range of BGCs. To date, only a small fraction of the predicted BGCs have their associated products identified.
The phylum of the cyanobacteria has been shown to be a prolific, but largely untapped source for natural products. Especially multicellular cyanobacterial genera, like Nostoc, harbor a high amount of BGCs in their genomes.
A main goal of this study was to develop new concepts for the discovery of natural products in cyanobacteria. Due to its diverse setup of orphan BGCs and its amenability to genetic manipulation, Nostoc punctiforme PCC 73102 (N. punctiforme) appeared to be a promising candidate to be established as a model organism for natural product discovery in cyanobacteria. By utilizing a combination of genome-mining, bioactivity-screening, variations of culture conditions, as well as metabolic engineering, not only two new polyketides were discovered, but also first-time insights into the regulation of the specialized metabolism in N. punctiforme were gained during this study.
The cultivation of N. punctiforme to very high densities by utilizing increasing light intensities and CO2 levels, led to an enhanced metabolite production, causing rather complex metabolite extracts. By utilizing a library of CFP reporter mutant strains, each strain reporting for one of the predicted BGCs, it was shown that eight out of 15 BGCs were upregulated under high density (HD) cultivation conditions. Furthermore, it could be demonstrated that the supernatant of an HD culture can increase the expression of four of the influenced BGCs, even under conventional cultivation conditions. This led to the hypothesis that a chemical mediator encoded by one of the affected BGCs is accumulating in the HD supernatant and is able to increase the expression of other BGCs as part of a cell-density dependent regulatory circuit. To identify which of the BGCs could be a main trigger of the presumed regulatory circuit, it was tried to activate four BGCs (pks1, pks2, ripp3, ripp4) selectively by overexpression of putative pathway-specific regulatory genes that were found inside the gene clusters. Transcriptional analysis of the mutants revealed that only the mutant strain targeting the pks1 BGC, called AraC_PKS1, was able to upregulate the expression of its associated BGC. From an RNA sequencing study of the AraC_PKS1 mutant strain, it was discovered that beside pks1, the orphan BGCs ripp3 and ripp4 were also upregulated in the mutant strain. Furthermore, it was observed that secondary metabolite production in the AraC_PKS1 mutant strain is further enhanced under high-light and high-CO2 cultivation conditions. The increased production of the pks1 regulator NvlA also had an impact on other regulatory factors, including sigma factors and the RNA chaperone Hfq. Analysis of the AraC_PKS1 cell and supernatant extracts led to the discovery of two novel polyketides, nostoclide and nostovalerolactone, both encoded by the pks1 BGC. Addition of the polyketides to N. punctiforme WT demonstrated that the pks1-derived compounds are able to partly reproduce the effects on secondary metabolite production found in the AraC_PKS1 mutant strain. This indicates that both compounds are acting as extracellular signaling factors as part of a regulatory network. Since not all transcriptional effects that were found in the AraC_PKS1 mutant strain could be reproduced by the pks1 products, it can be assumed that the regulator NvlA has a global effect and is not exclusively specific to the pks1 pathway.
This study was the first to use a putative pathway specific regulator for the specific activation of BGC expression in cyanobacteria. This strategy did not only lead to the detection of two novel polyketides, it also gave first-time insights into the regulatory mechanism of the specialized metabolism in N. punctiforme. This study illustrates that understanding regulatory pathways can aid in the discovery of novel natural products. The findings of this study can guide the design of new screening strategies for bioactive compounds in cyanobacteria and help to develop high-titer production platforms for cyanobacterial natural products.
Iron-sulfur clusters are essential enzyme cofactors. The most common and stable clusters are [2Fe-2S] and [4Fe-4S] that are found in nature. They are involved in crucial biological processes like respiration, gene regulation, protein translation, replication and DNA repair in prokaryotes and eukaryotes. In Escherichia coli, Fe-S clusters are essential for molybdenum cofactor (Moco) biosynthesis, which is a ubiquitous and highly conserved pathway. The first step of Moco biosynthesis is catalyzed by the MoaA protein to produce cyclic pyranopterin monophosphate (cPMP) from 5’GTP. MoaA is a [4Fe-4S] cluster containing radical S-adenosyl-L-methionine (SAM) enzyme. The focus of this study was to investigate Fe-S cluster insertion into MoaA under nitrate and TMAO respiratory conditions using E. coli as a model organism. Nitrate and TMAO respiration usually occur under anaerobic conditions, where oxygen is depleted. Under these conditions, E. coli uses nitrate and TMAO as terminal electron. Previous studies revealed that Fe-S cluster insertion is performed by Fe-S cluster carrier proteins. In E. coli, these proteins are known as A-type carrier proteins (ATC) by phylogenomic and genetic studies. So far, three of them have been characterized in detail in E. coli, namely IscA, SufA, and ErpA. This study shows that ErpA and IscA are involved in Fe-S cluster insertion into MoaA under nitrate and TMAO respiratory conditions. ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. SufA is not able to replace the functions of IscA or ErpA under nitrate respiratory conditions.
Nitrate reductase is a molybdoenzyme that coordinates Moco and Fe-S clusters. Under nitrate respiratory conditions, the expression of nitrate reductase is significantly increased in E. coli. Nitrate reductase is encoded in narGHJI genes, the expression of which is regulated by the transcriptional regulator, fumarate and nitrate reduction (FNR). The activation of FNR under conditions of nitrate respiration requires one [4Fe-4S] cluster. In this part of the study, we analyzed the insertion of Fe-S cluster into FNR for the expression of narGHJI genes in E. coli. The results indicate that ErpA is essential for the FNR-dependent expression of the narGHJI genes, a role that can be replaced partially by IscA and SufA when they are produced sufficiently under the conditions tested. This observation suggests that ErpA is indirectly regulating nitrate reductase expression via inserting Fe-S clusters into FNR.
Most molybdoenzymes are complex multi-subunit and multi-cofactor-containing enzymes that coordinate Fe-S clusters, which are functioning as electron transfer chains for catalysis. In E. coli, periplasmic aldehyde oxidoreductase (PaoAC) is a heterotrimeric molybdoenzyme that
consists of flavin, two [2Fe-2S], one [4Fe-4S] cluster and Moco. In the last part of this study, we investigated the insertion of Fe-S clusters into E. coli periplasmic aldehyde oxidoreductase (PaoAC). The results show that SufA and ErpA are involved in inserting [4Fe-4S] and [2Fe-2S] clusters into PaoABC, respectively under aerobic respiratory conditions.
Influenza A virus (IAV) is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. During the viral assembly process in the infected cells, the plasma membrane (PM) has to bend in localized regions into a vesicle towards the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. M1 is the most abundant protein in IAV particles. It plays an important role in virus assembly and budding at the PM. M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. However, the details of M1 interactions with the cellular PM, as well as M1-mediated membrane bending at the budozone, have not been clarified.
In this work, we used several experimental approaches to analyze M1-lipids and M1-M1 interactions. By performing SPR analysis, we quantified membrane association for full-length M1 and different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region). This allowed us to obtain novel information on the protein regions mediating M1 binding to membranes. By using fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), and three-dimensional (3D) tomography (cryo-ET), we showed that M1 is indeed able to cause membrane deformation on vesicles containing negatively-charged lipids, in the absence of other viral components. Further, sFCS analysis proved that simple protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1-M1 interactions and multimer formation are required to alter the bilayer three-dimensional structure through the formation of a protein scaffold.
Finally, to mimic the budding mechanism in cells that arise by the lateral organization of the virus membrane components on lipid raft domains, we created vesicles with lipid domains. Our results showed that local binding of M1 to spatial confined acidic lipids within membrane domains of vesicles led to local M1 inward curvature.
Sensing and Responding of Cardiomyocytes to Changes of Tissue Stiffness in the Diseased Heart
(2021)
Cardiomyocytes are permanently exposed to mechanical stimulation due to cardiac contractility. Passive myocardial stiffness is a crucial factor, which defines the physiological ventricular compliance and volume of diastolic filling with blood. Heart diseases often present with increased myocardial stiffness, for instance when fibrotic changes modify the composition of the cardiac extracellular matrix (ECM). Consequently, the ventricle loses its compliance, and the diastolic blood volume is reduced. Recent advances in the field of cardiac mechanobiology revealed that disease-related environmental stiffness changes cause severe alterations in cardiomyocyte cellular behavior and function. Here, we review the molecular mechanotransduction pathways that enable cardiomyocytes to sense stiffness changes and translate those into an altered gene expression. We will also summarize current knowledge about when myocardial stiffness increases in the diseased heart. Sophisticated in vitro studies revealed functional changes, when cardiomyocytes faced a stiffer matrix. Finally, we will highlight recent studies that described modulations of cardiac stiffness and thus myocardial performance in vivo. Mechanobiology research is just at the cusp of systematic investigations related to mechanical changes in the diseased heart but what is known already makes way for new therapeutic approaches in regenerative biology.
Trait variation among heterospecific and conspecific organisms may substantially affect community and food web dynamics. While the relevance of competition and feeding traits have been widely studied for different consumer species, studies on intraspecific differences are more scarce, partly owing to difficulties in distinguishing different clones of the same species. Here, we investigate how intraspecific trait variation affects the competition between the freshwater ciliates Euplotes octocarinatus and Coleps hirtus in a nitrogen-limited chemostat system. The ciliates competed for the microalgae Cryptomonas sp. (Cry) and Navicula pelliculosa (Nav), and the bacteria present in the cultures over a period of 33 days. We used monoclonal Euplotes and three different Coleps clones (Col 1, Col 2, and Col 3) in the experiment that could be distinguished by a newly developed rDNA-based molecular assay based on the internal transcribed spacer (ITS) regions. While Euplotes feeds on Cry and on bacteria, the Coleps clones cannot survive on bacteria alone but feed on both Cry and Nav with clone-specific rates. Experimental treatments comprised two-species mixtures of Euplotes and one or all of the three different Coleps clones, respectively. We found intraspecific variation in the traits "selectivity" and "maximum ingestion rate" for the different algae to significantly affect the competitive outcome between the two ciliate species. As Nav quickly escaped top-down control and likely reached a state of low food quality, ciliate competition was strongly determined by the preference of different Coleps clones for Cry as opposed to feeding on Nav. In addition, the ability of Euplotes to use bacteria as an alternative food source strengthened its persistence once Cry was depleted. Hence, trait variation at both trophic levels codetermined the population dynamics and the outcome of species competition.
Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing andmatching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the A(core) domains, yet domain interfaces and the flexible A(sub) domain remained untapped. Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches.
Dissecting the tree of life
(2021)
Social organisation in species with fluctuating population sizes can change with density. Therefore, information on (future) density obtained during early life stages may be associated with social behaviour. Olfactory cues may carry important social information. We investigated whether early life experience of different experimental densities was subsequently associated with differences in attraction to adult conspecific odours. We used common voles (Microtus arvalis), a rodent species undergoing extreme density fluctuations. We found that individuals originating from high experimental density populations kept in large outdoor enclosures invested more time in inspecting conspecific olfactory cues than individuals from low-density populations. Generally, voles from both treatments spent more time with the olfactory cues than expected by chance and did not differ in their latency to approach the odour samples. Our findings indicate either that early experience affects odour sensitivity or that animals evaluate the social information contained in conspecific odours differently, depending on their early life experience of conspecific density.
