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Oaks may contribute to the stabilization of European forests under climate change. We utilized two common gardens established in contrasting growth regimes, in Greece (Olympiada) and Germany (Schwanheim), to compare the diurnal photosynthetic performance of a Greek and an Italian provenance of two Mediterranean oaks (Quercus pubescens and Q. frainetto) during the 2019 growing season. Although the higher radiation in the southern common garden led to a strong midday depression of chlorophyll a fluorescence parameters (maximum quantum efficiency of PSII, performance index on absorption basis), comparable light-saturated net photosynthetic rates were achieved in both study areas. Moreover, both species and provenances exhibited analogous responses. Q. pubescens had enhanced chlorophyll a fluorescence traits but similar photosynthetic rates compared to Q. frainetto, whereas the provenances did not differ. These findings indicate the high photosynthetic efficiency of both oaks under the current climate in Central Europe and their suitability for assisted migration schemes.
The Brachybasidiaceae are a family of 22 known species of plant-parasitic microfungi belonging to Exobasidiales, Basidiomycota. Within this family, species of the largest genus Kordyana develop balls of basidia on top of stomatal openings. Basidial cells originate from fungal stroma filling substomatal chambers. Species of Kordyana typically infect species of Commelinaceae. During fieldwork in the neotropics, fungi morphologically similar to Kordyana spp. were found on Goeppertia spp. (syn. Calathea spp., Marantaceae), namely on G. panamensis in Panama and on G. propinqua in Bolivia. These specimens are proposed as representatives of a genus new to science, Marantokordyana, based on the distinct host family and molecular sequence data of ITS and LSU rDNA regions. The specimens on the two host species represent two species new to science, M. oberwinkleriana on G. panamensis and M. boliviana on G. propinqua. They differ by the size and shape of their basidia, molecular sequence data of ITS and LSU rDNA regions, and host plant species. In the past, the understanding of Brachybasidiaceae at order and family level was significantly improved by investigation realized by Franz Oberwinkler and his collaborators at the University of Tübingen, Germany. On species level, however, our knowledge is still very poor due to incomplete species descriptions of several existing names in literature, scarceness of specimens, as well as sequence data lacking for many taxa and for further barcode regions. Especially species of Kordyana and species of Dicellomyces are in need of revision.
Ischemic heart disease caused by occlusion of coronary vessels leads to the death of downstream tissues, resulting in a fibrotic scar that cannot be resolved. In contrast to the adult mammalian heart, the adult zebrafish heart can regenerate following injury, enabling the study of the underlying cellular and molecular mechanisms. One of the earliest responses that take place after cardiac injury in adult zebrafish is coronary revascularization. Previous transcriptomic data from our lab show that vegfc, a well-known regulator of lymphatic development, is upregulated early after injury and peaks at 96 hours post cryoinjury, coinciding with the peak of coronary endothelial cell proliferation. To test the hypothesis that vegfc is involved in coronary revascularization, I examined its expression pattern and found that it is expressed by coronary endothelial cells after cardiac damage. Using a loss-of-function approach to block Vegfc signaling, I found that it is required for coronary revascularization during cardiac regeneration. Notably, blocking Vegfc signaling resulted in a significant reduction in cardiomyocyte regeneration. Using transcriptomic analysis, I identified the extracellular matrix component gene emilin2a and the chemokine gene cxcl8a as effectors of Vegfc signaling. During cardiac regeneration, cxcl8a is expressed in epicardium-derived cells, while the gene encoding its receptor cxcr1 is expressed on coronary endothelial cells. I found that overexpressing emilin2a increases coronary revascularization, and induces cxcl8a expression. Using loss-of-function approaches, I observed that both cxcl8a and cxcr1 are required for coronary revascularization after cardiac injury.
Altogether, my findings indicate that Vegfc acts as an angiocrine factor that plays an important role in regulating cardiac regeneration in zebrafish. Mechanistically, Vegfc promotes the expression of emilin2a, which promotes coronary proliferation, at least in part by enhancing Cxcl8a-Cxcr1 signaling. This study helps in understanding the mechanisms underlying coronary revascularization during cardiac regeneration, with promising therapeutic applications for human heart regeneration.
Relationships among laurasiatherian clades represent one of the most highly disputed topics in mammalian phylogeny. In this study, we attempt to disentangle laurasiatherian interordinal relationships using two independent genome-level approaches: (1) quantifying retrotransposon presence/absence patterns, and (2) comparisons of exon datasets at the levels of nucleotides and amino acids. The two approaches revealed contradictory phylogenetic signals, possibly due to a high level of ancestral incomplete lineage sorting. The positions of Eulipotyphla and Chiroptera as the first and second earliest divergences were consistent across the approaches. However, the phylogenetic relationships of Perissodactyla, Cetartiodactyla, and Ferae, were contradictory. While retrotransposon insertion analyses suggest a clade with Cetartiodactyla and Ferae, the exon dataset favoured Cetartiodactyla and Perissodactyla. Future analyses of hitherto unsampled laurasiatherian lineages and synergistic analyses of retrotransposon insertions, exon and conserved intron/intergenic sequences might unravel the conflicting patterns of relationships in this major mammalian clade.
Tree bark constitutes an ideal habitat for microbial communities, because it is a stable substrate, rich in micro-niches. Bacteria, fungi, and terrestrial microalgae together form microbial communities, which in turn support more bark-associated organisms, such as mosses, lichens, and invertebrates, thus contributing to forest biodiversity. We have a limited understanding of the diversity and biotic interactions of the bark-associated microbiome, as investigations have mainly focused on agriculturally relevant systems and on single taxonomic groups. Here we implemented a multi-kingdom metabarcoding approach to analyze diversity and community structure of the green algal, bacterial, and fungal components of the bark-associated microbial communities of beech, the most common broadleaved tree of Central European forests. We identified the most abundant taxa, hub taxa, and co-occurring taxa. We found that tree size (as a proxy for age) is an important driver of community assembly, suggesting that environmental filtering leads to less diverse fungal and algal communities over time. Conversely, forest management intensity had negligible effects on microbial communities on bark. Our study suggests the presence of undescribed, yet ecologically meaningful taxa, especially in the fungi, and highlights the importance of bark surfaces as a reservoir of microbial diversity. Our results constitute a first, essential step toward an integrated framework for understanding microbial community assembly processes on bark surfaces, an understudied habitat and neglected component of terrestrial biodiversity. Finally, we propose a cost-effective sampling strategy to study bark-associated microbial communities across large spatial or environmental scales.
In Europe, the sugar refinery is largely based on sugar beets. This route for obtaining household sugar results in a large amount of biomass waste, consisting mainly of the insoluble beet resi-dues, e.g., cell wall fragments. To a vast moiety this debris consists of the polymer pectin (up to 20% in the dry total solids). The structure of pectin is based on a backbone of D-galacturonic acid units (GalA), but also contains various other sugar monomers, predominantly L-arabinose, D-galactose, L-rhamnose and D-xylose. The amount of GalA adds up to a moiety of up to 70% with-in this sugar cocktail. So far, this debris is only fed to cattle or simply burnt. In nature, pectin is a common substrate for various organisms. The degradation of pectin-rich biomass is often per-formed by filamentous fungi like Hypocrea jecorina (also known as Trichoderma reesei) and As-pergillus niger, which evolved pectinases to degrade the pectin backbone and pathways to con-sume the monomer GalA as a sole carbon source. The fungal catabolism of pectin residues starts with the reduction of GalA to L-galactonate (GalOA) by a GalA-reductase. Even though filamen-tous fungi are native hosts of the GalA-catabolism and certain engineering approaches have al-ready been demonstrated, this class of organisms remains challenging with regard to bioreactor cultivation and tedious genetic accessibility. In contrast, the yeast S. cerevisiae is well known in fermentation processes and easily modified by a versatile set of genetic tools. So far, first ap-proaches have already been conducted to transfer the GalA utilization pathways into S. cerevisiae, but these approaches indicated limitations regarding GalA-uptake and redox cofac-tor replenishment due to the relatively high oxidative state of GalA compared to other sugars like glucose and galactose. Furthermore, the generally strongly increased demand for redox co-factors must be met by GalA reduction by finding new cofactor sources or redirecting reactions of the core metabolism.
This work aimed at the production of GalOA, which is the first intermediate of the fungal GalA catabolism. This compound shows an interesting range of potential applications, for instance as a food and cosmetic additive. To overcome the oxidized character of GalA, the presence of a more reduced co-substrate as a redox donor and as a carbon and energy source was required. To further enhance the reduction of GalA, modulation of the redox-cofactor supply and enzyme engineering were performed.
Generally speaking, protein import into mitochondria and chloroplasts is a post-translational process during which the precursor proteins destined for mitochondria or chloroplasts are translated with cytosolic ribosomes and targeted. The previous results showed that the isolated chloroplasts can import in vitro synthesized proteins and the absence of ribosomes in the immediate area around chloroplasts in electron microscopy (EM) images. However, none of the EM images were recorded in the presence of a translation elongation inhibitor. Also, the observation showed that ribosomes stably bind to purified liver mitochondria in vitro, and the first indication of chloroplast localization of mRNAs encoding plastid proteins in Chlamydomonas rheinhardtii, which challenge the post-translational import and support the co-translational process. Therefore, in this study, the association of the ribosomes to the isolated chloroplasts were analyzed, a binding assay was established and showed that naked ribosomes are not considerably bound to chloroplasts. Additionally, mRNA localize in close vicinity to mitochondria also challenged post-translation protein import. Global analysis of transcripts bound to mitochondria in yeast or human revealed that around half of the transcripts of mitochondrial proteins displayed a high mitochondrial localization. The observed association of mRNAs with chloroplast fractions and the in vivo analysis of the distribution of mRNAs was used as base to formulate the hypothesis that mRNA can bind to chloroplast surface. Therefore, in this study, the mRNA binding assay was established and revealed that mRNAs coding for the mitochondrial cytochrome c oxidase copper chaperone COX17 showed unspecific binding to the chloroplasts. The mRNA coding for chloroplast outer envelope transport protein OEP24 and mRNA coding for the essential nuclear protein 1 (ENP1) showed specific binding, and OEP24 has a 3-fold higher affinity than ENP1 mRNA. Moreover, the BY2-L (Nicotiana tabacum non-green cell culture) could confer the highest enhancement of OEP24 mRNA binding efficiency than the COX17 and ENP1 mRNA and the preparation of the BY2-L was optimized. Afterwards, the feasibility to fix the interaction between mRNA and the proteins on the surface of chloroplasts was confirmed. OEP24 mRNA showed more efficiency in the UV-crosslinking. Following, the pull-down with antisense locked nucleic acid (LNA)/DNA oligonucleotides was established which could be used for the further investigation of the proteins involved in the mRNA binding to the chloroplasts.
Plastic pollution is a pervasive problem. In the environment, both the physical and chemical aspects of the material contribute to pollution. For instance, discarded plastic is useless waste that is fragmented upon degradation and so-called microplastics <5 mm are formed. Besides, the chemicals added into plastics are usually customized for specific functions, but these can easily transfer from the polymer into an ambient medium. This work examined both of these aspects. Moreover, the question of whether ecotoxicological effects are more likely to appear because of the microparticle properties or the chemicals transferring from the microplastics was addressed. A special focus was laid on the UV-weathering-induced chemical release.
