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Institute
- Institut für Biochemie und Biologie (117) (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.
Biodiversity decline causes a loss of functional diversity, which threatens ecosystems through a dangerous feedback loop: This loss may hamper ecosystems’ ability to buffer environmental changes, leading to further biodiversity losses. In this context, the increasing frequency of human-induced excessive loading of nutrients causes major problems in aquatic systems. Previous studies investigating how functional diversity influences the response of food webs to disturbances have mainly considered systems with at most two functionally diverse trophic levels. We investigated the effects of functional diversity on the robustness, that is, resistance, resilience, and elasticity, using a tritrophic—and thus more realistic—plankton food web model. We compared a non-adaptive food chain with no diversity within the individual trophic levels to a more diverse food web with three adaptive trophic levels. The species fitness differences were balanced through trade-offs between defense/growth rate for prey and selectivity/half-saturation constant for predators. We showed that the resistance, resilience, and elasticity of tritrophic food webs decreased with larger perturbation sizes and depended on the state of the system when the perturbation occurred. Importantly, we found that a more diverse food web was generally more resistant and resilient but its elasticity was context-dependent. Particularly, functional diversity reduced the probability of a regime shift toward a non-desirable alternative state. The basal-intermediate interaction consistently determined the robustness against a nutrient pulse despite the complex influence of the shape and type of the dynamical attractors. This relationship was strongly influenced by the diversity present and the third trophic level. Overall, using a food web model of realistic complexity, this study confirms the destructive potential of the positive feedback loop between biodiversity loss and robustness, by uncovering mechanisms leading to a decrease in resistance, resilience, and potentially elasticity as functional diversity declines.
Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix–helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers.
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.
Populations adapt to novel environmental conditions by genetic changes or phenotypic plasticity. Plastic responses are generally faster and can buffer fitness losses under variable conditions. Plasticity is typically modeled as random noise and linear reaction norms that assume simple one-to- one genotype–phenotype maps and no limits to the phenotypic response. Most studies on plasticity have focused on its effect on population viability. However, it is not clear, whether the advantage of plasticity depends solely on environmental fluctuations or also on the genetic and demographic properties (life histories) of populations. Here we present an individual-based model and study the relative importance of adaptive and nonadaptive plasticity for populations of sexual species with different life histories experiencing directional stochastic climate change. Environmental fluctuations were simulated using differentially autocorrelated climatic stochasticity or noise color, and scenarios of directiona
climate change. Nonadaptive plasticity was simulated as a random environmental effect on trait development, while adaptive plasticity as a linear, saturating, or sinusoidal reaction norm. The last two imposed limits to the plastic response and emphasized flexible interactions of the genotype with the environment. Interestingly, this assumption led to (a) smaller phenotypic than genotypic variance in the population (many-to- one genotype–phenotype map) and the coexistence of polymorphisms, and (b) the maintenance of higher genetic variation—compared to linear reaction norms and genetic determinism—even when the population was exposed to a constant environment for several generations. Limits to plasticity led to genetic accommodation, when costs were negligible, and to the appearance of cryptic variation when limits were exceeded. We found that adaptive plasticity promoted population persistence under red environmental noise and was particularly important for life histories with low fecundity. Populations produing more offspring could cope with environmental fluctuations solely by genetic changes or random plasticity, unless environmental change was too fast.
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.
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.
License to flower
(2021)
As sessile organisms, plants have evolved sophisticated ways to constantly gauge and adapt to changing environmental conditions including extremes that may be harmful to their growth and development and are thus perceived as stress. In nature, stressful events are often chronic or recurring and thus an initial stress may prime a plant to respond more efficiently to a subsequent stress event. An epigenetic basis of such stress memory was long postulated and in recent years it has been shown that this is indeed the case. High temperature stress has proven an excellent system to unpick the molecular basis of somatic stress memory, which includes histone modifications and nucleosome occupancy. This review discusses recent findings and pinpoints open questions in the field.
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.
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.
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.
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.