Reliably modelling the demographic and distributional responses of a species to environmental changes can be crucial for successful conservation and management planning. Process-based models have the potential to achieve this goal, but so far they remain underused for predictions of species' distributions. Individual-based models offer the additional capability to model inter-individual variation and evolutionary dynamics and thus capture adaptive responses to environmental change. We present RangeShiftR, an R implementation of a flexible individual-based modelling platform which simulates eco-evolutionary dynamics in a spatially explicit way. The package provides flexible and fast simulations by making the software RangeShifter available for the widely used statistical programming platform R. The package features additional auxiliary functions to support model specification and analysis of results. We provide an outline of the package's functionality, describe the underlying model structure with its main components and present a short example. RangeShiftR offers substantial model complexity, especially for the demographic and dispersal processes. It comes with elaborate tutorials and comprehensive documentation to facilitate learning the software and provide help at all levels. As the core code is implemented in C++, the computations are fast. The complete source code is published under a public licence, making adaptations and contributions feasible. The RangeShiftR package facilitates the application of individual-based and mechanistic modelling to eco-evolutionary questions by operating a flexible and powerful simulation model from R. It allows effortless interoperation with existing packages to create streamlined workflows that can include data preparation, integrated model specification and results analysis. Moreover, the implementation in R strengthens the potential for coupling RangeShiftR with other models.
Microcystis is the most commonly found toxic cyanobacterial genus around the world and has a negative impact on the ecosystem. As a predominant producer of the potent hepatotoxin microcystin (MC), the genus causes outbreaks in freshwaters worldwide. Standard analytical methods that are used for the detection of microcystin variants can only measure the free form of microcystin in cells. Since microcystin was found as free and proteinbound forms in the cells, a significant proportion of microcystin is underestimated with analytical methods. The aim of the study was to measure protein-bound microcystins and determine the environmental factors that affect the binding of microcystin to proteins. Samples were taken at depths of surface, 1 m, 5 m, 10 m, 15 m, and 18 m in Kucukcekmece Lagoon to analyze depth profiles of two different microcystin forms from June to September 2012 at regular monthly intervals. Our findings suggest that the most important parameter affecting proteinbound microcystin at surface water is high light. Due to favorable environmental conditions such as temperature, light, and physicochemical parameters, the higher microcystin contents, both free and protein-bound MCs, were found in summer periods.
Sensing and responding of cardiomyocytes to changes of tissue stiffness in the diseased heart
(2021)
Cardiomyocytes are permanently exposed to mechanical stimulation due to cardiac contractility. Passive myocardial stiffness is a crucial factor, which defines the physiological ventricular compliance and volume of diastolic filling with blood. Heart diseases often present with increased myocardial stiffness, for instance when fibrotic changes modify the composition of the cardiac extracellular matrix (ECM). Consequently, the ventricle loses its compliance, and the diastolic blood volume is reduced. Recent advances in the field of cardiac mechanobiology revealed that disease-related environmental stiffness changes cause severe alterations in cardiomyocyte cellular behavior and function. Here, we review the molecular mechanotransduction pathways that enable cardiomyocytes to sense stiffness changes and translate those into an altered gene expression. We will also summarize current knowledge about when myocardial stiffness increases in the diseased heart. Sophisticated in vitro studies revealed functional changes, when cardiomyocytes faced a stiffer matrix. Finally, we will highlight recent studies that described modulations of cardiac stiffness and thus myocardial performance in vivo. Mechanobiology research is just at the cusp of systematic investigations related to mechanical changes in the diseased heart but what is known already makes way for new therapeutic approaches in regenerative biology.
Floral volatiles and reward traits are major drivers for the behavior of mutualistic as well as antagonistic flower visitors, i.e., pollinators and florivores. These floral traits differ tremendously between species, but intraspecific differences and their consequences on organism interactions remain largely unknown. Floral volatile compounds, such as terpenoids, function as cues to advertise rewards to pollinators, but should at the same time also repel florivores. The reward composition, e.g., protein and lipid contents in pollen, differs between individuals of distinct plant families. Whether the nutritional value of rewards within the same plant species is linked to their chemotypes, which differ in their pattern of specialized metabolites, has yet not been investigated. In the present study, we compared Tanacetum vulgare plants of five terpenoid chemotypes with regard to flower production, floral headspace volatiles, pollen macronutrient and terpenoid content, and floral attractiveness to florivorous beetles. Our analyses revealed remarkable differences between the chemotypes in the amount and diameter of flower heads, duration of bloom period, and pollen nutritional quality. The floral headspace composition of pollen-producing mature flowers, but not of premature flowers, was correlated to that of pollen and leaves in the same plant individual. For two chemotypes, florivorous beetles discriminated between the scent of mature and premature flower heads and preferred the latter. In semi-field experiments, the abundance of florivorous beetles and flower tissue miners differed between T. vulgare chemotypes. Moreover, the scent environment affected the choice and beetles were more abundant in homogenous plots composed of one single chemotype than in plots with different neighboring chemotypes. In conclusion, flower production, floral metabolic composition and pollen quality varied to a remarkable extend within the species T. vulgare, and the attractiveness of floral scent differed also intra-individually with floral ontogeny. We found evidence for a trade-off between pollen lipid content and pollen amount on a per-plant-level. Our study highlights that chemotypes which are more susceptible to florivory are less attacked when they grow in the neighborhood of other chemotypes and thus gain a benefit from high overall chemodiversity.
There is an urgent need for screening of patients with a communicable viral disease to cut infection chains. Recently, we demonstrated that ion mobility spectrometry coupled with a multicapillary column (MCC-IMS) is able to identify influenza-A infections in patients' breath. With a decreasing influenza epidemic and upcoming SARS-CoV-2 infections we proceeded further and analyzed patients with suspected SARS-CoV-2 infections. In this study, the nasal breath of 75 patients (34 male, 41 female, aged 64.4 +/- 15.4 years) was investigated by MCC-IMS for viral infections. Fourteen were positively diagnosed with influenza-A infection and sixteen with SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR) of nasopharyngeal swabs. In one patient RT-PCR was highly suspicious of SARS-CoV-2 but initially inconclusive. The remaining 44 patients served as controls. Breath fingerprints for specific infections were assessed by a combination of cluster analysis and multivariate statistics. There were no significant differences in gender or age according to the groups. In the cross validation of the discriminant analysis 72 of the 74 clearly defined patients could be correctly classified to the respective group. Even the inconclusive patient could be mapped to the SARS-CoV-2 group by applying the discrimination functions. Conclusion: SARS-CoV-2 infection and influenza-A infection can be detected with the help of MCC-IMS in breath in this pilot study. As this method provides a fast non-invasive diagnosis it should be further developed in a larger cohort for screening of communicable viral diseases. A validation study is ongoing during the second wave of COVID-19.
Trial registration: ClinicalTrial.gov, NCT04282135 Registered 20 February 2020-Retrospectively registered,
Elaeidobius kamerunicus Faust. (Coleoptera: Curculionidae) is an essential insect pollinator in oil palm plantations. Recently, researches have been undertaken to improve pollination efficiency using this species. A fundamental understanding of the genes related to this pollinator behavior is necessary to achieve this goal. Here, we present the draft genome sequence, annotation, and simple sequence repeat (SSR) marker data for this pollinator. In total, 34.97 Gb of sequence data from one male individual (monoisolate) were obtained using Illumina short-read platform NextSeq 500. The draft genome assembly was found to be 269.79 Mb and about 59.9% of completeness based on Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment. Functional gene annotation predicted about 26.566 genes. Also, a total of 281.668 putative SSR markers were identified. This draft genome sequence is a valuable resource for understanding the population genetics, phylogenetics, dispersal patterns, and behavior of this species.
Seagrass beds are important habitats in coastal areas but increasingly decline in area and quality, thus conservation measures are urgently needed. Quantitative food webs, describing the biomass distribution and energy fluxes among trophic groups, reveal structural and functional aspects of ecosystems. Their knowledge can improve ecological conservation. For the recently discovered large warm-temperate seagrass (Zostera japonica) habitat in China's Yellow River Delta wetland, we used delta C-13 and delta N-15 measurements and a Bayesian isotope mixing model to construct its food web diagram with quantitative estimations of consumer diet compositions, comprising detritus and 14 living trophic groups from primary producers to fish. We then estimated the quantitative food web fluxes based on biomass measurements and calculated corresponding ecosystem functions. Pelagic producers were significantly C-13-depleted compared to benthic sources. Consumers (except zooplankton) were increasingly C-13-depleted with increasing trophic positions even though the consumed benthic production surpassed the pelagic one. Bivalves dominated consumer biomasses and fluxes and were the first to connect the pelagic and benthic pathways, whereas zooplankton and gastropods were specialized on the two pathways, respectively. We found flat biomass and production pyramids indicating low trophic transfer efficiencies. Generally, the energetic structure of the quantitative food web was consistent with the stable isotope analysis, and the estimated net primary production and most estimated production to biomass ratios of the trophic groups fell within literature ranges. This study provides a systematical understanding of the quantitative trophic ecology of a seagrass bed and facilitates synergistic knowledge on management, conservation, and restoration.
In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the bla(CTX-M15) gene in less than 1.6E-03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.
City mice and country mice
(2021)
The ability to produce innovative behaviour is a key determinant in the successful coping with environmental challenges and changes. The expansion of human-altered environments presents wildlife with multiple novel situations in which innovativeness could be beneficial. A better understanding of the drivers of within-species variation in innovation propensity and its consequences will provide insights into the traits enabling animals to thrive in the face of human-induced rapid environmental change. We compared problem-solving performance of 31 striped field mice, Apodemus agrarius, originating from rural or urban environments in a battery of eight foraging extraction tasks. We tested whether differences in problem-solving performance were mediated by the extent and duration of the animal's exploration of the experimental set-ups, the time required to solve the tasks, and their persistence. In addition, we tested the influence of the diversity of motor responses, as well as of behavioural traits boldness and activity on problem-solving performance. Urban individuals were better problem solvers despite rural individuals approaching faster and interacting longer with the test set-ups. Participation rates and time required to solve a task did not differ between rural and urban individuals. However, in case of failure to solve a task, rural mice were more persistent. The best predictors of solving success, aside from the area of origin, were the time spent exploring the set-ups and boldness, while activity and diversity of motor responses did not explain it. Problem-solving ability could thus be a contributing factor to the successful coping with the rapid and recent expansion of human-altered environments.
Das Fachwissen von Lehrkräften weist für die Ausprägung fachdidaktischer Expertise eine hohe Bedeutung auf. Welche Merkmale universitäre Lehrveranstaltungen aufweisen sollten, um Lehramtsstudierenden ein berufsspezifisches Fachwissen zu vermitteln, ist jedoch überwiegend noch unklar.