First, conventional and biodegradable plastics made from fossil and bio-based resources were chosen. The different materials (pre-production and recycled pellets as well as final products)were weathered and their leachates evaluated in vitro. The leachates were analyzed with nontarget screening in order to measure the number of transferred chemicals. Plastics identified as toxic were subjected to further investigations in vivo. A biodegradable shampoo bottle was processed to microplastics and the particles’ physical and chemical properties were assessed with the freshwater worm Lumbriculus variegatus. Here, commonly used endpoints such as mortality, reproduction and weight were tested via different exposure routes. Moreover, the freshwater shrimp Neocaridina palmata was exposed to microplastic beads and fragments to clarify if the shape of the particles affects the ingestion and egestion, respectively. Thereafter, two materials that displayed the strongest toxic responses in vitro within the first study were weathered and leached. Finally, the shrimps were exposed to the leachates and the locomotor behavior was used as an ecologically relevant but less frequently studied endpoint.
The results of the studies highlight that plastics are chemically complex mixtures, containing a wide range of chemicals in terms of the number and functionality. These chemicals induced oxidative stress, baseline toxicity and endocrine activities. This shows that pellets represent a processing state that comprises chemically heterogenous materials. Moreover, it was shown that a degradation initiator is not necessarily relevant to trigger inherent substances to leach out from plastics. Despite this, the UV-weathering resulted in increasingly released chemicals and exacerbated the in vitro toxicities. Even plastics assessed as toxicologically harmless prior to weathering released toxic chemical mixtures once they were weathered. One recycled and all of the biodegradable plastics were toxicologically most concerning. This means that such materials are currently not better than conventional, virgin plastics in terms of their toxicity.
To clarify the source of the microplastic toxicity, L. variegatus was exposed to biodegradable microplastics. The particles were ingested by the worms and adversely affected the examined endpoints. In comparison, microplastics that were depleted from their chemicals via a solvent treatment were less toxic. Kaolin as a natural particle control was evaluated alongside and positively affected the weight of the worms. This emphasizes the ecological relevance of fine-sized matter for the test species. The chemicals extracted from the microplastics induced a 100% mortality. A chemical analysis of the material revealed two ecotoxicologically relevant biocides. The physically-mediated effects of the microplastics seemed to be less of a concern for the worms, which is probably linked to their adaptation to high concentrations of naturally occurring particles in the environment. However, the effects related to the chemicals of plastic cannot be ignored, especially for materials that are claimed to be environmentally friendly.
In the third study, the role of the particle shape in the gut passaging of N. palmata was studied. While the particle size was a determinant factor for the ingestion, the ingestion and egestion of the beads and fragments did not differ, respectively. The shrimps ingested less fragments when food was provided than in the absence of food. As for the worms, the shrimps are known to ingest many naturally occurring particles. Their unselective feeding behavior towards the particle shape could indicate that microplastics as a physical pollutant are negligible for the shrimps. That is why the chemicals of the two most toxic in vitro materials were tested with N. palmata. However, no trend towards elevated or reduced movements of the shrimps was observed, even though the leachates contained baseline toxicants. This shows that the in vitro toxicities of plastics are not necessarily indicative for effects to occur at the in vivo level...
The European Community has set a milestone in the European water policy in 2000: all water directives and policies were united into one comprehensive document – the European Water Framework Directive (EU WFD). The EU WFD requires the monitoring of 45 priority substances, primarily in the water phase, which is not related to a substantial amount of chemicals available on the market worldwide (about 50,000). About 60% of these are human and environmentally toxic. Hence, the currently monitored 45 priority substances are not even close to being sufficient to provide a comprehensive picture of the actual chemical pollution in the aquatic environment.
Furthermore, the EU WFD in its original shape paid less attention to sediments as an important source and sink for chemical contamination. Under stable hydrological conditions, polluted old sediments are covered by less polluted younger sediments preventing erosion of deeper sediment layers and, therefore, the release of particle-bound contaminants. However, urbanization, deforestation, flooding, dredging, riverbed renaturation, and stormwater overflow basin releases can lead to an unpredictable release of particle-bound pollutants. Therefore, in 2008, sediments were added to the EU WFD as a monitoring matrix for substances that tend to accumulate there. As a result, after 18 years of the EU WFD, less than half of all European waterbodies reached a good ecological (40%) and chemical (38%) status.
One of the primary pollution sources in aquatic ecosystems are wastewater treatment plants (WWTPs). Advanced wastewater treatment by ozonation is promising to remove most micropollutants. However, the knowledge about the possible improvement of the receiving waterbody is rare. The latter aspects were the main reasons for the start of the DemO3AC project in 2014. The study area was located in the federal state of North Rhine-Westphalia (Germany). The study area included the Wurm River and its tributary, the Haarbach River. Both waterbodies act as receiving waterbodies for WWTPs. One of them is the Aachen-Soers WWTP (receiving waterbody: Wurm River), upgraded by full stream ozonation as an advanced effluent treatment. Therefore, the extensive investigation program within the DemO3AC project included an investigation of the ecological and chemical status of both receiving waterbodies and the investigation of a possible improvement of the Wurm River after implementing advanced effluent treatment.
The current study was a part of the DemO3AC project and covered the sediment toxicity and a possible impact of the ozonation on aquatic organisms in the receiving waterbody. Time-resolved sampling campaigns allowed investigations under different hydrological conditions, mainly determined by the weather. The first sampling campaign took place in June 2017 during a prolonged dry period with low water flow in the receiving waterbodies. The second sampling campaign was performed exactly one year later (June 2018) after a long rainy period and corresponding high-water levels. Full-stream ozonation at the Aachen-Soers WWTP had been in operation for half a year. Furthermore, a wide range of organic micropollutants was investigated in the effluent of the studied WWTPs to assess a possible hazard emerging from contaminants released into the receiving waterbody.
The study design was developed based on the holistic approach to assessing the ecotoxicological pollution of surface waterbodies. It included the detection of chemical compounds combined with effect-based methods to identify possible drivers of toxicity. The sediment's ecotoxicological assessment included studies on endocrine-disrupting activity, genotoxic and embryotoxic potentials. These endpoints were evaluated using in vitro and in vivo bioassays. In addition, sediments’ chemical profiling was performed using modern analytical chemistry techniques.
The genotoxic potential was investigated using the Ames fluctuation assay with Salmonella typhimurium bacterial strains TA98, TA100, YG1041, and YG1042, sensitive to different classes of compounds, and the Micronucleus assay as a eukaryotic assay with mammalian cells. A unique feature of the present study was the implementation of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay. Moreover, a comprehensive genotoxicity ranking of chemical compounds identified in sediments was used and combined with statistical analysis to identify the drivers of genotoxicity. The results of this study were published in Shuliakevich et al. (2022a) (see also Annex 1), describing the mutagenic potential of all sampling sites, which was primarily driven by polycyclic aromatic hydrocarbons, nitroarenes, aromatic amines, and polycyclic heteroarenes. In addition, the rainwater overflow basin was identified as a significant source for particle-bound pollutants from untreated wastewater, suggesting its role as a possible source of genotoxic potential. The present study showed high sensitivity and applicability of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay to assess the different classes of mutagenic compounds. A combination of effect-based methods and a chemical analysis was shown as a suitable tool for a genotoxic assessment of freshwater sediments.
The sediments' endocrine-disruptive activity was investigated using the cell-based reporter gene CALUX® assay. A simultaneous launch of the full-scale effluent ozonation at the Aachen-Soers WWTP was used for investigation of the entrance of the ozonated effluent into the Wurm River and the endocrine-disrupting activity in the water phase. A particular focus of the present study was the unique investigation of PAHs as possible drivers of the endocrine-disrupting activity in sediments of the Wurm River. The results of this study were laid down in the publication by Shuliakevich et al. (2022b) (see also Annex 2), describing variations in endocrine-disrupting activity in the Wurm River under different weather conditions. Briefly, under stable hydrological conditions in June 2017, the estrogenic and the antiandrogenic activities in sediments of the Wurm River were within the range of 0.03-0.1 ng E2 equivalents (eq.)/g dry weight sediment equivalents (dw SEQ) and 3.0-13.9 µg Flu eq./g dw SEQ, respectively. After extensive rain events in June 2018, the sediments' estrogenic and antiandrogenic activities were detected within the range of 0.06-0.2 ng E2 eq./g dw SEQ and 1.7-39.2 µg Flu eq./g de SEQ, respectively. Increased endocrine-disruptive activity (up to 0.2 ng E2 eq./g dw SEQ in ERα- and 39.2 µg Flu eq./g dw SEQ in anti-AR-CALUX® assays) in sediments downstream of the rainwater overflow basin suggested it as a possible source of pollution. A unique result of the second study was finding a positive correlation between measured particle-bound antiandrogenic activity and detected polyaromatic hydrocarbons (PAHs) ...
Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish
(2022)
The two diametrically opposing outcomes after tissue damage are regeneration and fibrotic scarring. After injury, adult mammals predominantly induce fibrotic scarring, which most often leads to patient lethality. Fibrotic scarring is the deposition of excessive extracellular matrix that matures and hinders tissue function. The scarring response is mainly orchestrated by myofibroblasts, which arise only upon tissue damage, from various cellular origins, including tissue resident fibroblasts, endothelial cells and circulating blood cells. On the contrary, species like zebrafish, possess the remarkable capacity to regenerate their damaged tissues. After injury, instead of inducing a myofibroblast-mediated fibrogenic gene program, cells in these species undergo regenerative reprogramming at the transcriptional level to activate vital cellular processes needed for regeneration, including proliferation, dedifferentiation, and migration. Several pro-regenerative mechanisms have been identified to date. Most of them, if not all, are also important for tissue homeostasis and hence, are not injury specific. Therefore, the central aim of this study is to identify injury-specific mechanisms that not only induce regeneration, but also limit fibrotic scarring.
To test the notion that fibrotic scarring limits regeneration, I first compared the scarring response in the regenerative zebrafish heart after cryoinjury with what is known in the non-regenerative adult mouse heart. I found that zebrafish display ~10-fold less myofibroblast differentiation compared to adult mouse after cardiac injury. With these findings, I hypothesized that zebrafish employ mechanisms to actively suppress scarring response. Using a novel comparative transcriptomic approach coupled with genetic loss-of-function analyses, I identified that Interleukin-6 (Il-6) cytokine family-mediated Stat3 is one such pro-regenerative pathway in zebrafish.
Il-6 cytokine family consists of Il-6, Interleukin-11 (Il-11), Ciliary neurotrophic factor, Leukemia inhibitory factor, Oncostatin M, and Cardiotrophin-like cytokine factor 1. Il-6 family ligands signal through their specific receptors and a common receptor subunit (Il6st or Gp130). Using gene expression analyses after adult heart and adult caudal fin injuries in zebrafish, I identified that both the Il-11 cytokine encoding paralogous genes (il11a and il11b) are the highest expressed and induced among the Il-6 family cytokines. Hence, I chose Il-11 signaling as a candidate pathway for further analysis. To investigate the role of Il-11 signaling, I generated genetic loss-of-function mutants for both the ligand (il11a and il11b) and the receptor (il11ra) encoding genes. Using various tissue regeneration models across developmental stages in these mutants, I identified that Il-11/Stat3 signaling is indispensable for global tissue regeneration in zebrafish.
To investigate the cellular and molecular mechanisms by which Il-11 signaling promotes regeneration, I performed transcriptomics comparing the non-regenerative il11ra mutant hearts and fins with that of the wild types, respectively. I identified that Il-11 signaling orchestrates both global and tissue-specific aspects of regenerative reprogramming at the transcriptional level. In addition, I also found that impaired regenerative reprogramming in the il11ra mutant hearts and fins resulted in defective cardiomyocyte and osteoblast repopulation of the injured area, respectively.