Trade-offs are inherent to biochemical networks governing diverse cellular functions, from gene expression to metabolism. Yet, trade-offs between fluxes of biochemical reactions in a metabolic network have not been formally studied. Here, we introduce the concept of absolute flux trade-offs and devise a constraint-based approach, termed FluTO, to identify and enumerate flux trade-offs in a given genome-scale metabolic network. By employing the metabolic networks of Escherichia coli and Saccharomyces cerevisiae, we demonstrate that the flux trade-offs are specific to carbon sources provided but that reactions involved in the cofactor and prosthetic group biosynthesis are present in trade-offs across all carbon sources supporting growth. We also show that absolute flux trade-offs depend on the biomass reaction used to model the growth of Arabidopsis thaliana under different carbon and nitrogen conditions. The identified flux trade-offs reflect the tight coupling between nitrogen, carbon, and sulphur metabolisms in leaves of C-3 plants. Altogether, FluTO provides the means to explore the space of alternative metabolic routes reflecting the constraints imposed by inherent flux trade-offs in large-scale metabolic networks.
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.
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.
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.
Reaction lumping in metabolic networks for application with thermodynamic metabolic flux analysis
(2021)
Thermodynamic metabolic flux analysis (TMFA) can narrow down the space of steady-state flux distributions, but requires knowledge of the standard Gibbs free energy for the modelled reactions. The latter are often not available due to unknown Gibbs free energy change of formation ,Delta fG0, of metabolites. To optimize the usage of data on thermodynamics in constraining a model, reaction lumping has been proposed to eliminate metabolites with unknown Delta fG0. However, the lumping procedure has not been formalized nor implemented for systematic identification of lumped reactions. Here, we propose, implement, and test a combined procedure for reaction lumping, applicable to genome-scale metabolic models. It is based on identification of groups of metabolites with unknown Delta fG0 whose elimination can be conducted independently of the others via: (1) group implementation, aiming to eliminate an entire such group, and, if this is infeasible, (2) a sequential implementation to ensure that a maximal number of metabolites with unknown Delta fG0 are eliminated. Our comparative analysis with genome-scale metabolic models of Escherichia coli, Bacillus subtilis, and Homo sapiens shows that the combined procedure provides an efficient means for systematic identification of lumped reactions. We also demonstrate that TMFA applied to models with reactions lumped according to the proposed procedure lead to more precise predictions in comparison to the original models. The provided implementation thus ensures the reproducibility of the findings and their application with standard TMFA.
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.
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.
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.
Plants are often challenged by an array of unfavorable environmental conditions. During cold exposure, many changes occur that include, for example, the stabilization of cell membranes, alterations in gene expression and enzyme activities, as well as the accumulation of metabolites. In the presented study, the carbohydrate metabolism was analyzed in the very early response of plants to a low temperature (2 degrees C) in the leaves of 5-week-old potato plants of the Russet Burbank cultivar during the first 12 h of cold treatment (2 h dark and 10 h light). First, some plant stress indicators were examined and it was shown that short-term cold exposure did not significantly affect the relative water content and chlorophyll content (only after 12 h), but caused an increase in malondialdehyde concentration and a decrease in the expression of NDA1, a homolog of the NADH dehydrogenase gene. In addition, it was shown that the content of transitory starch increased transiently in the very early phase of the plant response (3-6 h) to cold treatment, and then its decrease was observed after 12 h. In contrast, soluble sugars such as glucose and fructose were significantly increased only at the end of the light period, where a decrease in sucrose content was observed. The availability of the monosaccharides at constitutively high levels, regardless of the temperature, may delay the response to cold, involving amylolytic starch degradation in chloroplasts. The decrease in starch content, observed in leaves after 12 h of cold exposure, was preceded by a dramatic increase in the transcript levels of the key enzymes of starch degradation initiation, the alpha-glucan, water dikinase (GWD-EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD-EC 2.7.9.5). The gene expression of both dikinases peaked at 9 h of cold exposure, as analyzed by real-time PCR. Moreover, enhanced activities of the acid invertase as well as of both glucan phosphorylases during exposure to a chilling temperature were observed. However, it was also noticed that during the light phase, there was a general increase in glucan phosphorylase activities for both control and cold-stressed plants irrespective of the temperature. In conclusion, a short-term cold treatment alters the carbohydrate metabolism in the leaves of potato, which leads to an increase in the content of soluble sugars.