Innerhalb des Projekts PSI-Potsdam wurde auf theoretischer Grundlage das fachübergreifende Modell des erweiterten Fachwissens für den schulischen Kontext entwickelt. Als Ansatz zur Verbesserung des Biologie-Lehramtsstudiums diente dieses Modell als Konzeptionsgrundlage für eine additive Lehrveranstaltung. Hierbei werden Lerngelegenheiten geboten, um das universitär erworbene Fachwissen über zellbiologische Inhalte auf schulische Kontexte anzuwenden, z.B. durch die Dekonstruktion und anschließende Rekonstruktion von schulischen Lerntexten. Die Wirkung des Seminars wurde in mehreren Zyklen im Forschungsformat der Fachdidaktischen Entwicklungsforschung beforscht. Eine der zentralen Forschungsfragen lautet dabei: Wie kann eine Lerngelegenheit für Lehramtsstudierende der Biologie gestaltet sein, um ein erweitertes Fachwissen für den schulischen Kontext für den zellbiologischen Themenbereich „Struktur und Funktion der Biomembran“ zu fördern?
Anhand fallübergreifender Analysen (n = 29) wird im empirischen Teil aufgezeigt, welche Einstellungen zum Lehramtsstudium in der Stichprobe bestehen. Als ein wichtiges Ergebnis kann hierbei herausgestellt werden, dass sich das Fachinteresse hinsichtlich schulisch und universitär vermittelter Inhalte bei den untersuchten Studierenden auffallend unterscheidet, wobei dem Schulwissen ein deutlich höheres Interesse entgegengebracht wird. Die Berufsrelevanz fachlicher Inhalte wird seitens der Studierenden häufig am Schulwissen festgemacht.
Innerhalb konkreter Einzelfallanalysen (n = 6) wird anhand von Lernpfaden dargestellt, wie sich über mehrere Design-Experimente hinweg fachliche Konzepte entwickelt haben. Bei der Beschreibung wird vor allem auf Schlüsselstellen und Hürden im Lernprozess fokussiert. Aus diesen Ergebnissen folgend werden vorgenommene Iterationen für die einzelnen Zyklen beschrieben, die ebenfalls anhand der iterativen Entwicklung der Design-Prinzipien dargelegt werden.
Es konnte gezeigt werden, dass die Schlüsselstellen sehr individuell aufgrund der subjektiv fokussierten Inhalte zu Tage treten. Meist treten sie jedoch im Zusammenhang mit der Verknüpfung verschiedener fachlicher Konzepte oder durch kooperative Aufschlüsselungen von Konzepten auf. Fachliche Hürden konnten hingegen in Form von fachlich unangemessenen Vorstellungen fallübergreifend identifiziert werden. Dies betrifft unter anderem die Vorstellung der Biomembran als Wand, die mit den Vorstellungen einer Schutzfunktion und einer formgebenden Funktion der Biomembran einhergeht.
Weiterhin wird beleuchtet, wie das erweiterte Fachwissen für den schulischen Kontext zur Bearbeitung der Lernaufgaben angewendet wurde. Es hat sich gezeigt, dass sich bestimmte Lerngelegenheiten eigenen, um bestimmte Facetten des erweiterten Fachwissens zu fördern.
Insgesamt scheint das Modell des erweiterten Fachwissens für den schulischen Kontext äußerst geeignet zu sein, um anhand der Facetten und deren Beschreibungen Lerngelegenheiten oder Gestaltungsprinzipien für diese zu konzipieren. Für das untersuchte Lehr-Lernarrangement haben sich kleinere Adaptationen des Modells als sinnvoll erwiesen. Hinsichtlich der Methodologie konnten Ableitungen für die Anwendung der fachdidaktischen Entwicklungsforschung für additive fachliche Lehrveranstaltungen dieser Art herausgestellt werden.
Um den Professionsbezug der fachwissenschaftlichen Anteile im Lehramtsstudium zu verbessern, ist der weitere Einbezug des erweiterten Fachwissens für den schulischen Kontext in die fachwissenschaftlichen Studienanteile überaus wünschenswert.
There has been a growing awareness that graphing is an essential part of the science curriculum. While much research has focused on student conceptions and abilities regarding graphical representations, only few studies have investigated what teachers think about them and how they use graphs in science class. The purpose of this study is to explore educational beliefs, motivation, and teaching practices of German secondary biology teachers regarding graph construction. Via questionnaire surveys, 71 teachers from different regions in Germany rated their beliefs and motivation as well as the frequency of different graph construction activities in biology class. The teachers surveyed in this study were quite motivated in their teaching of graph construction. Furthermore, they tended to believe that graph construction should be practiced explicitly in biology class and that students should learn clear strategies for constructing graphs. We found that teaching subjects and own research experience make a difference in teachers' beliefs and motivation regarding graph construction in biology class. The self-report on classroom practices revealed that participants may provide limited opportunities for students to experience graphing as a social and iterative practice. Implications are drawn for teacher education and professional development as well as for further research in teacher education contexts.
The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs.
Purpose of review
The zebrafish embryo has emerged as a powerful model organism to investigate the mechanisms by which biophysical forces regulate vascular and cardiac cell biology during development and disease. A versatile arsenal of methods and tools is available to manipulate and analyze biomechanical signaling. This review aims to provide an overview of the experimental strategies and tools that have been utilized to study biomechanical signaling in cardiovascular developmental processes and different vascular disease models in the zebrafish embryo. Within the scope of this review, we focus on work published during the last two years.
Recent findings
Genetic and pharmacological tools for the manipulation of cardiac function allow alterations of hemodynamic flow patterns in the zebrafish embryo and various types of transgenic lines are available to report endothelial cell responses to biophysical forces. These tools have not only revealed the impact of biophysical forces on cardiovascular development but also helped to establish more accurate models for cardiovascular diseases including cerebral cavernous malformations, hereditary hemorrhagic telangiectasias, arteriovenous malformations, and lymphangiopathies.
Summary
The zebrafish embryo is a valuable vertebrate model in which in-vivo manipulations of biophysical forces due to cardiac contractility and blood flow can be performed. These analyses give important insights into biomechanical signaling pathways that control endothelial and endocardial cell behaviors. The technical advances using this vertebrate model will advance our understanding of the impact of biophysical forces in cardiovascular pathologies.
Deoxyribonucleic acid (DNA) nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions.
This dissertation covers three main projects. All of them use variations of functionalized DNA nanostructures that act as platform for oligovalent presentation of ligands. The purpose of this work was to evaluate the ability of DNA nanostructures to precisely display different types of functional molecules and to consequently enhance their efficacy according to the concept of multivalency. Moreover, functionalized DNA structures were examined for their suitability in functional screening assays. The developed DNA-based compound ligands were used to target structures in different biological systems.
One part of this dissertation attempted to bind pathogens with small modified DNA nanostructures. Pathogens like viruses and bacteria are known for their multivalent attachment to host cells membranes. By blocking their receptors for recognition and/or fusion with their targeted host in an oligovalent manner, the objective was to impede their ability to adhere to and invade cells. For influenza A, only enhanced binding of oligovalent peptide-DNA constructs compared to the monovalent peptide could be observed, whereas in the case of respiratory syncytial virus (RSV), binding as well as blocking of the target receptors led to an increased inhibition of infection in vitro.
In the final part, the ability of chimeric DNA-peptide constructs to bind to and activate signaling receptors on the surface of cells was investigated. Specific binding of DNA trimers, conjugated with up to three peptides, to EphA2 receptor expressing cells was evaluated in flow cytometry experiments. Subsequently, their ability to activate these receptors via phosphorylation was assessed. EphA2 phosphorylation was significantly increased by DNA trimers carrying three peptides compared to monovalent peptide. As a result of activation, cells underwent characteristic morphological changes, where they "round up" and retract their periphery.
The results obtained in this work comprehensively prove the capability of DNA nanostructures to serve as stable, biocompatible, controllable platforms for the oligovalent presentation of functional ligands. Functionalized DNA nanostructures were used to enhance biological effects and as tool for functional screening of bio-activity. This work demonstrates that modified DNA structures have the potential to improve drug development and to unravel the activation of signaling pathways.
In the zebrafish embryo, the onset of blood flow generates fluid shear stress on endocardial cells, which are specialized endothelial cells that line the interior of the heart. High levels of fluid shear stress activate both Notch and Klf2 signaling, which play crucial roles in atrioventricular valvulogenesis. However, it remains unclear why only individual endocardial cells ingress into the cardiac jelly and initiate valvulogenesis. Here, we show that lateral inhibition between endocardial cells, mediated by Notch, singles out Delta-like-4-positive endocardial cells. These cells ingress into the cardiac jelly, where they form an abluminal cell population. Delta-like-4-positive cells ingress in response to Wnt9a, which is produced in parallel through an Erk5Klf2-Wnt9a signaling cascade also activated by blood flow. Hence, mechanical stimulation activates parallel mechanosensitive signaling pathways that produce binary effects by driving endocardial cells toward either luminal or abluminal fates. Ultimately, these cell fate decisions sculpt cardiac valve leaflets.
Monoklonale Antikörper sind essenzielle Werkzeuge in der modernen Laboranalytik sowie in der medizinischen Therapie und Diagnostik. Die Herstellung monoklonaler Antikörper ist ein zeit- und arbeitsintensiver Prozess. Herkömmliche Methoden beruhen auf der Immunisierung von Labortieren, die mitunter mehrere Monate in Anspruch nimmt. Anschließend werden die Antikörper-produzierenden B-Lymphozyten bzw. deren Antikörpergene isoliert und in Screening-Verfahren untersucht, um geeignete Binder zu identifizieren.
Der Transfer der humoralen Immunantwort in eine in vitro Umgebung erlaubt eine Verkürzung des Prozesses und umgeht die Notwendigkeit der in vivo Immunisierung. Das komplexe Zusammenspiel aller involvierten Immunzellen in vitro abzubilden, stellt sich allerdings als schwierig dar. Der Schwerpunkt dieser Arbeit war deshalb die Realisierung einer vereinfachten In vitro Immunisierung, die sich auf die Protagonisten der Antikörper-Produktion konzentriert: die B-Lymphozyten. Darüber hinaus sollte eine permanente Zelllinie etabliert werden, die zur Antikörper-Herstellung eingesetzt werden und die Verwendung von Primärzellen ersetzen würde.
Im ersten Teil der Arbeit wurde ein Protokoll zur In vitro Immunisierung muriner BLymphozyten etabliert. In Vorversuchen wurden die optimalen Konditionen für die Antigenspezifische Aktivierung gereinigter Milz-B-Lymphozyten aus nicht-immunisierten Mäusen
determiniert. Dazu wurde der Einfluss verschiedener Stimuli auf die Produktion unspezifischer und spezifischer Antikörper untersucht. Eine Kombination aus dem Modellantigen VP1 (Hamster Polyomavirus Hüllprotein 1), einem Anti-CD40-Antikörper, Interleukin 4 (IL 4) und Lipopolysaccharid (LPS) oder IL 7 induzierte nachweislich eine Antigen-spezifische Antikörper-Antwort in vitro. Als Indikatoren einer erfolgreichen Aktivierung der B-Lymphozyten infolge der in vitro Stimulation wurden die rapide Proliferation und die Expression charakteristischer Aktivierungsmarker auf der Zelloberfläche nachgewiesen. In einer Zeitreihe über zehn Tage wurde am zehnten Tag der In vitro Immunisierung die verhältnismäßig höchste Konzentration Antigen-spezifischer IgG-Antikörper im Kulturüberstand der stimulierten Zellen nachgewiesen.