On the other hand, by deep phenotyping the scarring response in il11ra mutant hearts and fins, I identified that Il-11 signaling limits myofibroblast differentiation. Furthermore, I found that cardiac endothelial cells and fibroblasts are one of the major responders to injury-induced Il-11 signaling. Using lineage tracing, I found that both the endothelial and fibroblast lineages in the non-regenerative il11ra mutants commit to a myofibroblast fate, spearheading the scarring response. In addition, using cell type specific manipulations, I showed that Il-11 signaling in cardiac endothelial cells allows cardiomyocyte repopulation of the injured area. Finally, using human endothelial cells in culture, I uncovered a novel feedback mechanism by which Il-11 signaling limits fibrogenic gene expression by inhibiting its parent activator and a master regulator of tissue fibrosis, TGF-β signaling.
Overall, I identified Interleukin-11/Stat3 signaling as the first global regulator of regeneration in zebrafish. Briefly, I showed that Interleukin-11 signaling promotes regeneration by regulating two crucial cellular aspects in response to injury – (1) it promotes regenerative reprogramming, thereby allowing cell repopulation of the injured area and (2) it limits mammalian-like fibrotic scarring by inhibiting myofibroblast differentiation and TGF-β signaling. Altogether, these zebrafish data, together with the contradicting mammalian data strongly indicate that the secrets of tissue regeneration lie downstream of IL-11 signaling, in the differences between regenerative and non-regenerative species. Furthermore, I establish the non-regenerative il11ra mutant as an invaluable zebrafish model to study mammalian tissue fibrosis.
Premise: Both universal and family-specific targeted sequencing probe kits are becoming widely used for reconstruction of phylogenetic relationships in angiosperms. Within the pantropical Ochnaceae, we show that with careful data filtering, universal kits are equally as capable in resolving intergeneric relationships as custom probe kits. Furthermore, we show the strength in combining data from both kits to mitigate bias and provide a more robust result to resolve evolutionary relationships.
Methods: We sampled 23 Ochnaceae genera and used targeted sequencing with two probe kits, the universal Angiosperms353 kit and a family-specific kit. We used maximum likelihood inference with a concatenated matrix of loci and multispecies-coalescence approaches to infer relationships in the family. We explored phylogenetic informativeness and the impact of missing data on resolution and tree support.
Results: For the Angiosperms353 data set, the concatenation approach provided results more congruent with those of the Ochnaceae-specific data set. Filtering missing data was most impactful on the Angiosperms353 data set, with a relaxed threshold being the optimum scenario. The Ochnaceae-specific data set resolved consistent topologies using both inference methods, and no major improvements were obtained after data filtering. Merging of data obtained with the two kits resulted in a well-supported phylogenetic tree.
Conclusions: The Angiosperms353 data set improved upon data filtering, and missing data played an important role in phylogenetic reconstruction. The Angiosperms353 data set resolved the phylogenetic backbone of Ochnaceae as equally well as the family specific data set. All analyses indicated that both Sauvagesia L. and Campylospermum Tiegh. as currently circumscribed are polyphyletic and require revised delimitation.
The heart is the first functional organ that develops in the embryo. To become a functional organ, it undergoes several morphogenetic processes. These morphogenetic events involve different cell types, that interact with each other and respond to the surrounding extracellular matrix, as well as intrinsic and extrinsic mechanical forces, assuming different behaviors. Additionally, transcription factor networks, conserved among vertebrates, control the development.
To have a better understanding of cell behavior during development, it is necessary to find a model system that allows the investigation in vivo and at single-cell resolution. Thanks to the common evolutionary origin of the different cardiac structures, together with the conserved molecular pathways, the two-chambered zebrafish heart offers many advantages to study cell behavior during cardiac morphogenesis. Here, using the zebrafish heart as a model system, I uncovered the cell behavior behind two of the main cardiac morphogenetic events: cardiac wall maturation and cardiac valve formation.
In the first part of this study, I investigated how the cardiac wall is maintained at the molecular level. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required for myocardial wall integrity. Global loss of snai1b leads to the extrusion of CMs away from the cardiac lumen, a process we show is dependent on cardiac contractility. Examining CM junctions in snai1b mutants, we observed that N-cadherin localization was compromised, thereby likely weakening cell-cell adhesion. In addition, extruding CMs exhibit increased actomyosin contractility basally, as revealed by the specific enrichment of canonical markers of actomyosin tension - phosphorylated myosin light chain (active myosin) and the α-catenin epitope α-18. By comparing the transcriptome of wild-type and snai1b mutant hearts at the early stages of CM extrusion, we found the dysregulation of intermediate filament genes in mutants including the upregulation of desmin b. We tested the role of desmin b in myocardial wall integrity and found that CM-specific desmin b overexpression led to CM extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 is a critical regulator of intermediate filament gene expression in CMs and that it maintains the integrity of the myocardial epithelium during embryogenesis, at least in part by repressing desmin b expression.
In the second part of this study, I focused on the behavior of valve cells during cardiac development. Using the zebrafish atrioventricular valve, I focus on the valve interstitial cells which confer biomechanical strength to the cardiac valve leaflets. We find that initially AV endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the extracellular matrix (ECM) between the two EC monolayers, undergo an endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a pro-migratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b, a well-known regulator of epithelial-to-mesenchymal transition. This study shows for the first time that Nfatc1 regulates zebrafish VICs formation regulating valve EMT in part by regulating twist1b expression. Moreover, it proposes the zebrafish valve as an excellent model to study the cellular and molecular process that regulate VIC development and dysfunction.
In conclusion, my work: 1) identified an unsuspected role of Snai1 in maintaining the integrity of the myocardial epithelium, opening new avenues in its role in regulating cellular contractility; 2) uncovered the function of Nfatc1 in the establishment of the VIC, establishing a new model to study valve development and function.
The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
Despite all advancements in cancer research and clinical practice, cancer remains a life- threatening disease with an increasing incidence. According to a 2018 WHO forecast, cancer incidence will double to approximately 37 million new cancer cases by 2040. Today, clinical management of cancer is based on a "one-fits-all" strategy. Most cancers are still treated by surgical therapy followed by adjuvant or neoadjuvant chemotherapy based on rather strict guidelines (S3 guidelines in Europe) which are based on studies of large cohorts of patients with the same tumor entity. While this approach has led to substantial increases in progression-free survival and overall patient survival, most patients do not benefit from the administered treatment regimen. One reason for this is intra-tumor heterogeneity, which results from clonal evolution between cancer cells and their environment. This means that cancer patients may respond differently to a particular drug due to the different mutation patterns of their tumor cells. Therefore, patients should be screened in advance for reliable cancer biomarkers that definitively predict whether they will respond to a particular therapy. This would increase the probability of a successful treatment.
Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer deaths worldwide. The main cause of death in CRC is a metastatic disease, which is presented in 20 % of patients and eventually develops in more than 30 % of early-stage patients. Despite the significant increase (to more than 30 months) in median survival with the development of cytotoxic agents and the introduction of targeted therapy, the progression-free survival in the first-line setting has remained largely unchanged over the past decade.
The heterogeneity in CRC is characterized by alterations in multiple signaling pathways that affect cellular functions such as cell proliferation or apoptosis. Commonly affected signaling pathways include the mitogen-activated protein kinase (MAPK)- and the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP)-pathway. Alterations in the TGF-β/BMP pathway, due to mutations in the SMAD4 gene (mothers against decapentaplegic homolog 4), are associated with different drug response and promote resistance to chemotherapy. In addition, they are associated with a higher recurrence rate.
SMAD4 is one of the most common cancer driver genes, and mutations occur in up to 15 % of CRC cases. Therefore, there is an urgent need for therapeutic agents that can specifically target SMAD4-mutated tumors.
The aim of the present study was the identification of the clinical relevance of the SMAD4 gene and the investigation of its suitability as a potential biomarker in CRC.
For this purpose, I investigated sibling patient-derived organoids (PDOs) derived from different regions of a chemo-naïve CRC tumor. PDOs are 3D cell cultures that reliably recapitulate the architecture of the tissue of origin, as well as preserve the genomic background and intra-tumor heterogeneity. The sibling PDOs (R1R361H and R4wt) shared the most common CRC mutations, such as KRASG12D (kirsten rat sarcoma), PIK3CAH1047R (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha), and TP53C242F (tumor protein 53), but differed in a SMAD4R361H mutation and showed a different drug response. The single nucleotide variant R361H of the SMAD4 gene is among the most common pathogenic alterations in various cancers, including CRC.
The sibling PDOs showed significant differences in response to the MEK-inhibitors cobimetinib, trametinib, and selumetinib. MEK-inhibitors are antineoplastic agents that inhibit the function of MEK1 and MEK2, preventing phosphorylation of transcription factors, which leads to inhibition of tumor cell proliferation. MEK-inhibitors are approved for the treatment of malignant melanoma. Currently, they are in phase-III clinical trials for the treatment of patients with metastatic CRC.
To investigate whether SMAD4R361H is responsible for sensitivity to MEK-inhibitors, Iestablished three syngeneic PDOs harboring a SMAD4R361H mutation using the CRISPR/Cas9 genome editing system. All CRISPR-PDOs were significantly more sensitive to the MEK-inhibitors, compared to R4wt. I have shown that the SMAD4R361H mutation is responsible for sensitivity to MEK inhibition in CRC models and may be a predictive biomarker.
To test this hypothesis, I examined 62 CRC PDO models and treated them with the MEK-inhibitors cobimetinib, trametinib, and selumetinib. All models that had a pathogenic mutation or deletion in the SMAD4 gene (15 %) were sensitive to cobimetinib, 10 % of models were sensitive to trametinib, and 8 % were sensitive to selumetinib.
I performed transcriptome (RNA sequencing) and proteome analyses using the DigiWest® method to investigate the mechanism underlying MEK-inhibitor sensitivity.
DigiWest® is a Luminex® bead-based analysis that allows the simultaneous analysis of over 100 (phospho-)proteins. The transcriptome and proteome data support the observation that MEK inhibition primarily affects SMAD4R361H PDOs. Furthermore, I have shown that activation of the BMP signaling pathway in organoids with wild-type SMAD4 appears to be responsible for resistance to MEK-inhibitors. Thus, a genetic alteration in the BMP signaling pathway, beyond SMAD4, could lead to sensitivity to MEK-inhibitors.
I identified four genes involved in the TGF-β/BMP signaling pathway that are frequently mutated in CRC and grouped them into the so-called SFAB-signature (SMAD4, FBXW7 (F-box/WD repeat-containing protein 7), ARID1A (AT-rich interactive domain-containing protein 1A), or BMPR2 (Bone morphogenetic protein receptor type II). Clinical data show that approximately 36 % of CRC patients have at least one pathogenic mutation in these genes.
I tested all 62 CRC PDO models and found a significant positive prediction for sensitivity to cobimetinib (95 %) and selumetinib (70 %) for the SFAB-signature. Trametinib and the newly approved MEK-inhibitor binimetinib showed a similar trend. Therefore, the SFAB-signature has high predictive power for response to MEK-inhibitors and could be used as a predictive biomarker panel.
The current clinically used biomarkers for CRC are based on the mutation status of driver genes KRAS and BRAF, which are present in up to 50 % and 10 % of CRC, respectively. Investigation of molecular alterations in CRC revealed that mutations in the KRAS gene, which is downstream of EGFR (epidermal growth factor receptor) in the MAPK-pathway, interfere with an anti-EGFR-antibody therapy (e.g., cetuximab). Therefore, cetuximab is only relevant for RAS wild-type tumors. However, approximately 40 % of patients with RAS wild-type status do not respond to this treatment.