Large-scale biochemical models are of increasing sizes due to the consideration of interacting organisms and tissues. Model reduction approaches that preserve the flux phenotypes can simplify the analysis and predictions of steady-state metabolic phenotypes. However, existing approaches either restrict functionality of reduced models or do not lead to significant decreases in the number of modelled metabolites. Here, we introduce an approach for model reduction based on the structural property of balancing of complexes that preserves the steady-state fluxes supported by the network and can be efficiently determined at genome scale. Using two large-scale mass-action kinetic models of Escherichia coli, we show that our approach results in a substantial reduction of 99% of metabolites. Applications to genome-scale metabolic models across kingdoms of life result in up to 55% and 85% reduction in the number of metabolites when arbitrary and mass-action kinetics is assumed, respectively. We also show that predictions of the specific growth rate from the reduced models match those based on the original models. Since steady-state flux phenotypes from the original model are preserved in the reduced, the approach paves the way for analysing other metabolic phenotypes in large-scale biochemical networks.
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.
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.
L-2,L-1-norm regularized multivariate regression model with applications to genomic prediction
(2021)
Motivation:
Genomic selection (GS) is currently deemed the most effective approach to speed up breeding of agricultural varieties. It has been recognized that consideration of multiple traits in GS can improve accuracy of prediction for traits of low heritability. However, since GS forgoes statistical testing with the idea of improving predictions, it does not facilitate mechanistic understanding of the contribution of particular single nucleotide polymorphisms (SNP).
Results:
Here, we propose a L-2,L-1-norm regularized multivariate regression model and devise a fast and efficient iterative optimization algorithm, called L-2,L-1-joint, applicable in multi-trait GS. The usage of the L-2,L-1-norm facilitates variable selection in a penalized multivariate regression that considers the relation between individuals, when the number of SNPs is much larger than the number of individuals. The capacity for variable selection allows us to define master regulators that can be used in a multi-trait GS setting to dissect the genetic architecture of the analyzed traits. Our comparative analyses demonstrate that the proposed model is a favorable candidate compared to existing state-of-the-art approaches. Prediction and variable selection with datasets from Brassica napus, wheat and Arabidopsis thaliana diversity panels are conducted to further showcase the performance of the proposed model.
Ribosome biogenesis is tightly associated to plant metabolism due to the usage of ribosomes in the synthesis of proteins necessary to drive metabolic pathways. Given the central role of ribosome biogenesis in cell physiology, it is important to characterize the impact of different components involved in this process on plant metabolism. Double mutants of the Arabidopsis thaliana cytosolic 60S maturation factors REIL1 and REIL2 do not resume growth after shift to moderate 10 degrees C chilling conditions. To gain mechanistic insights into the metabolic effects of this ribosome biogenesis defect on metabolism, we developed TC-iReMet2, a constraint-based modelling approach that integrates relative metabolomics and transcriptomics time-course data to predict differential fluxes on a genome-scale level. We employed TC-iReMet2 with metabolomics and transcriptomics data from the Arabidopsis Columbia 0 wild type and the reil1-1 reil2-1 double mutant before and after cold shift. We identified reactions and pathways that are highly altered in a mutant relative to the wild type. These pathways include the Calvin-Benson cycle, photorespiration, gluconeogenesis, and glycolysis. Our findings also indicated differential NAD(P)/NAD(P)H ratios after cold shift. TC-iReMet2 allows for mechanistic hypothesis generation and interpretation of system biology experiments related to metabolic fluxes on a genome-scale level.
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.
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.
Motivation:
Constraint-based modeling approaches allow the estimation of maximal in vivo enzyme catalytic rates that can serve as proxies for enzyme turnover numbers. Yet, genome-scale flux profiling remains a challenge in deploying these approaches to catalogue proxies for enzyme catalytic rates across organisms.
Results:
Here, we formulate a constraint-based approach, termed NIDLE-flux, to estimate fluxes at a genome-scale level by using the principle of efficient usage of expressed enzymes. Using proteomics data from Escherichia coli, we show that the fluxes estimated by NIDLE-flux and the existing approaches are in excellent qualitative agreement (Pearson correlation > 0.9). We also find that the maximal in vivo catalytic rates estimated by NIDLE-flux exhibits a Pearson correlation of 0.74 with in vitro enzyme turnover numbers. However, NIDLE-flux results in a 1.4-fold increase in the size of the estimated maximal in vivo catalytic rates in comparison to the contenders. Integration of the maximum in vivo catalytic rates with publically available proteomics and metabolomics data provide a better match to fluxes estimated by NIDLE-flux. Therefore, NIDLE-flux facilitates more effective usage of proteomics data to estimate proxies for kcatomes.
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.