Als nächster Schritt sollte eine permanente Zelllinie hergestellt werden, die statt primärer BLymphozyten für die zuvor etablierte In vitro Immunisierung eingesetzt werden könnte. Zu diesem Zweck wurden retrovirale Vektoren hergestellt, die durch den Transfer verschiedener Onkogene in murine B-Lymphozyten bzw. deren Vorläuferzellen das Proliferationsverhalten der Zellen manipulieren sollen. Es wurden Retroviren mit Doxycyclin-induzierbaren Expressionskassetten mit den Onkogenen cmyc, Bcl2, BclxL und dem Fusionsgen NUP98HOXB4 generiert. Eine Testzelllinie wurde erfolgreich mit den hergestellten Retroviren transduziert und die Funktionalität der hergestellten Viren anhand verschiedener Assays belegt. Die transferierten Gene konnten in der Testzelllinie auf DNAEbene nachgewiesen oder die Überexpression der entsprechenden Proteine im Western Blot detektiert werden. Es wurden schließlich B-Lymphozyten bzw. unreife Vorläuferzellen derselben mit den generierten Retroviren transduziert und mit Knochenmark-ähnlichen Stromazellen co-kultiviert. Aus keinem der transduzierten Ansätze konnte bisher eine Zelllinie oder eine Langzeit-Kultur etabliert werden.
Im letzten Teil der Arbeit wurde die Effektivität und Übertragbarkeit des zuvor etablierten Protokolls zur In vitro Immunisierung muriner B-Lymphozyten anhand verschiedener Antigene gezeigt. Es konnten in vitro spezifische IgG-Antworten gegen VP1, Legionella pneumophila und das Protein Mip, von dem ein Peptid in das zur Immunisierung eingesetzte VP1 integriert wurde, induziert werden. Die stimulierten B-Lymphozyten wurden durch Fusion mit Myelomzellen in permanente Antikörper-produzierende Zelllinien transformiert.
Dabei konnten mehrere Hybridomzelllinien generiert werden, die spezifische IgGAntikörper gegen VP1 oder Mip produzieren. Die generierten Antikörper konnten sowohl im Western Blot als auch im ELISA (Enzyme-Linked Immunosorbent Assay) das entsprechende Antigen spezifisch binden.
Die hier etablierte In vitro Immunisierung bietet eine effektive Alternative zu bisherigen Verfahren zur Herstellung spezifischer Antikörper. Sie ersetzt die Immunisierung von Versuchstieren und reduziert den Zeitaufwand erheblich. In Kombination mit der Hybridomtechnologie können die in vitro immunisierten Zellen, wie hier demonstriert, zur Generation von Hybridomzelllinien und zur Herstellung monoklonaler Antikörper genutzt werden. Um die Verwendung von Versuchstieren in dieser Methode durch eine adäquate permanente Zelllinie zu ersetzen, muss die genetische Veränderung von B-Lymphozyten und unreifen hämatopoetischen Zellen optimiert werden. Die Ergebnisse bieten eine Basis für eine universelle, Spezies-unabhängige Methodik zur Antikörperherstellung und für die
Etablierung einer idealen, tierfreien In vitro Immunisierung.
Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.
Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.
Generating a monoclonal antibody to date is a time intense process that requires immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings enables a shortening of this process and circumvents the necessity of in vivo immunization. However, to orchestrate the complex interplay of dendritic cells, T and B lymphocytes in vitro is very challenging. We therefore aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes alone in vitro by using combinations of antigen and stimuli. We were able to induce a specific antibody response within ten days of culture against a viral coat protein as model antigen. Antibodies were of both IgM and IgG subclass. The stimulated B lymphocytes were transformed into permanently antibody-producing hybridomas by cell fusion technology. We furthermore used this method to induce a specific antibody response against L. pneumophila in vitro. We thus established a useful and effective in vitro protocol to generate monoclonal antibodies. By overcoming the necessity of in vivo immunization this protocol may be the first step towards a universal strategy to generate antibodies from various species.
Photodynamic therapy (PDT) is a mild but effective method to treat certain types of cancer upon irradiation with visible light. Here, three isomeric methylpyridinium alkynylanthracenes 1op were evaluated as sensitizers for PDT. Upon irradiation with blue or green light, all three compounds show the ability to initiate strand breaks of plasmid DNA. The mayor species responsible for cleavage is singlet oxygen (O-1(2)) as confirmed by scavenging reagents. Only isomers 1m and 1p can be incorporated into HeLa cells, whereas isomer 1o cannot permeate through the membrane. While isomer 1m targets the cell nucleus, isomer 1p assembles in the cellular cytoplasm and impacts the cellular integrity. This is in accordance with a moderate toxicity of 1p in the dark, whereas 1m exhibits no dark toxicity. Both isomers are suitable as PDT reagents, with a CC50 of 3 mu m and 75 nm, for 1p and 1m, respectively. Thus, derivative 1m, which can be easily synthesized, becomes an interesting candidate for cancer therapy.
Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical Sadenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression. <br /> IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.
Northern range margin populations of the European fire-bellied toad (Bombina bombina) have rapidly declined during recent decades. Extensive agricultural land use has fragmented the landscape, leading to habitat disruption and loss, as well as eutrophication of ponds. In Northern Germany (Schleswig-Holstein) and Southern Sweden (Skåne), this population decline resulted in decreased gene flow from surrounding populations, low genetic diversity, and a putative reduction in adaptive potential, leaving populations vulnerable to future environmental and climatic changes. Previous studies using mitochondrial control region and nuclear transcriptome-wide SNP data detected introgressive hybridization in multiple northern B. bombina populations after unreported release of toads from Austria. Here, we determine the impact of this introgression by comparing the body conditions (proxy for fitness) of introgressed and nonintrogressed populations and the genetic consequences in two candidate genes for putative local adaptation (the MHC II gene as part of the adaptive immune system and the stress response gene HSP70 kDa). We detected regional differences in body condition and observed significantly elevated levels of within individual MHC allele counts in introgressed Swedish populations, associated with a tendency toward higher body weight, relative to regional nonintrogressed populations. These differences were not observed among introgressed and nonintrogressed German populations. Genetic diversity in both MHC and HSP was generally lower in northern than Austrian populations. Our study sheds light on the potential benefits of translocations of more distantly related conspecifics as a means to increase adaptive genetic variability and fitness of genetically depauperate range margin populations without distortion of local adaptation.
Domestic cattle were brought to Spain by early settlers and agricultural societies. Due to missing Neolithic sites in the Spanish region of Galicia, very little is known about this process in this region. We sampled 18 cattle subfossils from different ages and different mountain caves in Galicia, of which 11 were subject to sequencing of the mitochondrial genome and phylogenetic analysis, to provide insight into the introduction of cattle to this region. We detected high similarity between samples from different time periods and were able to compare the time frame of the first domesticated cattle in Galicia to data from the connecting region of Cantabria to show a plausible connection between the Neolithization of these two regions. Our data shows a close relationship of the early domesticated cattle of Galicia and modern cow breeds and gives a general insight into cattle phylogeny. We conclude that settlers migrated to this region of Spain from Europe and introduced common European breeds to Galicia.
Domestic cattle were brought to Spain by early settlers and agricultural societies. Due to missing Neolithic sites in the Spanish region of Galicia, very little is known about this process in this region. We sampled 18 cattle subfossils from different ages and different mountain caves in Galicia, of which 11 were subject to sequencing of the mitochondrial genome and phylogenetic analysis, to provide insight into the introduction of cattle to this region. We detected high similarity between samples from different time periods and were able to compare the time frame of the first domesticated cattle in Galicia to data from the connecting region of Cantabria to show a plausible connection between the Neolithization of these two regions. Our data shows a close relationship of the early domesticated cattle of Galicia and modern cow breeds and gives a general insight into cattle phylogeny. We conclude that settlers migrated to this region of Spain from Europe and introduced common European breeds to Galicia.
Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.
Spectral detection enables multi-color fluorescence fluctuation spectroscopy studies in living cells
(2021)
Evidence of female preference for odor of distant over local males in a bat with female dispersal
(2021)
Geographic variation of sexually selected male traits is common in animals. Female choice also varies geographically and several studies found female preference for local males, which is assumed to lead to local adaptation and, therefore, increases fitness. As females are the nondispersing sex in most mammalian taxa, this preference for local males might be explained by the learning of male characteristics. Studies on the preference of females in female-dispersing species are lacking so far. To find out whether such females would also show preferences for local males, we conducted a study on greater sac-winged bats (Saccopteryx bilineata), a species where females disperse and males stay in their natal colony. Male greater sac-winged bats possess a wing pouch that is filled with odoriferous secretion and fanned toward females during courtship display. In a combination of chemical analysis and behavioral preference tests, we analyzed whether the composition of wing sac secretion varies between two geographically distinct populations (300 km), and whether females show a preference for local or distant male scent. Using gas chromatography, we found significant differences in the composition of the wing sac odors between the two geographically distinct populations. In addition, the behavioral preference experiments revealed that females of both populations preferred the scent of geographically distant males over local males. The wing sac odor might thus be used to guarantee optimal outbreeding when dispersing to a new colony. This is-to our knowledge-the first study on odor preference of females of a species with female-biased dispersal.
Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.
Finding sufficient natural fodder resources to feed livestock has become a challenge for herders in the Sahel zone of Burkina Faso. Despite the existence of pastoral reserves, the issue of fodder shortage remains unsolved. This article highlights the changes in behaviour and the evolution of pastoral practices caused by the scarcity of forage resources. These changes are defined and classified as resilience strategies. Thus, this paper aims to analyse these strategies using new semantics that calls for other forms of perceptions or approach to the questions of pastoralists' resilience strategies. Interviews (semi-structured and casual conversations), ethnographic observations and ethnobotanical surveys were used to collect data. In rangelands, such high value fodder species as Andropogon gayanus, Pennisetum pedicellatum and Dactyloctenium aegyptium that were abundant herbaceous plants during the last decades are disappearing. Concomitantly, species with lower forage value, such as Senna obtusifolia, which are more resilient to ecological disturbance factors, are colonizing rangelands. Faced with these ecological changes, pastoralists are trying to redefine and reconfigure their practices, and this implies a redefinition of their identity. They use resilience strategies such as mowing grasses, building up fodder bundles, conserving crop residues, exploiting Senna obtusifolia (a previously neglected species), using woody fodder and adapting the type of livestock and the size of the herds to the ability of pastoralists to feed them. Strategies that are older than these are the integration of agriculture with livestock and decollectivized transhumance. It is these resilience strategies that this article exposes and analyses as defence mechanisms of Sahelian pastoralists in the face of the depletion of forage resources in their environments.