About 53 % of CRC PDO models carry a pathogenic RAS mutation, about 10 % harbor a pathogenic BRAF mutation. Both, the RAS and RAF status alone as well as the combination of RAS and RAF status with SFAB-signature did not provide a better prediction of sensitivity to MEK inhibition.
Eine große Gruppe von Aptameren sind die Guanosintriphosphat (GTP) Aptamere. Diese zeigt sehr eindrücklich, wie RNA unterschiedliche Strategien nutzt, um denselben Liganden zu erkennen. Die komplette Struktur des GTP Klasse II Aptamers wird in der ersten Publikation gezeigt. Interessanterweise zeichnet die Struktur ein stabil protoniertes Adenine unterhalb der GTP-Bindestelle aus. Dieses wurde durch eine Kombination aus weiterführenden NMR- und ITC-Experimente untersucht und charakterisiert. Es zeigte sich, dass die protonierte Base einen pKs-Wert hat, der weit von der Neutralität verschoben ist. Die Protonierung ist auch noch bei sehr basischen Puffern stabil.
Eine Art der funktionellen Protonierung wird von den zyklischen di-Nukleotiden (CDN) bindenden Riboswitches genutzt, um zwei CDN mit ähnlicher Affinität zu binden. c-di-GMP Riboswitches wurden als regulatorische Einheit beschrieben und deren Kristallstruktur aufgeklärt. Mutationsexperimente führten dazu, dass bei einer G-zu-A Mutation an der Gα-Bindestelle die Selektivität des Riboswitches verändert wurde. Die Mutante bindet sowohl c-di-GMP als auch cGAMP mit ähnlichen Bindungsaffinitäten. Riboswitche, die cGAMP binden wurden auch in der bakteriellen Genomen gefunden. Hierbei ist die Promiskuität unterschiedlich stark ausgeprägt. Die Untersuchung des Bindungsmodus und der damit verbundenen Promiskuität ist in der zweiten Publikation beschrieben. Hier wurde gezeigt, dass die Riboswitche beide Liganden nur binden können, wenn zur Bindung von c-di-GMP das Ligand bindende A protoniert vorliegt. Auch diese Protonierung konnte mit weiterführenden NMR- und ITC-Experimenten charakterisiert werden. Die Untersuchungen einer solch großen RNA sind mit NMR Spektroskopie herausfordernd. Hierbei wurde ausgenutzt, dass die Kristallstruktur bereits bekannt war, welche allerdings die Protonierung nicht zeigte. Auch diese Protonierung zeigt einen pKs-Wert, der weit von der Neutralität verschoben ist und außerdem bei unterschiedlichen pH stabil ist.
In den beiden untersuchten Beispielen wurden zwei verschiedene Arten von Protonierung gezeigt: eine strukturelle und eine funktionelle. Das GTP Klasse II Aptamer benutzt die Protonierung als strukturelle Basis für die Basis der Ligandenbindungsstelle. Hierbei werden durch die Protonierung des Adenines mehr nutzbare Wasserstoffbrücken ausgebildet und damit die Tertiärstruktur stabilisiert. Im Unterschied dazu nutzen die promiskuitiven CDN Ribsowicthes die Protonierung, um verschiedene Liganden binden zu können und es kommt damit zu einer Verschiebung der Funktionalität. Der regulatorische Nutzen dafür ist allerdings noch unbekannt.
Auch bei den SAM Riboswitches wurde ein promiskuitiver Vertreter beschrieben. SAM Riboswitches gehören zu den am längsten bekannten Klassen der Riboswitches. Bis heute sind hier die meisten unterschiedlichen Klassen bekannt. SAM wird häufig als Donor für funktionelle Gruppen benutzt, besonders häufig als Methlygruppendonor für die Methylierung einer Reihe unterschiedlicher Substrate (z.B. DNA, Proteine, Metabolite etc.). Bei dieser Reaktion entsteht SAH als Nebenprodukt. Zusätzlich ist SAH zelltoxisch, da es affin an Methyltransferasen bindet und damit diese essenzielle Reaktion inhibiert. Eine enge Kontrolle der SAH-Konzentration ist daher kritisch. SAM bindende Riboswitches haben zu SAM eine bis zu 1000-fach höhere Bindungsaffinität im Vergleich zu SAH. Die Beschreibung eines translationalen OFF-Riboswitches, der SAM und SAH mit ähnlicher Affinität bindet, ist daher überraschend. Zumal seine Genassoziation fast ausschließlich zu SAM Synthetasen ist, deren Regulation durch SAH wenig sinnvoll erscheint. Um ein besseres Verständnis für die Funktion des SAM/SAH Riboswitches zu erhalten, wurde seine 3D-Struktur mittels NMR-Spektroskopie aufgeklärt, wie in der vierten Publikation beschrieben. Dafür mussten zunächst alle Resonanzen der Sequenz und dem Liganden zugeordnet werden, wie in der dritten Publikation beschrieben. Dabei wurde als Ligand SAH gewählt, da dieser chemisch stabiler und damit für die teils tagelangen NMR-Messungen besser geeignet ist. Zusätzlich wurden Mutanten bzw. verwandte Liganden mittels ITC Experimente auf ihre Bindungseigenschaften untersucht, um die Bedeutung der Linkerlänge, einzelner Basenpaare und funktionelle Gruppen des Liganden zu untersuchen. Bei anderen bekannten SAM Riboswitches umschließt die RNA den Liganden fast komplett. Dabei wird zum einem das Sulfoniumion spezifisch durch die Carboxylgruppen verschiedener Uracil-Nukleotide erkennt und koordiniert. Außerdem bildet sich eine Bindetasche aus, die genug Platz für die stabile Bindung der Methylgruppe hat. Beim SAH Riboswitch wird die Selektivität für SAH dadurch erreicht, dass die Bindetasche sterisch keinen Platz für die Methylgruppe von SAM bereitstellt.
Zusammenfassend wurden in dieser Arbeit drei verschiedene Ligand bindende RNA-Strukturen untersucht, die alle sehr unterschiedliche Strategien zur Bindung der Liganden nutzen. Obwohl Portionierungen bei Aptameren und Riboswitches selten beschrieben wurden, haben sie eine maßgebliche Funktion in den beiden zuerst untersuchten Strukturen. Obwohl bisher im Hinblick auf alle bekannten RNA Strukturen eher selten beschrieben, gibt es doch neben den genannten zwei, einige Beispiele für strukturelle oder funktionelle Protonierungen. Auch in Hinblick auf zukünftige bzw. Verbesserung bestehender RNA-Strukturvorhersage-Programme ähnlich wie sie für Proteine schon lange nutzt werden, müssen protonierte Nukleobasen ernsthaft in Betracht gezogen werden. Außerdem konnte gezeigt werden, dass zwei der untersuchten Riboswitches zwei Liganden mit ähnlicher Affinität binden. Die genutzte Strategie ist hierbei unterschiedlich. Während bei den promiskuitiven CDN Riboswitches der regulatorische Nutzen noch unbekannt ist, konnte für den SAM/SAH Ribsowitch gezeigt werden, dass SAH nur zufällig aufgrund der wahrscheinlich sehr niedrigen intrazellulären Konzentration gebunden wird und dieser daher wahrscheinlich später in der evolutionären Entwicklung entstanden ist. Riboswitches halten es weiterhin spannend.
The intensive use of the North Sea area through offshore activities, sand mining, and the spreading of dredged material is leading to increasing pollution of the ecosystem by chemicals such as hydrophobic organic contaminants (HOCs). Due to their toxicological properties and their ability to accumulate in the environment, HOCs are of particular concern. The contaminants partition between aqueous (pore water, overlying water) and solid phases (sediment, suspended particulate matter, and biota) within these systems. The accumulated contaminants in the sediment are of major concern for benthic organisms, who are in close contact with sediment and interstitial water. It is thus particularly important to better understand how contaminants interact with biota, as these animals may contribute to trophic transfer through the food web. Furthermore, sediments are a crucial factor for the water quality of aquatic systems. They not only represent a sink for contaminants but also determine environmental fate, bioavailability, and toxicity. The Marine Strategy Framework Directive (MSFD) was introduced to protect our marine environment across Europe and includes the assessment of pollutant concentrations in the total sediment, which, however, rarely reflects the actual exposure situation. The consideration of the pollutant concentrations in the pore water is not implemented, although this is needed for the evaluation of bioavailability and risk assessment. For this reason, special attention is given to further development, implementation, and validation of pollutant monitoring methods that can determine the bioavailable fraction in sediment pore water. For risk assessment purposes, it is furthermore important to use biological indicators in addition to classical analytics to determine the effect of pollutants on organisms. The main objective of this thesis was to gain insight into the pollution load and the potential risk of hydrophobic organic chemicals (HOCs) in the sediment of the North Sea and to evaluate these results with regard to possible risks for benthic organisms and the ecosystem. The following five aims are covered within these studies to gain a holistic assessment of sediment contamination:
1. Assessment of the pore water concentrations of PAHs and PCBs
2. Determination of the bioturbation potential by macrofauna analysis
3. Application of the SPME method on biological tissue
4. Assessment of recreated environmental mixtures in passive dosing bioassays
5. Development of SPME method for DDT in sediments
The thesis is comprised of three main studies supported by three additional studies ...
Echolocation behavior, a navigation strategy based on acoustic signals, allows scientists to explore neural processing of behaviorally relevant stimuli. For the purpose of orientation, bats broadcast echolocation calls and extract spatial information from the echoes. Because bats control call emission and thus the availability of spatial information, the behavioral relevance of these signals is undiscussable. While most neurophysiological studies, conducted in the past, used synthesized acoustic stimuli that mimic portions of the echolocation signals, recent progress has been made to understand how naturalistic echolocation signals are encoded in the bat brain. Here, we review how does stimulus history affect neural processing, how spatial information from multiple objects and how echolocation signals embedded in a naturalistic, noisy environment are processed in the bat brain. We end our review by discussing the huge potential that state-of-the-art recording techniques provide to gain a more complete picture on the neuroethology of echolocation behavior.
Marine oomycetes are highly diverse, globally distributed, and play key roles in marine food webs as decomposers, food source, and parasites. Despite their potential importance in global ocean ecosystems, marine oomycetes are comparatively little studied. Here, we tested if the primer pair cox2F_Hud and cox2-RC4, which is already well-established for phylogenetic investigations of terrestrial oomycetes, can also be used for high-throughput community barcoding. Community barcoding of a plankton sample from Brudenell River (Prince Edward Island, Canada), revealed six distinct oomycete OTU clusters. Two of these clusters corresponded to members of the Peronosporaceae—one could be assigned to Peronospora verna, an obligate biotrophic pathogen of the terrestrial plant Veronica serpyllifolia and related species, the other was closely related to Globisporangium rostratum. While the detection of the former in the sample is likely due to long-distance dispersal from the island, the latter might be a bona fide marine species, as several cultivable species of the Peronosporaceae are known to withstand high salt concentrations. Two OTU lineages could be assigned to the Saprolegniaceae. While these might represent marine species of the otherwise terrestrial genus, it is also conceivable that they were introduced on detritus from the island. Two additional OTU clusters were grouped with the early-diverging oomycete lineages but could not be assigned to a specific family. This reflects the current underrepresentation of cox2 sequence data which will hopefully improve with the increasing interest in marine oomycetes.