Ecological niche models (ENMs) are often used to investigate how climatic variables from known occurrence records can estimate potential species range distribution. Although climate-based ENMs provide critical baseline information, the inclusion of non-climatic predictors related to vegetation cover might generate more realistic scenarios. This assumption is particularly relevant for species with life-history traits related to forest habitats and sensitive to habitat loss and fragmentation. Here, we developed ENMs for 36 Atlantic Forest endemic birds considering two sets of predictor variables: (i) climatic variables only and (ii) climatic variables combined with the percentage of remaining native vegetation. We hypothesized that the inclusion of native vegetation data would decrease the potential range distribution of forest-dependent species by limiting their occurrence in regions harboring small areas of native vegetation habitats, despite otherwise favorable climatic conditions. We also expected that habitat restriction in the climate-vegetation models would be more pronounced for highly forest-dependent birds. The inclusion of vegetation data in the modeling procedures restricted the final distribution ranges of 22 out of 36 modeled species, while the 14 remaining presented an expansion of their ranges. We observed that species with high and medium forest dependency showed higher restriction in range size predictions between predictor sets than species with low forest dependency, which showed no alteration or range expansion. Overall, our results suggest that ENMs based on climatic and landscape variables may be a useful tool for conservationists to better understand the dynamic of bird species distributions in threatened and highly fragmented regions such as the Atlantic Forest hotspot.(c) 2021 Associacao Brasileira de Cie circumflex accent ncia Ecol ogica e Conservacao. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
Membrane sterol composition in Arabidopsis thaliana affects root elongation via auxin biosynthesis
(2021)
Plant membrane sterol composition has been reported to affect growth and gravitropism via polar auxin transport and auxin signaling. However, as to whether sterols influence auxin biosynthesis has received little attention. Here, by using the sterol biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) and sterol application, we reveal that cycloeucalenol, a CPI1 substrate, and sitosterol, an end-product of sterol biosynthesis, antagonistically affect auxin biosynthesis. The short root phenotype of cpi1-1 was associated with a markedly enhanced auxin response in the root tip. Both were neither suppressed by mutations in polar auxin transport (PAT) proteins nor by treatment with a PAT inhibitor and responded to an auxin signaling inhibitor. However, expression of several auxin biosynthesis genes TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) was upregulated in cpi1-1. Functionally, TAA1 mutation reduced the auxin response in cpi1-1 and partially rescued its short root phenotype. In support of this genetic evidence, application of cycloeucalenol upregulated expression of the auxin responsive reporter DR5:GUS (beta-glucuronidase) and of several auxin biosynthesis genes, while sitosterol repressed their expression. Hence, our combined genetic, pharmacological, and sterol application studies reveal a hitherto unexplored sterol-dependent modulation of auxin biosynthesis during Arabidopsis root elongation.
Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the virus structural stability. M1 organizes virion assembly and budding at the plasma membrane (PM), where it interacts with other viral components. The recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin [HA], neuraminidase, matrix protein 2 [M2]) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e., scanning fluorescence cross-correlation spectroscopy and number and brightness) to quantify the oligomeric state of M1 and its interactions with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the event that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins.
RangeShiftR
(2021)
Reliably modelling the demographic and distributional responses of a species to environmental changes can be crucial for successful conservation and management planning. Process-based models have the potential to achieve this goal, but so far they remain underused for predictions of species' distributions. Individual-based models offer the additional capability to model inter-individual variation and evolutionary dynamics and thus capture adaptive responses to environmental change. We present RangeShiftR, an R implementation of a flexible individual-based modelling platform which simulates eco-evolutionary dynamics in a spatially explicit way. The package provides flexible and fast simulations by making the software RangeShifter available for the widely used statistical programming platform R. The package features additional auxiliary functions to support model specification and analysis of results. We provide an outline of the package's functionality, describe the underlying model structure with its main components and present a short example. RangeShiftR offers substantial model complexity, especially for the demographic and dispersal processes. It comes with elaborate tutorials and comprehensive documentation to facilitate learning the software and provide help at all levels. As the core code is implemented in C++, the computations are fast. The complete source code is published under a public licence, making adaptations and contributions feasible. The RangeShiftR package facilitates the application of individual-based and mechanistic modelling to eco-evolutionary questions by operating a flexible and powerful simulation model from R. It allows effortless interoperation with existing packages to create streamlined workflows that can include data preparation, integrated model specification and results analysis. Moreover, the implementation in R strengthens the potential for coupling RangeShiftR with other models.
In the light of climate change, rising demands for agricultural products and the intensification and specialization of agricultural systems, ensuring an adequate and reliable supply of food is fundamental for food security. Maintaining diversity and redundancy has been postulated as one generic principle to increase the resilience of agricultural production and other ecosystem services. For example, if one crop fails due to climate instability and extreme events, others can compensate the losses. Crop diversity might be particularly important if different crops show asynchronous production trends. Furthermore, spatial heterogeneity has been suggested to increase stability at larger scales as production losses in some areas can be buffered by surpluses in undisturbed ones. Besides systematically investigating the mechanisms underlying stability, identifying transformative pathways that foster them is important.
In my thesis, I aim at answering the following questions: (i) How does yield stability differ between nations, regions and farms, and what is the effect of crop diversity on yield stability in relation to agricultural inputs, climate heterogeneity, climate instability and time at the national, regional or farm level? (ii) Is asynchrony between crops a better predictor of production stability than crop diversity? (iii) What is the effect of asynchrony between and within crops on stability and how is it related to crop diversity and space, respectively? (iv) What is the state of the art and what are knowledge gaps in exploring resilience and its multidimensionality in ecological and social-ecological systems with agent-based models and what are potential ways forward?
In the first chapter, I provide the theoretical background for the subsequent analyses. I stress the need to better understand the resilience of social-ecological systems and particularly the stability of agricultural production. Moreover, I introduce diversity and spatial heterogeneity as two prominently discussed resilience mechanisms and describe approaches to assess resilience.
In the second chapter, I combined agriculture and climate data at three levels of organization and spatial extents to investigate yield stability patterns and their relation to crop diversity, fertilizer, irrigation, climate heterogeneity and instability and time of nations globally, regions in Europe and farms in Germany using statistical analyses. Yield stability decreased from the national to the farm level. Several nations and regions substantially contributed to larger-scale stability. Crop diversity was positively associated with yield stability across all three levels of organization. This effect was typically more profound at smaller scales and in variable climates. In addition to crop diversity, climate heterogeneity was an important stabilizing mechanism especially at larger scales. These results confirm the stabilizing effect of crop diversity and spatial heterogeneity, yet their importance depends on the scale and agricultural management.
Building on the findings of the second chapter, I deepened in the third chapter my research on the effect of crop diversity at the national level. In particular, I tested if asynchrony between crops, i.e. between the temporal production patterns of different crops, better predicts agricultural production stability than crop diversity. The stabilizing effect of asynchrony was multiple times higher than the effect of crop diversity, i.e. asynchrony is one important property that can explain why a higher diversity supports the stability of national food production. Therefore, strategies to stabilize agricultural production through crop diversification also need to account for the asynchrony of the crops considered.
The previous chapters suggest that both asynchrony between crops and spatial heterogeneity are important stabilizing mechanisms. In the fourth chapter, I therefore aimed at better understanding the relative importance of asynchrony between and within crops, i.e. between the temporal production patterns of different crops and between the temporal production patterns of different cultivation areas of the same crop. Better understanding their relative importance is important to inform agricultural management decisions, but so far this has been hardly assessed. To address this, I used crop production data to study the effect of asynchrony between and within crops on the stability of agricultural production in regions in Germany and nations in Europe. Both asynchrony between and within crops consistently stabilized agricultural production. Adding crops increased asynchrony between crops, yet this effect levelled off after eight crops in regions in Germany and after four crops in nations in Europe. Combining already ten farms within a region led to high asynchrony within crops, indicating distinct production patters, while this effect was weaker when combining multiple regions within a nation. The results suggest, that both mechanisms need to be considered in agricultural management strategies that strive for more resilient farming systems.
The analyses in the foregoing chapters focused at different levels of organization, scales and factors potentially influencing agricultural stability. However, these statistical analyses are restricted by data availability and investigate correlative relationships, thus they cannot provide a mechanistic understanding of the actual processes underlying resilience. In this regard, agent-based models (ABM) are a promising tool. Besides their ability to measure different properties and to integrate multiple situations through extensive manipulation in a fully controlled system, they can capture the emergence of system resilience from individual interactions and feedbacks across different levels of organization. In the fifth chapter, I therefore reviewed the state of the art and potential knowledge gaps in exploring resilience and its multidimensionality in ecological and social-ecological systems with ABMs. Next, I derived recommendations for a more effective use of ABMs in resilience research. The review suggests that the potential of ABMs is not utilized in most models as they typically focus on a single dimension of resilience and are mostly limited to one reference state, disturbance type and scale. Moreover, only few studies explicitly test the ability of different mechanisms to support resilience. To solve real-world problems related to the resilience of complex systems, ABMs need to assess multiple stability properties for different situations and under consideration of the mechanisms that are hypothesized to render a system resilient.
In the sixth chapter, I discuss the major conclusions that can be drawn from the previous chapters. Moreover, I showcase the use of simulation models to identify management strategies to enhance asynchrony and thus stability, and the potential of ABMs to identify pathways to implement such strategies.
The results of my thesis confirm the stabilizing effect of crop diversity, yet its importance depends on the scale, agricultural management and climate. Moreover, strategies to stabilize agricultural production through crop diversification also need to account for the asynchrony of the crops considered. As spatial heterogeneity and particularly asynchrony within crops strongly enhances stability, integrated management approaches are needed that simultaneously address multiple resilience mechanisms at different levels of organization, scales and time horizons. For example, the simulation suggests that only increasing the number of crops at both the pixel and landscape level avoids trade-offs between asynchrony between and within crops. If their potential is better exploited, agent-based models have the capacity to systematically assess resilience and to identify comprehensive pathways towards resilient farming systems.
Anthropogenic activities such as continuous landscape changes threaten biodiversity at both local and regional scales. Metacommunity models attempt to combine these two scales and continuously contribute to a better mechanistic understanding of how spatial processes and constraints, such as fragmentation, affect biodiversity. There is a strong consensus that such structural changes of the landscape tend to negatively effect the stability of metacommunities. However, in particular the interplay of complex trophic communities and landscape structure is not yet fully understood.
In this present dissertation, a metacommunity approach is used based on a dynamic and spatially explicit model that integrates population dynamics at the local scale and dispersal dynamics at the regional scale. This approach allows the assessment of complex spatial landscape components such as habitat clustering on complex species communities, as well as the analysis of population dynamics of a single species. In addition to the impact of a fixed landscape structure, periodic environmental disturbances are also considered, where a periodical change of habitat availability, temporally alters landscape structure, such as the seasonal drying of a water body.
On the local scale, the model results suggest that large-bodied animal species, such as predator species at high trophic positions, are more prone to extinction in a state of large patch isolation than smaller species at lower trophic levels.
Increased metabolic losses for species with a lower body mass lead to increased energy limitation for species on higher trophic levels and serves as an explanation for a predominant loss of these species. This effect is particularly pronounced for food webs, where species are more sensitive to increased metabolic losses through dispersal and a change in landscape structure.
In addition to the impact of species composition in a food web for diversity, the strength of local foraging interactions likewise affect the synchronization of population dynamics. A reduced predation pressure leads to more asynchronous population dynamics, beneficial for the stability of population dynamics as it reduces the risk of correlated extinction events among habitats. On the regional scale, two landscape aspects, which are the mean patch isolation and the formation of local clusters of two patches, promote an increase in $\beta$-diversity. Yet, the individual composition and robustness of the local species community equally explain a large proportion of the observed diversity patterns.
A combination of periodic environmental disturbance and patch isolation has a particular impact on population dynamics of a species. While the periodic disturbance has a synchronizing effect, it can even superimpose emerging asynchronous dynamics in a state of large patch isolation and unifies trends in synchronization between different species communities.
In summary, the findings underline a large local impact of species composition and interactions on local diversity patterns of a metacommunity. In comparison, landscape structures such as fragmentation have a negligible effect on local diversity patterns, but increase their impact for regional diversity patterns. In contrast, at the level of population dynamics, regional characteristics such as periodic environmental disturbance and patch isolation have a particularly strong impact and contribute substantially to the understanding of the stability of population dynamics in a metacommunity. These studies demonstrate once again the complexity of our ecosystems and the need for further analysis for a better understanding of our surrounding environment and more targeted conservation of biodiversity.
Hantaviruses are emerging pathogens that occasionally cause deadly outbreaks in the human population. While the structure of the viral envelope has been characterized with high precision, protein-protein interactions leading to the formation of new virions in infected cells are not fully understood. We used quantitative fluorescence microscopy (i.e., number and brightness analysis and fluorescence fluctuation spectroscopy) to monitor the interactions that lead to oligomeric spike complex formation in the physiological context of living cells. To this aim, we quantified protein-protein interactions for the glycoproteins Gn and Gc from Puumala and Hantaan orthohantaviruses in several cellular models. The oligomerization of each protein was analyzed in relation to subcellular localization, concentration, and the concentration of its interaction partner. Our results indicate that, when expressed separately, Gn and Gc form, respectively, homo-tetrameric and homo-dimeric complexes, in a concentration-dependent manner. Site-directed mutations or deletion mutants showed the specificity of their homotypic interactions. When both glycoproteins were coexpressed, we observed in the Golgi apparatus clear indication of GnGc interactions and the formation of Gn-Gc multimeric protein complexes of different sizes, while using various labeling schemes to minimize the influence of the fluorescent tags. Such large glycoprotein multimers may be identified as multiple Gn viral spikes interconnected via Gc-Gc contacts. This observation provides the possible first evidence for the initial assembly steps of the viral envelope within this organelle, and does so directly in living cells. <br /> IMPORTANCE In this work, we investigate protein-protein interactions that drive the assembly of the hantavirus envelope. These emerging pathogens have the potential to cause deadly outbreaks in the human population. Therefore, it is important to improve our quantitative understanding of the viral assembly process in infected cells, from a molecular point of view. By applying advanced fluorescence microscopy methods, we monitored the formation of viral spike complexes in different cell types. Our data support a model for hantavirus assembly according to which viral spikes are formed via the clustering of hetero-dimers of the two viral glycoproteins Gn and Gc. Furthermore, the observation of large Gn-Gc hetero-multimers provide the possible first evidence for the initial assembly steps of the viral envelope, directly in the Golgi apparatus of living cells.
TusA is a versatile protein that links translation efficiency to cell division in Escherichia coli
(2021)
To enable accurate and efficient translation, sulfur modifications are introduced posttranscriptionally into nucleosides in tRNAs. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems for the incorporation of sulfur atoms in different nucleosides of tRNA. One of the proteins that is involved in inserting the sulfur for 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modifications in tRNAs is the TusA protein. TusA, however, is a versatile protein that is also involved in numerous other cellular pathways. Despite its role as a sulfur transfer protein for the 2-thiouridine formation in tRNA, a fundamental role of TusA in the general physiology of Escherichia coli has also been discovered. Poor viability, a defect in cell division, and a filamentous cell morphology have been described previously for tusA-deficient cells. In this report, we aimed to dissect the role of TusA for cell viability. We were able to show that the lack of the thiolation status of wobble uridine (U-34) nucleotides present on Lys, Gln, or Glu in tRNAs has a major consequence on the translation efficiency of proteins; among the affected targets are the proteins RpoS and Fis. Both proteins are major regulatory factors, and the deregulation of their abundance consequently has a major effect on the cellular regulatory network, with one consequence being a defect in cell division by regulating the FtsZ ring formation. <br /> IMPORTANCE More than 100 different modifications are found in RNAs. One of these modifications is the mnm(5)s(2)U modification at the wobble position 34 of tRNAs for Lys, Gln, and Glu. The functional significance of U34 modifications is substantial since it restricts the conformational flexibility of the anticodon, thus providing translational fidelity. We show that in an Escherichia coli TusA mutant strain, involved in sulfur transfer for the mnm(5)s(2)U34 thio modifications, the translation efficiency of RpoS and Fis, two major cellular regulatory proteins, is altered. Therefore, in addition to the transcriptional regulation and the factors that influence protein stability, tRNA modifications that ensure the translational efficiency provide an additional crucial regulatory factor for protein synthesis.
The two important mechanisms influencing the response of phytoplankton communities to alterations of abiotic factors in their environment are difficult to distinguish: species sorting resulting from a change in interspecific competitive pressure, and phenotypic plasticity (here explicitly physiological plasticity i.e. species-specific physiological adjustment). A shift in species composition as well as physiological adjustments in species can lead to changes in fatty acid composition that determine the food quality for zooplankton consumers. We used phytoplankton communities consisting of five species and exposed them to two different light intensities, two light conditions (constant and variable), and two levels of phosphorus supply. Changes in fatty acid and species composition were analyzed. We compared community pairs differing in one factor by calculating the Bray-Curtis similarity index for the composition of both variables. Comparing the Bray-Curtis similarity index of the species composition with the index of the fatty acid composition was used to estimate the effects of species sorting and physiological plasticity. Changes in nutrient supply influenced fatty acid responses based on species sorting and physiological plasticity the most. On one hand, the relevance of physiological plasticity was highest at cultivation in different nutrient supplies but the same light environment. Conversely with low nutrients species sorting appeared to dominate the response to changes in light, while at high nutrients physiological plasticity appeared to influence the response. Overall, under low phosphorus supply the communities showed a lower total fatty acid content per carbon and had increased proportions of saturated and monounsaturated fatty acids. Instead, communities in low light produced more of eicosapentaenoic acid. Our results suggest that the relevance of species sorting and physiological plasticity in shaping the community response highly depends on the environmental factors that influence the system. Nutrient supply had the largest effect, while light had more limited conditional effects. However, all of these factors are important in shaping the food quality of the phytoplankton community for higher trophic levels.
Plants have evolved numerous molecular strategies to cope with perturbations in environmental temperature, and to adjust growth and physiology to limit the negative effects of extreme temperature. One of the strategies involves alternative splicing of primary transcripts to encode alternative protein products or transcript variants destined for degradation by nonsense-mediated decay. Here, we review how changes in environmental temperature-cold, heat, and moderate alterations in temperature-affect alternative splicing in plants, including crops. We present examples of the mode of action of various temperature-induced splice variants and discuss how these alternative splicing events enable favourable plant responses to altered temperatures. Finally, we point out unanswered questions that should be addressed to fully utilize the endogenous mechanisms in plants to adjust their growth to environmental temperature. We also indicate how this knowledge might be used to enhance crop productivity in the future.
Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.
Averting today's loss of biodiversity and ecosystem services can be achieved through conservation efforts, especially of keystone species. Giraffes (Giraffa camelopardalis) play an important role in sustaining Africa's ecosystems, but are 'vulnerable' according to the IUCN Red List since 2016. Monitoring an animal's behavior in the wild helps to develop and assess their conservation management. One mechanism for remote tracking of wildlife behavior is to attach accelerometers to animals to record their body movement. We tested two different commercially available high-resolution accelerometers, e-obs and Africa Wildlife Tracking (AWT), attached to the top of the heads of three captive giraffes and analyzed the accuracy of automatic behavior classifications, focused on the Random Forests algorithm. For both accelerometers, behaviors of lower variety in head and neck movements could be better predicted (i.e., feeding above eye level, mean prediction accuracy e-obs/AWT: 97.6%/99.7%; drinking: 96.7%/97.0%) than those with a higher variety of body postures (such as standing: 90.7-91.0%/75.2-76.7%; rumination: 89.6-91.6%/53.5-86.5%). Nonetheless both devices come with limitations and especially the AWT needs technological adaptations before applying it on animals in the wild. Nevertheless, looking at the prediction results, both are promising accelerometers for behavioral classification of giraffes. Therefore, these devices when applied to free-ranging animals, in combination with GPS tracking, can contribute greatly to the conservation of giraffes.
Tire-wear particles (TWPs) are being released into the environment by wearing down during car driving, and are considered an important microplastic pollution source. The chemical additive leaching from these polymer-based materials and its potential effects are likely temporally dynamic, since amounts of potentially toxic compounds can gradually increase with contact time of plastic particles with surrounding media. In the present study, we conducted soil toxicity tests using the soil nematode Caenorhabditis elegans with different soil pre-incubation (30 and 75 days) and exposure (short-term exposure, 2 days; lifetime exposure, 10 days) times. Soil pre-incubation increased toxicity of TWPs, and the effective concentrations after the pre-incubation were much lower than environmentally relevant concentrations. The lifetime of C. elegans was reduced faster in the TWP treatment groups, and the effective concentration for lifetime exposure tests were 100- to 1,000-fold lower than those of short-term exposure tests. Water-extractable metal concentrations (Cr, Cu, Ni, Pb, and Zn) in the TWP-soils showed no correlation with nominal TWP concentrations or pre-incubation times, and the incorporated metals in the TWPs may be not the main reason of toxicity in this study. Our results show that toxic effects of TWPs can be time-dependent, both in terms of the microplastic particles themselves and their interactions in the soil matrix, but also because of susceptibility of target organisms depending on developmental stage. It is vital that future works consider these aspects, since otherwise effects of microplastics and TWPs could be underestimated.
The Anthropocene is the era of urbanization. The accelerating expansion of cities occurs at the expense of natural reservoirs of biodiversity and presents animals with challenges for which their evolutionary past might not have prepared them. Cognitive and behavioral adjustments to novelty could promote animals’ persistence under these altered conditions. We investigated the structure of, and covariance between, different aspects of responses to novelty in rural and urban small mammals of two non-commensal rodent species. We ran replicated experiments testing responses to three novelty types (object, food, or space) of 47 individual common voles (Microtus arvalis) and 41 individual striped field mice (Apodemus agrarius). We found partial support for the hypothesis that responses to novelty are structured, clustering (i) speed of responses, (ii) intensity of responses, and (iii) responses to food into separate dimensions. Rural and urban small mammals did not differ in most responses to novelty, suggesting that urban habitats do not reduce neophobia in these species. Further studies investigating whether comparable response patters are found throughout different stages of colonization, and along synurbanization processes of different duration, will help illuminate the dynamics of animals’ cognitive adjustments to urban life.