Correction to: Apidologie (2020) 51:1182–1198
https://doi.org/10.1007/s13592-020-00796-9
The article Insights into Ethiopian honey bee diversity based on wing geomorphometric and mitochondrial DNA analyses, written by Hailu, T.G., D’Alvise, P., Tofilski, A. et al., was originally published Online First without Open Access. After publication in volume 51, issue 6, page 1182-1198, the author decided to opt for Open Choice and to make the article an Open Access publication. Therefore, the copyright of the article has been changed to © The Author(s) 2020 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution, and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article is included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Open Access funding enabled and organized by Projekt DEAL.
The acetogenic model bacterium Acetobacterium woodii is well-known to produce acetate by homoacetogenesis from sugars, but under certain conditions minor amounts of ethanol are produced in addition. Here, we have aimed to identify physiological conditions that increase electron and carbon flow towards ethanol production. Ethanol was only produced from fructose but not from H2 + CO2, formate, pyruvate, lactate or alanine. In the absence of Na+, the Wood–Ljungdahl pathway (WLP) of acetate formation is not functional. Therefore, the ethanol yield increased to 0.42 mol/mol (ethanol/fructose) with an ethanol/acetate ratio of 0.28 mol/mol. The presence of bicarbonate/CO2 stimulated electron and carbon flow through the WLP and led to less ethanol produced. Of the 11 potential alcohol dehydrogenase genes, the most upregulated during ethanologenesis was adh4. A deletion of adh4 led to an increase in ethanol production by 100% to a yield of 0.79 mol/mol (ethanol/fructose); this correlated with an increase in transcript abundance of adh6. In sum, our studies revealed low Na+ and bicarbonate/CO2 as factors that trigger ethanol formation and that a deletion of adh4 drastically increased ethanol formation in A. woodii.
Invasive alien species are a well-known and pervasive threat to global biodiversity and human well-being. Despite substantial impacts of invasive alien species, quantitative syntheses of monetary costs incurred from invasions in national economies are often missing. As a consequence, adequate resource allocation for management responses to invasions has been inhibited, because cost-benefit analysis of management actions cannot be derived. To determine the economic cost of invasions in Germany, a Central European country with the 4th largest GDP in the world, we analysed published data collected from the first global assessment of economic costs of invasive alien species. Overall, economic costs were estimated at US$ 9.8 billion between 1960 and 2020, including US$ 8.9 billion in potential costs. The potential costs were mostly linked to extrapolated costs of the American bullfrog Lithobates catesbeianus, the black cherry Prunus serotina and two mammals: the muskrat Ondatra zibethicus and the American mink Neovison vison. Observed costs were driven by a broad range of taxa and mostly associated with control-related spending and resource damages or losses. We identified a considerable increase in costs relative to previous estimates and through time. Importantly, of the 2,249 alien and 181 invasive species reported in Germany, only 28 species had recorded economic costs. Therefore, total quantifications of invasive species costs here should be seen as very conservative. Our findings highlight a distinct lack of information in the openly-accessible literature and governmental sources on invasion costs at the national level, masking the highly-probable existence of much greater costs of invasions in Germany. In addition, given that invasion rates are increasing, economic costs are expected to further increase. The evaluation and reporting of economic costs need to be improved in order to deliver a basis for effective mitigation and management of invasions on national and international economies.
Traditional beekeeping has been playing important socio-economic roles in Ethiopia for millennia. The country is situated in northeast Africa, where ranges of major evolutionary lineages of Apis mellifera adjoin. However, studies on the classification and distribution of subspecies and lineages of honey bees in the country are partly inconsistent, either proposing multiple subspecies and lineages or a unique A. m. simensis. This study was conducted with the aim of elucidating Ethiopian honey bees in reference to African subspecies and major global lineages using wing geometric morphometrics and COI-COII mitochondrial DNA analyses. For this purpose, 660 worker bees were collected from 66 colonies representing highland, midland, and lowland zones in different locations. Both methods indicated that the samples from this study form a distinct cluster together with A. m. simensis reference. In addition, forewing venation patterns showed that most of the Ethiopian samples are separate from all reference subspecies, except A. m. simensis. Analysis of COI-COII sequences revealed five DraI haplotypes (Y2, Y1, A1, and O5’), of which one was new denoted as Y3. Moreover, centroid size strongly associated with elevation. In conclusion, the results supported that Ethiopian honey bees are distinct both at lineage and subspecies levels; however, there is an indication of lineage O in the north.
Autism spectrum disorder (ASD) is a common neurodevelopmental disorder with a multifarious clinical presentation. Even though many genetic risk factors have been identified and studied in mouse models, the neurophysiological mechanisms underlying the autistic phenotype are still unclear. Based on the high rates of comorbidity with epilepsy, it was hypothesized that the balance between excitation and inhibition in neural circuits may be disrupted in autistic individuals.
In this dissertation, synaptic and network activity was measured in three different genetically modified mouse models that exhibit the characteristic behavioral abnormalities of the disorder: the Neurobeachin (Nbea) haploinsufficient mouse, the Neuroligin-3 (Nlgn3) knockout (KO) mouse, and the Neuroligin-4 (Nlgn4) KO mouse. Each of the affected proteins is involved in the formation and/or function of synapses in the central nervous system. Therefore, it was posited that the reduction or deletion of these proteins might alter the balance of excitatory to inhibitory synaptic transmission in individual neurons and in neural circuits. Extracellular recordings in the hippocampal dentate gyrus of anesthetized mice revealed that the excitation-inhibition (E-I) balance was reduced in Nbea haploinsufficient and Nlgn4 KO mice, but unchanged in Nlgn3 KO mice despite a reduction in excitatory synaptic transmission to dentate granule cells. Unexpectedly, the intrinsic excitability of dentate granule cells was altered in all three mouse models. These results imply that a homeostatic increase in the intrinsic excitability is able to compensate for the decreased excitatory transmission in Nlgn3 KO mice, whereas the decreased intrinsic excitability in the Nbea haploinsufficient and Nlgn4 KO mice leads to a reduction in the E-I balance. Taken together, these findings suggest that the influence of genetic factors on the E-I balance might be a potential common mechanism underlying the development of ASD.
The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.
The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5′-untranslated region (5′-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.
Accurate determination of the evolutionary relationships between genes is a foundational challenge in biology. Homology—evolutionary relatedness—is in many cases readily determined based on sequence similarity analysis. By contrast, whether or not two genes directly descended from a common ancestor by a speciation event (orthologs) or duplication event (paralogs) is more challenging, yet provides critical information on the history of a gene. Since 2009, this task has been the focus of the Quest for Orthologs (QFO) Consortium. The sixth QFO meeting took place in Okazaki, Japan in conjunction with the 67th National Institute for Basic Biology conference. Here, we report recent advances, applications, and oncoming challenges that were discussed during the conference. Steady progress has been made toward standardization and scalability of new and existing tools. A feature of the conference was the presentation of a panel of accessible tools for phylogenetic profiling and several developments to bring orthology beyond the gene unit—from domains to networks. This meeting brought into light several challenges to come: leveraging orthology computations to get the most of the incoming avalanche of genomic data, integrating orthology from domain to biological network levels, building better gene models, and adapting orthology approaches to the broad evolutionary and genomic diversity recognized in different forms of life and viruses.
Peronospora belbahrii is one of the most destructive downy mildew diseases that has emerged throughout the past two decades. Due to the lack of quarantine regulations and its possible seed-borne nature, it has spread globally and is now present in most areas in which basil is produced. While most obligate biotrophic, plant parasitic oomycetes are highly host-specific, there are a few that have a wider host range, e.g. Albugo candida, Bremia tulasnei, and Pseudoperonospora cubensis. Recently, it was shown that Peronospora belbahrii is able to infect Rosmarinus, Nepetia, and Micromeria in Israel in cross-infection trials, hinting an extended host range for also this pathogen. In this study, a newly occurring downy mildew pathogen on lavender was investigated with respect to its morphology and phylogeny, and it is shown that it belongs to Peronospora belbahrii as well. Thus, it seems that Peronospora belbahrii is currently extending its host range to additional members of the tribe Mentheae and Ocimeae. Therefore, it seems advisable to scrutinise all commonly used members of these tribes in order to avoid further spread of virulent genotypes.
In Zeiten der globalen Klimaerwärmung und des Klimawandels werden Strategien zur Vermeidung, Reduzierung oder Wiederverwertung von CO2-Emissionen sowie die Abkehr von fossilen Energieträgern immer wichtiger. Aus diesem Grund finden Technologien zur Bindung, Speicherung und Wiederverwertung von CO2 immer größere Aufmerksamkeit und diverse chemische als auch biologische Ansätze werden verfolgt. Eine dieser Möglichkeiten umfasst die Reduktion von CO2 mit Hilfe von molekularem Wasserstoff. Im Prozess der direkten Hydrogenierung von CO2 zu Ameisensäure bzw. Formiat wird nicht nur CO2 gebunden, sondern ebenfalls H2 in flüssiger Form gespeichert. Die Ameisensäure weist gegenüber dem hochflüchtigen Wasserstoffgas verschiedene Vorteile auf und zählt zu der Gruppe der flüssigen, organischen Wasserstoffspeicherverbindungen. Daneben ist das Einsatzgebiet von Ameisensäure als Ausgangstoff für Chemikalien oder als mikrobielle Kohlenstoffquelle sehr vielseitig und die Verbindung erfreut sich zunehmenden Interesses.
Die Natur hält biologische Katalysatoren (Enzyme) für die Reduktion von CO2 bereit. Die Gruppe der obligat anaeroben, acetogenen Bakterien verwendet so genannte Formiatdehydrogenasen als CO2-Reduktasen, um CO2 im Wood-Ljungdahl-Weg (WLP) der Bakterien fixieren zu können. Diese Enzyme katalysieren die reversible 2-Elektronen Reduktion von CO2 zu Ameisensäure. Kürzlich konnte aus den beiden Vertretern A. woodii (mesophil) und T. kivui (thermophil) ein neuartiger, cytoplasmatischer Enzymkomplex isoliert werden. Dieser Enzymkomplex koppelt die Reduktion von CO2 direkt an die Oxidation von H2 und wird deshalb als Wasserstoff-abhängige CO2-Reduktase bezeichnet (engl. hydrogen-dependent CO2 reductase, HDCR). Die HDCR katalysiert dabei die reversible Hydrogenierung von CO2 zu Formiat mit annähernd gleicher Kinetik und gleichen Umsatzraten. Die bei der CO2 Reduktion erreichten Umsatzraten übertrafen dabei bisherige chemische als auch biologische Katalysatoren um mehre Größenordnungen.