The Anthropocene is the era of urbanization. The accelerating expansion of cities occurs at the expense of natural reservoirs of biodiversity and presents animals with challenges for which their evolutionary past might not have prepared them. Cognitive and behavioral adjustments to novelty could promote animals’ persistence under these altered conditions. We investigated the structure of, and covariance between, different aspects of responses to novelty in rural and urban small mammals of two non-commensal rodent species. We ran replicated experiments testing responses to three novelty types (object, food, or space) of 47 individual common voles (Microtus arvalis) and 41 individual striped field mice (Apodemus agrarius). We found partial support for the hypothesis that responses to novelty are structured, clustering (i) speed of responses, (ii) intensity of responses, and (iii) responses to food into separate dimensions. Rural and urban small mammals did not differ in most responses to novelty, suggesting that urban habitats do not reduce neophobia in these species. Further studies investigating whether comparable response patters are found throughout different stages of colonization, and along synurbanization processes of different duration, will help illuminate the dynamics of animals’ cognitive adjustments to urban life.
Simple and robust
(2021)
A spectrum of 7562 publications on Molecularly Imprinted Polymers (MIPs) has been presented in literature within the last ten years (Scopus, September 7, 2020). Around 10 % of the papers published on MIPs describe the recognition of proteins. The straightforward synthesis of MIPs is a significant advantage as compared with the preparation of enzymes or antibodies. MIPs have been synthesized from only one up to six functional monomers while proteins are made up of 20 natural amino acids. Furthermore, they can be synthesized against structures of low immunogenicity and allow multi-analyte measurements via multi-target synthesis. Electrochemical methods allow simple polymer synthesis, removal of the template and readout. Among the different sensor configurations electrochemical MIP-sensors provide the broadest spectrum of protein analytes. The sensitivity of MIP-sensors is sufficiently high for biomarkers in the sub-nanomolar region, nevertheless the cross-reactivity of highly abundant proteins in human serum is still a challenge. MIPs for proteins offer innovative tools not only for clinical and environmental analysis, but also for bioimaging, therapy and protein engineering.
Pathogens and animal pests (P&A) are a major threat to global food security as they directly affect the quantity and quality of food. The Southern Amazon, Brazil's largest domestic region for soybean, maize and cotton production, is particularly vulnerable to the outbreak of P&A due to its (sub)tropical climate and intensive farming systems. However, little is known about the spatial distribution of P&A and the related yield losses. Machine learning approaches for the automated recognition of plant diseases can help to overcome this research gap. The main objectives of this study are to (1) evaluate the performance of Convolutional Neural Networks (ConvNets) in classifying P&A, (2) map the spatial distribution of P&A in the Southern Amazon, and (3) quantify perceived yield and economic losses for the main soybean and maize P&A. The objectives were addressed by making use of data collected with the smartphone application Plantix. The core of the app's functioning is the automated recognition of plant diseases via ConvNets. Data on expected yield losses were gathered through a short survey included in an "expert" version of the application, which was distributed among agronomists. Between 2016 and 2020, Plantix users collected approximately 78,000 georeferenced P&A images in the Southern Amazon. The study results indicate a high performance of the trained ConvNets in classifying 420 different crop-disease combinations. Spatial distribution maps and expert-based yield loss estimates indicate that maize rust, bacterial stalk rot and the fall armyworm are among the most severe maize P&A, whereas soybean is mainly affected by P&A like anthracnose, downy mildew, frogeye leaf spot, stink bugs and brown spot. Perceived soybean and maize yield losses amount to 12 and 16%, respectively, resulting in annual yield losses of approximately 3.75 million tonnes for each crop and economic losses of US$2 billion for both crops together. The high level of accuracy of the trained ConvNets, when paired with widespread use from following a citizen-science approach, results in a data source that will shed new light on yield loss estimates, e.g., for the analysis of yield gaps and the development of measures to minimise them.
Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.
Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.
Boon and bane
(2021)
Semi-natural habitats (SNHs) in agricultural landscapes represent important refugia for biodiversity including organisms providing ecosystem services. Their spill-over into agricultural fields may lead to the provision of regulating ecosystem services such as biological pest control ultimately affecting agricultural yield. Still, it remains largely unexplored, how different habitat types and their distributions in the surrounding landscape shape this provision of ecosystem services within arable fields. Hence, in this thesis I investigated the effect of SNHs on biodiversity-driven ecosystem services and disservices affecting wheat production with an emphasis on the role and interplay of habitat type, distance to the habitat and landscape complexity.
I established transects from the field border into the wheat field, starting either from a field-to-field border, a hedgerow, or a kettle hole, and assessed beneficial and detrimental organisms and their ecosystem functions as well as wheat yield at several in-field distances. Using this study design, I conducted three studies where I aimed to relate the impacts of SNHs at the field and at the landscape scale on ecosystem service providers to crop production.
In the first study, I observed yield losses close to SNHs for all transect types. Woody habitats, such as hedgerows, reduced yields stronger than kettle holes, most likely due to shading from the tall vegetation structure. In order to find the biotic drivers of these yield losses close to SNHs, I measured pest infestation by selected wheat pests as potential ecosystem disservices to crop production in the second study. Besides relating their damage rates to wheat yield of experimental plots, I studied the effect of SNHs on these pest rates at the field and at the landscape scale. Only weed cover could be associated to yield losses, having their strongest impact on wheat yield close to the SNH. While fungal seed infection rates did not respond to SNHs, fungal leaf infection and herbivory rates of cereal leaf beetle larvae were positively influenced by kettle holes. The latter even increased at kettle holes with increasing landscape complexity suggesting a release of natural enemies at isolated habitats within the field interior.
In the third study, I found that also ecosystem service providers benefit from the presence of kettle holes. The distance to a SNH decreased species richness of ecosystem service providers, whereby the spatial range depended on species mobility, i.e. arable weeds diminished rapidly while carabids were less affected by the distance to a SNH. Contrarily, weed seed predation increased with distance suggesting that a higher food availability at field borders might have diluted the predation on experimental seeds. Intriguingly, responses to landscape complexity were rather mixed: While weed species richness was generally elevated with increasing landscape complexity, carabids followed a hump-shaped curve with highest species numbers and activity-density in simple landscapes. The latter might give a hint that carabids profit from a minimum endowment of SNHs, while a further increase impedes their mobility. Weed seed predation was affected differently by landscape complexity depending on weed species displayed. However, in habitat-rich landscapes seed predation of the different weed species converged to similar rates, emphasising that landscape complexity can stabilize the provision of ecosystem services. Lastly, I could relate a higher weed seed predation to an increase in wheat yield even though seed predation did not diminish weed cover. The exact mechanisms of the provision of weed control to crop production remain to be investigated in future studies.
In conclusion, I found habitat-specific responses of ecosystem (dis)service providers and their functions emphasizing the need to evaluate the effect of different habitat types on the provision of ecosystem services not only at the field scale, but also at the landscape scale. My findings confirm that besides identifying species richness of ecosystem (dis)service providers the assessment of their functions is indispensable to relate the actual delivery of ecosystem (dis)services to crop production.
Background
Over the last 20 years, a decreasing trend in external skeletal robusticity and an increasing trend in overweight and obesity was observed worldwide in adults and children as modern lifestyles in nutritional and activity behavior have changed. However, body mass index (BMI) as a measure for overweight is not an ideal predictor of % body fat (%BF) either in children and adolescents or in adults. On the contrary, it disguises a phenomenon called “hidden obesity”.
Objectives
We aim to approximate %BF by combining skeletal robusticity and BMI and develop an estimation-based tool to identify normal weight obese children and adolescents.
Sample and Methods
We analyzed cross-sectional data on height, weight, elbow breadth, and skinfold thickness (triceps and subscapular) of German children aged 6 to 18 years (N=15,034). We used modified Hattori charts and multiple linear regression to develop a tool, the “%BF estimator”, to estimate %BF by using BMI and skeletal robusticity measured as Frame Index.
Results
Independent of sex and age an increase in BMI is associated with an increase in %BF, an increase in Frame Index is associated with a decrease in %BF. The developed tool “%BF estimator” allows the estimation of %BF per sex and age group after calculation of BMI and Frame Index.
Conclusion
The “%BF estimator” is an easily applicable tool for the estimation of %BF in respect of body composition for clinical practice, screening, and public health research. It is non-invasive and has high accuracy. Further, it allows the identification of normal weight obese children and adolescents.
Twenty-three scientists met at Krobielowice, Poland to discuss the role of growth, nutrition and economy on body size. Contrasting prevailing concepts, re-analyses of studies in Indonesian and Guatemalan school children with high prevalence of stunting failed to provide evidence for an association between nutritional status and body height. Direct effects of parental education on growth that were not transmitted via nutrition were shown in Indian datasets using network analysis and novel statistical methods (St. Nicolas House Analysis) that translate correlation matrices into network graphs. Data on Polish children suggest significant impact of socioeconomic sensitivity on child growth, with no effect of maternal money satisfaction. Height and maturation tempo affect the position of a child among its peers. Correlations also exist between mood disorders and height. Secular changes in height and weight varied across decades independent of population size. Historic and recent Russian data showed that height of persons whose fathers performed manual work were on average four cm shorter than persons whose fathers were high-degree specialists. Body height, menarcheal age, and body proportions are sensitive to socioeconomic variables. Additional topics included delayed motherhood and its associations with newborn size; geographic and socioeconomic indicators related to low birth weight, prematurity and stillbirth rate; data on anthropometric history of Brazil, 1850-1950; the impact of central nervous system stimulants on the growth of children with attention-deficit/hyperactivity disorder; and pituitary development and growth hormone secretion. Final discussions debated on reverse causality interfering between social position, and adolescent growth and developmental tempo.
Cellulose is the most abundant biopolymer on Earth and cell wall (CW) synthesis is one of the major carbon consumers in the plant cell. Structure and several interaction partners of plasma membrane (PM)-bound cellulose synthase (CESA) complexes, CSCs, have been studied extensively, but much less is understood about the signals that activate and translocate CESAs to the PM and how exactly cellulose synthesis is being regulated during the diel cycle. The literature describes CSC regulation possibilities through interactions with accessory proteins upon stress conditions (e.g. CC1), post-translational modifications that regulate CSC speed and their possible anchoring in the PM (e.g. with phosphorylation and S-acylation, respectively). In this thesis, 13CO2 labeling and imaging techniques were employed in the same Arabidopsis seedling growth system to elucidate how and when new carbon is incorporated into cell wall (CW) sugars and UDP-glucose, and to follow CSC behavior during the diel cycle. Additionally, an ubiquitination analysis was performed to investigate a possible mechanism to affect CSC trafficking to and/or from the PM. Carbon is being incorporated into CW glucose at a 3-fold higher rate during the light period in comparison to the night in wild-type seedlings. Furthermore, CSC density at the PM, as an indication of active cellulose synthesizing machinery, is increasing in the light and falling during the night, showing that CW biosynthesis is more active in the light. Therefore, CW synthesis might be regulated by the carbon status of the cell. This regulation is broken in the starchless pgm mutant where light and dark carbon incorporation rates into CW glucose are similar, possibly due to the high soluble sugar content in pgm during the first part of the night. Strikingly, pgm CSC abundance at the PM is constantly low during the whole diel cycle, indicating little or no cellulose synthesis, but can be restored with exogenous sucrose or a longer photoperiod. Ubiquitination was explored as a possible regulating mechanism for translocation of primary CW CSCs from the PM and several potential ubiquitination sites have been identified.. The approach in this thesis enabled to study cellulose/CW synthesis from different angles but in the same growth system, allowing direct comparison of those methodologies, which could help understand the relationship between the amount of available carbon in a plant cell and the cells capacity to synthesize cellulose/CW. Understanding which factors contribute to cellulose synthesis regulation and addressing those fundamental questions can provide essential knowledge to manage the need for increased crop production.