Im Hinblick auf die besonderen katalytischen Eigenschaften der HDCRs wurde in dieser Arbeit die biotechnologische Anwendbarkeit der Enzyme als Biokatalysatoren zur Speicherung und Sequestrierung von H2 und CO2 in Form von Ameisensäure untersucht. Im Speziellen wurde ein HDCR-basiertes Ganz-Zell-System für das thermophile Bakterium T. kivui entwickelt. Um eine Ganz-Zell basierte Umwandlung von H2 und CO2 zu Formiat zu gewährleisten, wurde zuvor die Weiterverwertung des Formiats zu Acetat im WLP gestoppt. Durch eine Reduktion des zellulären ATP-Gehalts konnte eine weitere Prozessierung des aus der HDCR-Reaktion gebildeten Formiats im Zellstoffwechsel des Bakteriums unterbunden werden. Die Formiatbildung aus H2 und CO2 wurde in Zellsuspensionen von T. kivui untersucht und charakterisiert. Hier zeigten T. kivui Zellen die höchste spezifische Formiatbildungsrate, die bis dato in der Literatur genannt wurde. Ebenfalls wurde in dieser Arbeit die Umwandlung von Synthesegas (H2 + CO2 und CO) und CO zu Formiat geprüft. Bioenergetisch entkoppelte und auf CO-adaptierte T. kivui Zellen konnten in der Tat Synthesegas exklusiv zu Formiat umsetzen. Um die CO-Verwertung zu Acetat und Formiat im Stoffwechsel der Rnf- (A. woodii) und Ech-Acetogenen (T. kivui) verstehen zu können, wurden Mutanten von Δhdcr, ΔcooS, ΔhydBA, Δrnf and Δech2 von A. woodii und T. kivui zur Hilfe genommen. In beiden Organismen war die CO-basierte Formiatbildung vom Vorhandensein eines funktionalen HDCR-Enzymkomplexes abhängig.
Für eine mögliche biotechnologische Anwendung wurde die Maßstabsvergrößerung des Ganz-Zell-Systems angestrebt und hin zum Bioreaktormaßstab mit kontrollierten Prozessbedingungen skaliert. Diese Arbeit demonstriert die effiziente Umwandlung von H2 und CO2 zu Formiat und vice versa unter Verwendung eines Rührkesselreaktors. Der Prozess zeigte eine Effizienz von 100% für die Umwandlung von CO2 zu Formiat und spezifische Raten von 48.3 mmol g-1 h-1 wurden von A. woodii Zellen erreicht. Die spezifische H2-Produktionsrate (qH2) aus der Ameisensäureoxidation betrug 27.6 mmol g-1 h-1 und mehr als 2.12 M Ameisensäure konnte über einen Zeitraum von 195 h oxidiert werden. Wichtige Parameter der Enzymkatalyse wie Wechselzahl (engl. turnover frequency, TOF) und katalytische Produktivität (engl. turnover number, TON) wurden ebenfalls im Versuch bestimmt. Basierend auf dem generierten Prozessverständnis und der effizienten Reversibilität der katalysierten Reaktionen wurde abschließend ein Ganz-Zell-basierter Bioreaktoraufbau gewählt, der die vielfache Speicherung und Freisetzung von H2 in einem einzigen Rührkesselreaktor und unter Verwendung des gleichen Katalysators ermöglicht. Über eine Prozesszeit von 2 Wochen und 15 CO2 Reduktions-/Formiat Oxidations-Zyklen konnte so im Mittel 330 mM Formiat produziert und oxidiert werden.
Zusammenfassend thematisiert diese Arbeit die biotechnologische Anwendbarkeit eines Ganz-Zell-Systems zur Speicherung und Sequestrierung von H2 und CO2 in Form von Formiat und vice versa. Die katalytische Aktivität der betrachteten Organismen fußt dabei auf der Aktivität eines neuartigen Enzymkomplexes, der erstmals in der Gruppe der acetogenen Bakterien entdeckt wurde. Der als Wasserstoff-abhängige CO2-Reduktase bezeichnete Enzymkomplex könnte die zukünftige Konzipierung Enzym-inspirierter und effizienter chemischer Katalysatoren vorantreiben. Auch der Einsatz des Enzyms/der Zellen in so genannten Hydrogelen oder die Etablierung elektrochemischer Prozesse sind vorstellbar. Diese Arbeit stellt somit eine Basis für mögliche zukünftige Anwendungen des etablierten Ganz-Zell-Systems von A. woodii und T. kivui im Bereich der Wasserstoffökonomie dar.
The increasing demand of the high value ω-3 fatty acids due to its beneficial role for human health, explains the huge need for alternative production ways of ω-3 fatty acids. The oleaginous alga Phaeodactylum tricornutum is a prominent candidate and has been investigated as biofactory for ω-3 fatty acids, e.g. the synthesis of eicosapentaenoic acid (EPA). In general, the growth and the lipid content of diatoms can be enhanced by genetic engineering or are influenced by environmental factors, e.g. nutrients, light or temperature.
In this study, the potential of P. tricornutum as biofactory was improved by heterologously expressing the hexose uptake protein 1 (HUP1) from the Chlorophyte Chlorella kessleri.
An in situ localization study revealed that only the full length HUP1 protein fused to eGFP was correctly targeted to the plasma membrane, whereas the N-terminal sequence of the protein is only sufficient to enter the ER. Protein and gene expression data displayed that the gene-promoter combination was relevant for the expression level of HUP1, while only cells expressing the protein under the light-inducible fcpA promoter showed a significant expression. In these mutants an efficient glucose uptake was detectable under mixotrophic growth condition, low light intensities and low glucose concentrations leading to an increased cell dry weight.
In a second approach, the growth and lipid content of wildtype cells were analyzed in a small 1l photobioreactor. Here, a commercial F/2 medium and a common culture medium, ASP and modified versions were compared. There was neither a significant impact on the growth and lipid content in P. tricornutum cells due to the supplemention of trace elements nor due to elevated salt concentrations in the media. In a modified version of ASP medium, with adapted nitrate and phosphate concentration a constantly high biomass productivity was achieved, yielding the highest value of 82 mg l-1 d-1 during the first three days. This was achieved even though light intensity was reduced by 40%. The differences in biomass productivity as well as the lipid content and the lipid composition underlined the importance of the choice of culture medium and the harvest time for enhanced growth and EPA yields in P. tricornutum.
Non-technical summary: There has been a long history of conflicts, studies, and debate over how to both protect rivers and develop them sustainably. With a pause in new developments caused by the global pandemic, anticipated further implementation of the Paris Agreement and high-level global climate and biodiversity meetings in 2021, now is an opportune moment to consider the current trajectory of development and policy options for reconciling dams with freshwater system health. Technical summary: We calculate potential loss of free-flowing rivers (FFRs) if proposed hydropower projects are built globally. Over 260,000 km of rivers, including Amazon, Congo, Irrawaddy, and Salween mainstem rivers, would lose free-flowing status if all dams were built. We propose a set of tested and proven solutions to navigate trade-offs associated with river conservation and dam development. These solution pathways are framed within the mitigation hierarchy and include (1) avoidance through either formal river protection or through exploration of alternative development options; (2) minimization of impacts through strategic or system-scale planning or re-regulation of downstream flows; (3) restoration of rivers through dam removal; and (4) mitigation of dam impacts through biodiversity offsets that include restoration and protection of FFRs. A series of examples illustrate how avoiding or reducing impacts on rivers is possible – particularly when implemented at a system scale – and can be achieved while maintaining or expanding benefits for climate resilience, water, food, and energy security. Social media summary: Policy solutions and development pathways exist to navigate trade-offs to meet climate resilience, water, food, and energy security goals while safeguarding FFRs.
The ORCID iDs are missing for the second, fifth, and sixth authors. Please see the authors’ respective ORCID iDs here:
Author Christine Hertler’s ORCID iD is: 0000-0002-8252-9674 (https://orcid.org/0000-0002-8252-9674).
Author Jan Ole Berndt’s ORCID iD is: 0000-0001-7241-3291 (https://orcid.org/0000-0001-7241-3291).
Author Ingo J. Timm’s ORCID iD is: 0000-0002-3369-813X (https://orcid.org/0000-0002-3369-813X).
Neuro-vascular communication is essential to synchronize central nervous system development. Here, we identify angiopoietin/Tie2 as a neuro-vascular signaling axis involved in regulating dendritic morphogenesis of Purkinje cells (PCs). We show that in the developing cerebellum Tie2 expression is not restricted to blood vessels, but it is also present in PCs. Its ligands angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are expressed in neural cells and endothelial cells (ECs), respectively. PC-specific deletion of Tie2 results in reduced dendritic arborization, which is recapitulated in neural-specific Ang1-knockout and Ang2 full-knockout mice. Mechanistically, RNA sequencing reveals that Tie2-deficient PCs present alterations in gene expression of multiple genes involved in cytoskeleton organization, dendritic formation, growth, and branching. Functionally, mice with deletion of Tie2 in PCs present alterations in PC network functionality. Altogether, our data propose Ang/Tie2 signaling as a mediator of intercellular communication between neural cells, ECs, and PCs, required for proper PC dendritic morphogenesis and function.
Aim: Knowledge concerning species distribution is important for biodiversity conservation and environmental management. Fungi form a large and diverse group of species and play a key role in nutrient cycling and carbon storage. However, our understanding of fungal diversity and distribution remains limited, particularly at large spatial scales. Here, we predicted the diversity and distribution of ectomycorrhizal and saprotrophic macrofungi at relatively fine spatial resolution at a continental scale and examined the importance of variables that affect the distribution of these two functional groups. Location: Europe. Time period: 1990–2018. Major taxa studied: Macrofungi. Methods: From observations of 1,845 macrofungal species, we predicted the diversity and distribution of two functional groups of macrofungi at a resolution of 5 km across eight European countries based on 25 environmental variables using the MAXENT model. We determined the importance of variables that affect the distribution of these two functional groups of macrofungi using the built-in jackknife test in the model. Results: Analysis of the modelling results showed that eastern Denmark and southern Sweden are biodiversity hotspots for both functional groups of macrofungal species. Tree species and human disturbance (i.e., the human footprint index) were found to be the two most important predictor variables explaining the distribution of ectomycorrhizal and saprotrophic macrofungi. Main conclusions: Overall, our study demonstrates that tree species and human disturbance have played a more important role than climatic factors in determining the diversity and distribution of macrofungi at the continental scale. Our study suggests that fungal diversity and distribution might change considerably if the strongest predictors (i.e., tree species) were to be affected by climate change and/or human activity. Changes in fungal diversity might, in turn, influence other processes, because fungi are important in driving ecosystem processes, such as nutrient and carbon cycling.
An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
With 5-10 newly diagnosed patients per 100,000 people every year, glioblastoma is the most common malignant primary brain tumor. Despite extensive research activity in the last decades, clinical effectiveness of the currently available therapy standard of surgery, radiochemotherapy and tumor-treating fields is still limited and mean survival rates in unselected collectives are only about one year. Accordingly, there is an urgent need to explore new therapeutic options. The current standard of care includes surgery followed by radiation therapy in combination with the alkylating chemotherapeutic agent Temozolomide. Even with successful initial therapy, tumor recurrence is still inevitable. Currently, there are no defined recommendations for clinical management of the disease in the event of tumor recurrence. Only 20-30% of patients qualify for a second surgical resection, while other options include retreatment with Temozolomide, CCNU (Lomustine) or Regorafenib and enrollment in a clinical trial.
The development of immunotherapies for glioblastoma, in particular, has been the focus of intense preclinical and clinical efforts. However, low numbers of mutations and a highly immunosuppressive tumor microenvironment result in glioblastoma being considered an immunologically “cold” tumor. Strategies successfully established in mutagen-induced tumors with antibodies directed against the PD-1, PD-L1 or CTLA-A4 immune checkpoints have therefore failed in glioblastoma.