Background
There is a recurring and seamless interaction between the biology of human development and the social-economic-political-emotional (SEPE) environment. The SEPE environment influences the quality of the material conditions for human biology and, simultaneously, human growth in height and other dimensions provide social and moral signals that provide information to community networks.
Objectives
This article reviews the role of SEPE factors in human growth, especially skeletal growth.
Sample and Methods
The meaning of SEPE is defined and shown to be related to individual and group prestige, to social identity, and to ego and task motivation. These influence dominance or subordination of communities and the material and moral conditions of societies. Historical and contemporary examples of SEPE effects on skeletal size are presented.
Results
Membership in a SEPE community impacts skeletal size in height and breadth. Higher SEPE classes are taller, lower SEPE classes are broader. In elite level sport the winners have more growth stimulation via the hormone IGF-1 even before the contest. These findings are explained in terms of dominance versus subordination and the Community Effect in Height hypothesis.
Conclusions
SEPE factor regulation of human growth is shown to be a more comprehensive explanation for plasticity in height than traditional concepts such as socioeconomic status and simple-minded genetic determinism. People belonging to upper SEPE class communities, the elites, know that they are superior and are treated as such by the non-elites. The material and moral condition for life operating through these community social networks provide positive stimulation for the elites and negative stimulation for the lower SEPE classes. These differences maintain the gradients in height between SEPE communities in human societies.
Background
Nicotine consumption during pregnancy and advanced maternal age are well known independent risk factors for poor pregnancy outcome and therefore serious public health problems.
Objectives
Considering the ongoing trend of delaying childbirth in our society, this study investigates potential additive effects of nicotine consumption during pregnancy and advanced maternal age on foetal growth.
Sample and Methods
In a medical record-based study, we analysed the impact of maternal age and smoking behaviour before and during pregnancy on newborn size among 4142 singleton births that took place in Vienna, Austria between 1990 and 1995.
Results
Birth weight (H=82.176, p<0.001), birth length (H=91.525, p<0.001) and head circumference (H=42.097, p<0.001) differed significantly according to maternal smoking behaviour. For birth weight, the adjusted mean differences between smokers and non-smokers increased from 101.8g for the < 18-year-old mothers to 254.8g for >35 year olds, with the respective values for birth length being 0.6 cm to 0.7cm, for head circumference from 0.3 cm to 0.6 cm.
Conclusion
Increasing maternal age amplified the negative effects of smoking during pregnancy on newborn parameters. Our findings identify older smoking mothers as a high-risk group which should be of special interest for public health systems.
Background
Subjective Social Status is used as an important predictor for psychological and physiological findings, most commonly measured with the MacArthur Scale (Ladder Test). Previous studies have shown that this test fits better in Western cultures. The idea of a social ladder itself and ranking oneself “higher” or “lower” is a concept that accords to the Western thinking.
Objectives
We hypothesize that in a culture where only the elites have adapted to a Western lifestyle, the test results reflect a higher level of accuracy for this stratum. We also expect that self-perception differs per sex.
Sample and Methods
We implemented the Ladder Test in a study of Indonesian schoolchildren aged between 5 and 13 years (boys N = 369, girls N= 364) from non-private and private schools in Kupang in 2020.
Results
Our analysis showed that the Ladder Test results were according to the Western expectations only for the private school, as the Ladder Scores significantly decreased with age (LM: p = 0.04). The Ladder Test results are best explained by “Education Father” for the non-private school pupils (p = 0.01) and all boys (p = 0.04), by “School Grades” for the private school cohort (p = 0.06) and by “Household Score” for girls (p =0.09).
Conclusion
This finding indicates that the concept of ranking oneself “high” or “low” on a social ladder is strongly implicated with Western ideas. A ladder implies social movement by “climbing” up or down. According to that, reflection of self-perception is influenced by culture.
Groundwater arsenic contamination in the Bengal Delta Plain is an important public health issue
(2021)
There is a close association between human biology, epidemiology and public health. Exposure to toxic elements is one area of such associations and global concerns. The Bengal Delta Plain (BDP) is a region where contamination of ground water by arsenic has assumed epidemic proportions. Apart from dermatological manifestations, chronic exposure to arsenic causes a heavy toll through several carcinogenic and non-carcinogenic disorders. This article provides a global overview of groundwater arsenic contamination in the BDP region, especially the sources, speciation, and mobility of arsenic, and critically reviews the effects of arsenic on human health. The present review also provides a summary of comprehensive knowledge on various measures required for mitigation and social consequences of the problem of arsenic contaminated groundwater in the BDP region.
Settings Perspective
(2021)
The energy required to drive photochemical reactions is derived from charge separation across the thylakoid membrane. As the consequence of difference in proton concentration between chloroplasts stroma and thylakoid lumen, a proton motive force (pmf) is generated. The pmf is composed out of the proton gradient (ΔpH) and membrane potential (ΔΨ), and together they drive the ATP synthesis. In nature, the amount of energy fueling photosynthesis varies due to frequent changes in the light intensity. Thylakoid ion transport can adapt the energy flow through a photosynthetic apparatus to the light availability by adjusting the pmf composition. Dissipation of ΔΨ reduces the charge recombination at the photosystem II, allowing for an increase in ΔpH component to trigger a feedback downregulation of photosynthesis. K+ Exchange Antiporter 3 (KEA3) driven K+/H+ antiport reduces the ΔpH fraction of pmf, thereby dampening a non-photochemical quenching (NPQ). As a result, it increases the photosynthesis efficiency during the transition to lower light intensity. This thesis aimed to find the answers for questions concerning KEA3 activity regulation and its role in plant development. Presented data shows that in plants lacking chloroplast ATP synthase assembly factor CGL160 with decreased ATP synthase activity, KEA3 has a pivotal role in photosynthesis regulation and plant growth during steady-state conditions. Lack of KEA3 in cgl160 mutant results in a strong growth impairment, as photosynthesis is limited due to increased pH-dependent NPQ and decreased electron flow through cytochrome b6f complex. Overexpression of KEA3 in cgl160 mutant increases charge recombination at photosystem II, promoting photosynthesis. Thus, during periods of low ATP synthase activity, plants benefit from KEA3 activity. The KEA3 undergoes dimerization via its regulatory C-terminus (RCT). The RCT responds to changes in light intensity as the plants expressing KEA3 without this domain show reduced photo-protective mechanism in light intensity transients. However, those plants fix more carbon during the photosynthesis induction phase as a trade-off for a long-term photoprotection, showing KEA3 regulatory role in plant development. The KEA3 RCT is facing thylakoid stroma, thus its regulation depends on light-induced changes in the stromal environment. KEA3 activity regulation overlaps with the stromal pH changes occurring during light fluctuations. The ATP and ADP has shown to have an affinity towards heterologously expressed KEA3 RCT. Such interaction causes conformational changes in RCT structure. The fold change of RCT-ligand interaction depends on the environmental pH value. With a combination of bioinformatics and in vitro approach, the ATP binding site at RCT was located. Introduction of binding site point mutation in planta KEA3 RCT resulted in antiporter activity deregulation during transition to low light. Together, the data presented in this thesis allowed us to assess more broadly a KEA3 role in photosynthesis adjustment and propose the models of KEA3 activity regulation throughout transition in light intensity.
The role of the monoaminergic system in the feeding behavior of neonatal chicks has been reported, but the functional relationship between the metabolism of monoamines and appetite-related neuropeptides is still unclear. This study aimed to investigate the changes in catecholamine and indolamine metabolism in response to the central action of neuropeptide Y (NPY) in different feeding statuses and the underlying mechanisms. In Experiment 1, the diencephalic concentrations of amino acids and monoamines following the intracerebroventricular (ICV) injection of NPY (375 pmol/10 mu l/chick), saline solution under ad libitum, and fasting conditions for 30 min were determined. Central NPY significantly decreased L-tyrosine concentration, the precursor of catecholamines under feeding condition, but not under fasting condition. Central NPY significantly increased dopamine metabolites, including 3,4-dihydroxyphenylacetic acid and homovanillic acid (HVA). The concentration of 3-methoxy-4-hydroxyphenylglycol was significantly reduced under feeding condition, but did not change under fasting condition by NPY. However, no effects of NPY on indolamine metabolism were found in either feeding status. Therefore, the mechanism of action of catecholamines with central NPY under feeding condition was elucidated in Experiment 2. Central NPY significantly attenuated diencephalic gene expression of catecholaminergic synthetic enzymes, such as tyrosine hydroxylase, L-aromatic amino acid decarboxylase, and GTP cyclohydrolase I after 30 min of feeding. In Experiment 3, co-injection of alpha-methyl-L-tyrosine, an inhibitor of tyrosine hydroxylase with NPY, moderately attenuated the orexigenic effect of NPY, accompanied by a significant positive correlation between food intake and HVA levels. In Experiment 4, there was a significant interaction between NPY and clorgyline, an inhibitor of monoamine oxidase A with ICV co-injection which implies that co-existence of NPY and clorgyline enhances the orexigenic effect of NPY. In conclusion, central NPY modifies a part of catecholamine metabolism, which is illustrated by the involvement of dopamine transmission and metabolism under feeding but not fasting conditions.
Heteromeric HSFA2/HSFA3 complexes drive transcriptional memory after heat stress in Arabidopsis
(2021)
Adaptive plasticity in stress responses is a key element of plant survival strategies. For instance, moderate heat stress (HS) primes a plant to acquire thermotolerance, which allows subsequent survival of more severe HS conditions. Acquired thermotolerance is actively maintained over several days (HS memory) and involves the sustained induction of memory-related genes. Here we show that FORGETTER3/ HEAT SHOCK TRANSCRIPTION FACTOR A3 (FGT3/HSFA3) is specifically required for physiological HS memory and maintaining high memory-gene expression during the days following a HS exposure. HSFA3 mediates HS memory by direct transcriptional activation of memory-related genes after return to normal growth temperatures. HSFA3 binds HSFA2, and in vivo both proteins form heteromeric complexes with additional HSFs. Our results indicate that only complexes containing both HSFA2 and HSFA3 efficiently promote transcriptional memory by positively influencing histone H3 lysine 4 (H3K4) hyper-methylation. In summary, our work defines the major HSF complex controlling transcriptional memory and elucidates the in vivo dynamics of HSF complexes during somatic stress memory. Moderate heat stress primes plants to acquire tolerance to subsequent, more severe heat stress. Here the authors show that the HSFA3 transcription factor forms a heteromeric complex with HSFA2 to sustain activated transcription of genes required for acquired thermotolerance by promoting H3K4 hyper-methylation.