Cellular immunotherapies based on chimeric antigen receptor (CAR)-technology have emerged as an alternative powerful option to tackle immunologically “cold” tumors. Several CAR-T cell products targeting glioma antigens have been developed and some evidence of clinical activity has been demonstrated. Natural killer (NK) cells as carriers of CAR constructs have several advantages over T cells, including a much lower risk of neurotoxicity and better interaction with immune cells in the microenvironment. Based on the human NK cell line NK-92, a clinical-grade product, suitable as an off-the-shelf therapeutic, has been developed. The NK-92/5.28.z clone (CAR-NK) expresses a CAR based on the HER2-specific antibody FRP5 in addition to signal-enhancing CD28 and CD3ζ domains. Similar to several other tumor entities, overexpression of the growth factor receptor HER2 is often found in glioblastoma patients. Because of its substantial role in the regulation of cell proliferation, survival, differentiation, angiogenesis and invasion, this receptor is classified as an oncogene. HER2 overexpression plays a major role in the malignant transformation of cells and its oncogenic potential has been studied in detail in breast cancer. However, HER2 expression was also found in up to 80% of glioblastomas, which correlates with an impaired probability of survival. Under physiological conditions, HER2 is not expressed in the adult central nervous system, making it a promising target antigen for glioblastoma immunotherapy.
In previous projects, it has already been shown that these CAR-NK cells exhibit a high and specific lytic activity towards HER2+ glioblastoma cells. While repetitive intratumoral injections of CAR-NK cells already significantly extended symptom-free survival in murine orthotopic xenograft models, CAR-NK cell therapy in immunocompetent mice promotes an endogenous anti-tumor immune response which improves tumor control and provides persisting anti-tumor immunity after therapy of early-stage tumors. However, in more advanced tumor models, efficacy is limited and induction of the checkpoint-molecule PD-L1 in response to CAR-NK-cell therapy was identified as a key mechanism of therapy resistance.
Immunotherapy employing the intravenous administration of checkpoint inhibitors has already revolutionized the treatment of various malignant diseases such as melanoma or lung cancer. In particular, the approach of cancer immunotherapy has focused on the systemic administration of antibodies directed against immune checkpoints such as PD-1, PD-L1 and CTLA-4. In glioblastoma, both tumor cells and microglia, the brain-resident macrophages, express PD-L1, which hinders the activation of CD8+ and CD4+ T cells. Therefore, immunotherapy directed against the PD-1/PD-L1 axis represents a promising approach for the treatment of glioblastoma. One problem, however, is the severe toxicity caused by the systemic effects of checkpoint inhibitors, since the immune response is stimulated not only in tumor tissue but also in healthy organs. Serious side effects such as colitis, hepatitis, pancreatitis or hypophysitis, including numerous deaths, have been reported.
This study aimed to improve the efficacy of CAR-NK cell therapy by combining it with adeno-associated virus (AAV)-mediated transfer of anti-PD-1 antibodies as a strategy to enable local combination therapy to control intracranial tumors.
AAVs carrying a payload coding for an anti-PD-1 immunoadhesin (aPD-1) retargeted to HER2-expressing cells by fusion of so-called Designed Ankyrin Repeat Proteins (DARPins) with a viral capsid protein were employed for this to focus checkpoint inhibitor therapy to the tumor area, resulting in high intratumoral and low systemic drug concentrations. ...
We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. In our vector concepts, transgenes are accompanied by two of four clearly distinguishable transformation markers that are embedded in interweaved, but incompatible Lox site pairs. Following Cre-mediated recombination, the genotypes of single and double transgenic individuals were successfully identified by specific marker combinations in 461 scorings.
The ingestion of microplastics (MPs) is well documented for various animals and spherical MPs (beads) in many studies. However, the retention time and egestion of MPs have been examined less, especially for irregular MPs (fragments) which are predominantly found in the environment. Furthermore, the accumulation of such particles in the gastrointestinal tract is likely to determine whether adverse effects are induced. To address this, we investigated if the ingestion and egestion of beads are different to those of fragments in the freshwater shrimp Neocaridina palmata. Therefore, organisms were exposed to 20–20,000 particles L−1 of either polyethylene (PE) beads (41 μm and 87 μm) or polyvinyl chloride (PVC) fragments (<63 μm). Moreover, shrimps were exposed to 20,000 particles L−1 of either 41 μm PE and 11 μm polystyrene (PS) beads or the PVC fragments for 24 h, followed by a post-exposure period of 4 h to analyze the excretion of particles. To simulate natural conditions, an additional fragment ingestion study was performed in the presence of food. After each treatment, the shrimps were analyzed for retained or excreted particles. Our results demonstrate that the ingestion of beads and fragments were concentration-dependent. Shrimps egested 59% of beads and 18% of fragments within 4 h. Particle shape did not significantly affect MP ingestion or egestion, but size was a relevant factor. Medium- and small-sized beads were frequently ingested. Furthermore, fragment uptake decreased slightly when co-exposed to food, but was not significantly different to the treatments without food. Finally, the investigations highlight that the assessment of ingestion and egestion rates can help to clarify whether MPs remain in specific organisms and, thereby, become a potential health threat.
Growing amounts of genomic data and more efficient assembly tools advance organelle genomics at an unprecedented scale. Genomic resources are increasingly used for phylogenetic analyses of many plant species, but are less frequently used to investigate within-species variability and phylogeography. In this study, we investigated genetic diversity of Fagus sylvatica, an important broadleaved tree species of European forests, based on complete chloroplast genomes of 18 individuals sampled widely across the species distribution. Our results confirm the hypothesis of a low cpDNA diversity in European beech. The chloroplast genome size was remarkably stable (158,428 ± 37 bp). The polymorphic markers, 12 microsatellites (SSR), four SNPs and one indel, were found only in the single copy regions, while inverted repeat regions were monomorphic both in terms of length and sequence, suggesting highly efficient suppression of mutation. The within-individual analysis of polymorphisms showed >9k of markers which were proportionally present in gene and non-gene areas. However, an investigation of the frequency of alternate alleles revealed that the source of this diversity originated likely from nuclear-encoded plastome remnants (NUPTs). Phylogeographic and Mantel correlation analysis based on the complete chloroplast genomes exhibited clustering of individuals according to geographic distance in the first distance class, suggesting that the novel markers and in particular the cpSSRs could provide a more detailed picture of beech population structure in Central Europe.
The effect of the extreme summer drought and heatwave 2018 in Central Europe on wood properties of oaks at four sandy valley river sites (Quercus robur L.) and one south-exposed schist slope (Qu. petraea (Matt.) Liebl.) in the middle Rhine and lower Main valley were studied and compared to well-watered trees from a riparian stand. While properties of the 2018 tree rings mostly resembled those of the previous (wet) year, significant decreases in Δ13C, wood density and ring width occurred in 2019 at most drought-prone sites. In the sandy sites, ring widths correlated with previous-year precipitation from June to August over a 20-year period. In organs formed in 2018, in general, decreasing Δ13C values were obtained in the order leaves, twigs, wood and acorns, with the values from acorns often resembling those from 2019-year rings. The observed changes indicated an increased intrinsic water use efficiency and lack of starch reserve formation during the unprecedented hot and dry summer 2018. Qu. petraea revealed quite different values from Qu. robur (lower Δ13C, wider and denser year rings), but qualitatively showed the same reaction to the drought in 2018, except for an enhanced formation of tyloses in recent-year tree rings.
Acinetobacter baumannii is outstanding for its ability to cope with low water activities which significantly contributes to its persistence in hospital environments. The vast majority of bacteria are able to prevent loss of cellular water by amassing osmoactive compatible solutes or their precursors into the cytoplasm. One such precursor of an osmoprotectant is choline that is taken up from the environment and oxidized to the compatible solute glycine betaine. Here, we report the identification of the osmotic stress operon betIBA in A. baumannii. This operon encodes the choline oxidation pathway important for the production of the solute glycine betaine. The salt-sensitive phenotype of a betA deletion strain could not be rescued by addition of choline, which is consistent with the role of BetA in choline oxidation. We found that BetA is a choline dehydrogenase but also mediates in vitro the oxidation of glycine betaine aldehyde to glycine betaine. BetA was found to be associated with the membrane and to contain a flavin, indicative for BetA donating electrons into the respiratory chain. The choline dehydrogenase activity was not salt dependent but was stimulated by the compatible solute glutamate.
The original version of this Article contained errors where Table S5 and Table S6 were incorrectly cited. As the result, in the Methods section, under the subheading ‘Germline transformation, crossing setups and insertion junction sequencing’, “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S4).” now reads: “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S5).” And, under the subheading ‘Light sheet-based fluorescence microscopy’, “Metadata for the three datasets are provided in Table S5.” now reads: “Metadata for the three datasets are provided in Table S6.” In Data availability section, “Microscopy data can be accessed as described in Table S5.” now reads: “Microscopy data can be accessed as described in Table S6.” Additionally, in the Supplementary Information 8 file, the “Data Access” row was omitted in Table S6. The “Data Access” row now reads: Dataset (DS) DS0001 DS0002 DS0003 Dataset Access DOI: 10.5281/zenodo.4892363 DOI: 10.5281/zenodo.4892373 DOI: 10.5281/zenodo.4892381 The original Supplementary Information 8 file is provided below. Finally, the Supplementary Information 1 and 5 files published with this Article contained tracked changes, these have now been removed. The original Article and accompanying Supplementary Information files have been corrected.
Mosquito breeding sites are complex aquatic environments with wide microbial diversity and physicochemical parameters that can change over time during the development of immature insect stages. Changes in biotic and abiotic conditions in water can alter life-history traits of adult mosquitos but this area remains understudied. Here, using microbial genomic and metabolomics analyses, we explored the metabolites associated with Aedes aegypti breeding sites as well as the potential contribution of Klebsiella sp., symbiotic bacteria highly associated with mosquitoes. We sought to address whether breeding sites have a signature metabolic profile and understand the metabolite contribution of the bacteria in the aquatic niches where Ae. aegypti larvae develop. An analysis of 32 mosquito-associated bacterial genomes, including Klebsiella, allowed us to identify gene clusters involved in primary metabolic pathways. From them, we inferred metabolites that could impact larval development (e.g., spermidine), as well as influence the quality assessment of a breeding site by a gravid female (e.g., putrescine), if produced by bacteria in the water. We also detected significant variance in metabolite presence profiles between water samples representing a decoupled oviposition event (oviposition by single females and manually deposited eggs) versus a control where no mosquito interactions occurred (PERMANOVA: p < 0.05; R2 = 24.64% and R2 = 30.07%). Five Klebsiella metabolites were exclusively linked to water samples where oviposition and development occurred. These data suggest metabolomics can be applied to identify compounds potentially used by female Ae. aegypti to evaluate the quality of a breeding site. Elucidating the physiological mechanisms by which the females could integrate these sensory cues while ovipositing constitutes a growing field of interest, which could benefit from a more depurated list of candidate molecules.
Background: Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results: A fed-batch system with plant biomass-derived sugars D-xylose, L-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and L-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion: This work has identified two additional CAZyme inducers for T. aurantiacus, L-arabinose and cellobiose, along with D-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.
RATIONALE: RBPs (RNA-binding proteins) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP SRSF4 (serine/arginine-rich splicing factor 4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. OBJECTIVE: To investigate the role of SRSF4 in the heart. METHODS AND RESULTS: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long noncoding RNAs, including GAS5 (growth arrest-specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotide- resolution cross-linking and immuno-precipitation. GAS5 is a repressor of the GR (glucocorticoid receptor) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. CONCLUSIONS: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in patients with Cushing syndrome.
Aim: The identification of the mechanisms determining spatial variation in biological diversity along elevational gradients is a central objective in ecology and biogeography. Here, we disentangle the direct and indirect effects of abiotic drivers (climatic conditions, and land use) and biotic drivers (vegetation structure and food resources) on functional diversity and composition of bird and bat assemblages along a tropical elevational gradient. Location: Southern slopes of Mt. Kilimanjaro, Tanzania, East Africa. Methods: We counted birds and recorded bat sonotypes on 58 plots distributed in near-natural and anthropogenically modified habitats from 700 to 4,600 m above sea level. For the recorded taxa, we compiled functional traits related to movement, foraging and body size from museum specimens and databases. Further, we recorded mean annual temperature, precipitation, vegetation complexity as well as the number of fruits, flowers, and insect biomass as measures of resource availability on each study site. Results: Using path analyses, we found similar responses of bird and bat functional diversity to the variation in abiotic and biotic drivers along the elevational gradient. In contrast, the functional composition of both taxa showed distinct responses to abiotic and biotic drivers. For both groups, direct temperature effects were most important, followed by resource availability, precipitation and vegetation complexity. Main Conclusions: Our findings indicate that physiological and metabolic constraints imposed by temperature and resource availability determine the functional diversity of bird and bat assemblages, whereas the composition of individual functional traits is driven by taxon-specific processes. Our study illustrates that distinct filtering mechanisms can result in similar patterns of functional diversity along broad environmental gradients. Such differences need to be taken into account when it comes to conserving the functional diversity of flying vertebrates on tropical mountains.
Toxicogenomic differentiation of functional responses to fipronil and imidacloprid in Daphnia magna
(2021)
Active substances of pesticides, biocides or pharmaceuticals can induce adverse side effects in the aquatic ecosystem, necessitating environmental hazard and risk assessment prior to substance registration. The freshwater crustacean Daphnia magna is a model organism for acute and chronic toxicity assessment representing aquatic invertebrates. However, standardized tests involving daphnia are restricted to the endpoints immobility and reproduction and thus provide only limited insights into the underlying modes-of-action. Here, we applied transcriptome profiling to a modified D. magna Acute Immobilization test to analyze and compare gene expression profiles induced by the GABA-gated chloride channel blocker fipronil and the nicotinic acetylcholine receptor (nAChR) agonist imidacloprid. Daphnids were expose to two low effect concentrations of each substance followed by RNA sequencing and functional classification of affected gene ontologies and pathways. For both insecticides, we observed a concentration-dependent increase in the number of differentially expressed genes, whose expression changes were highly significantly positively correlated when comparing both test concentrations. These gene expression fingerprints showed virtually no overlap between the test substances and they related well to previous data of diazepam and carbaryl, two substances targeting similar molecular key events. While, based on our results, fipronil predominantly interfered with molecular functions involved in ATPase-coupled transmembrane transport and transcription regulation, imidacloprid primarily affected oxidase and oxidoreductase activity. These findings provide evidence that systems biology approaches can be utilized to identify and differentiate modes-of-action of chemical stressors in D. magna as an invertebrate aquatic non-target organism. The mechanistic knowledge extracted from such data will in future contribute to the development of Adverse Outcome Pathways (AOPs) for read-across and prediction of population effects.
Sleep is one of the fundamental requirements of all animals from nematodes to humans. It appears in different formats with shared features such as reduced muscle activities and reduced responsiveness to the environment. Despite the long history of sleep research, why a brain must be taken offline for a large portion of each day remains unknown. Moreover, sleep research focused on mammals and birds reveals two stages, rapid-eye-movement (REM) and slow-wave (SW) sleep, alternating during sleep. Whether these two stages of sleep exist in other vertebrates, particularly reptiles, is debated, as is the evolution of sleep in general.
Recordings from the brain of a lizard, the Australian bearded dragon Pogona vitticeps, indicate the presence of two electrophysiological states and provides a better picture of their sleep. Local field potential (LFP) signals, head velocity, eye movements, and heart rate during sleep match the pattern of REM and SW sleep in mammals. The SW and REM sleep patterns that we observed in lizards oscillated continuously for 6 to 10 hours with a period of 80-100 seconds when the ambient temperature was ~27°C. Lizard SW dynamics closely resemble those observed in rodent hippocampal CA1, yet originated from a brain area, the dorsal ventricular ridge (DVR), that does not correspond anatomically or transcriptomically to the mammalian hippocampus. This finding pushes back the probable evolution of these dynamics to the emergence of amniotes, at least 300 million years ago.
Unlike mammals and birds, REM and SW sleep in lizards occupy an almost equal amount of time during sleep. The clock-like alternation between these two sleep states was found initially by measuring the power modulation of two frequency bands, delta and beta. I recorded the full-band LFP and found an infra-slow oscillation (ISO) in the frequency range between 5 and 20 milli-Hz during sleep. The magnitude of ISO increased during sleep and decreased during both wakefulness and arousal during sleep. The up- and down-states of ISO were synchronized with the sleep state alternating rhythm but with a significant time lag dependent on the locations of the recording electrodes. Multi-site LFP recordings indicated that this ISO is a putative propagation wave sweeping extremely slowly, 30-67 µm/sec, from the posterior-dorsal pole to the anterior-ventral pole of the DVR.
Previous studies in other animals showed that brainstem areas such as the locus coeruleus, laterodorsal tegmentum, and periaqueductal gray are involved in sleep states regulation. It is sadly impossible to carry out in vivo recordings in the lizard brainstem without severely affecting them and their quality of life. I thus carried out ex vivo recordings in both DVR and brainstem. Pharmacological stimulation of the brainstem could reversibly silence one distinct EEG pattern characteristic of SW sleep, the sharp-wave and ripple complex, in DVR. An ISO could be recorded simultaneously in both DVR and brainstem. From data collected in both intact and split ex vivo brains, I concluded that there are independent ISO generators in at least two areas, the brainstem and the telencephalon. Their signals may normally be synchronized by long-range connections. The DVR ISO leads the brainstem ISO by ~29 sec. Optogenetic stimulation of brainstem neurons was able to disrupt the ISO in DVR reversibly.
In conclusion, the lizard brain offers a relatively simple model system to study sleep. Despite a diversity of results in different lizard species, my results revealed a number of new findings. Relevant for sleep research in general: 1) REM and SW sleep exist in a reptile. Since they also exist in birds and mammals, they probably existed in their common amniote ancestor, if not earlier. 2) REM and SW occupy equal amounts of time during sleep (50% duty cycle), a unique feature among all described sleep electrophysiological patterns, suggesting the possible existence of a simple central pattern generator of sleep, possibly ancestral. 3) I discovered the existence, in the local field potential, of an infra slow oscillation with extremely slow propagation, locked to the SW-REM alternating rhythm. The causes and mechanisms of this ISO remain to be understood. To my knowledge, the correlation between sleep states and a slow rhythm has only been reported in human scalp EEG recordings so far.
Recently, the potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and its potential transformation products 4-methyl-7-ethylaminocoumarin (C47T1) and 4-methyl-7-aminocoumarin (C47T2) were identified as novel environmental contaminants. We assessed for the first time the sources, distribution, and fate of these compounds in aquatic systems using the Holtemme River (Saxony-Anhalt, Germany), which is a hotspot for these contaminants. To this end, wastewater-treatment plant (WWTP) influent and effluent samples, surface water samples over 3 years, and the longitudinal profiles in water, sediment, and gammarids were analyzed. From the longitudinal profile of the river stretch, the WWTP of Silstedt was identified as the sole point source for these compounds in the River Holtemme, and exposure concentrations in the low micrograms per liter range could be recorded continuously over 3 years. Analysis of WWTP influent and effluent showed a transformation of approximately half of the C47 into C47T1 and C47T2 but no complete removal. A further attenuation of the three coumarins after discharge into the river could be largely attributed to dilution, while transformation was only approximately 20%, thus suggesting a significant persistence in aquatic systems. Experimentally derived partitioning coefficients between water and sediment organic carbon exceeded those predicted using the OPERA quantitative structure–activity relationship tools and polyparameter linear free-energy relationships by up to 93-fold, suggesting cation binding as a significant factor for their sorption behavior. Near-equilibrium conditions between water and sediment were not observed close to the emitting WWTP but farther downstream in the river. Experimental and predicted bioaccumulation factors for gammarids were closely matching, and the concentrations in field-sampled gammarids were close to steady state with exposure concentrations in the water phase of the river. Environ Toxicol Chem 2021;40:3078–3091. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains (N/CDDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of “designer” NRPS from proteins that normally do not interact. The multiprotein “peptide-antimicrobial-Xenorhabdus” (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small CDD of PaxA cooperates with its preceding thiolation (T1) domain to bind the NDD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.
The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.
Acetogenic bacteria are already established as biocatalysts for production of high-value compounds from C1 substrates such as H2 + CO2 or CO. However, little is known about the physiology, biochemistry and bioenergetics of acetogenesis from formate, an interesting feedstock for biorefineries. Here, we analysed formate metabolism in the model acetogen Acetobacterium woodii. Cells grew optimally on 200 mM formate to an optical density of 0.6. Formate was exclusively converted to acetate (and CO2) with a ratio of 4.4:1. Transcriptome analyses revealed genes/enzymes involved in formate metabolism. Strikingly, A. woodii has two genes potentially encoding a formyl-THF synthetase, fhs1 and fhs2. fhs2 forms an operon with a gene encoding a potential formate transporter, fdhC. Deletion of fhs2/fdhC led to a reduced growth rate, formate consumption and optical densities. Acetogenesis from H2 + CO2 was accompanied by transient formate production; strikingly, formate reutilization was completely abolished in the Δfhs2/fdhC mutant. Take together, our studies gave the first detailed insights into the formatotrophic lifestyle of A. woodii.
Non-ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural products, including many clinically used drugs. Efficient engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) is an ongoing challenge. Here we describe a cloning and co-expression strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 220 mg L−1. Extending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into independently expressed and translated polypeptide chains. These synthetic type of NRPS (type S) enables easier access to engineering, overcomes cloning limitations, and provides a simple and rapid approach to building peptide libraries via the combination of different NRPS subunits.
Methanol is the simplest of all alcohols, is universally distributed in anoxic sediments as a result of plant material decomposition and is constantly attracting attention as an interesting substrate for anaerobes like acetogens that can convert bio-renewable methanol into value-added chemicals. A major drawback in the development of environmentally friendly but economically attractive biotechnological processes is the present lack of information on biochemistry and bioenergetics during methanol conversion in these bacteria. The mesophilic acetogen Eubacterium callanderi KIST612 is naturally able to consume methanol and produce acetate as well as butyrate. To grasp the full potential of methanol-based production of chemicals, we analysed the genes and enzymes involved in methanol conversion to acetate and identified the redox carriers involved. We will display a complete model for methanol-derived acetogenesis and butyrogenesis in Eubacterium callanderi KIST612, tracing the electron transfer routes and shed light on the bioenergetics during the process.
The constitution and regulation of effector repertoires shape host–microbe interactions. Ustilago maydis and Sporisorium reilianum are two closely related smut fungi, which both infect maize but cause distinct disease symptoms. Understanding how effector orthologs are regulated in these two pathogens can therefore provide insights into the evolution of different infection strategies. We tracked the infection progress of U. maydis and S. reilianum in maize leaves and used two distinct infection stages for cross-species RNA-sequencing analyses. We identified 207 of 335 one-to-one effector orthologs as differentially regulated during host colonization, which might reflect the distinct disease development strategies. Using CRISPR-Cas9-mediated gene conversion, we identified two differentially expressed effector orthologs with conserved function between two pathogens. Thus, differential expression of functionally conserved genes might contribute to species-specific adaptation and symptom development. Interestingly, another differentially expressed orthogroup (UMAG_05318/Sr10075) showed divergent protein function, providing a possible case for neofunctionalization. Collectively, we demonstrated that the diversification of effector genes in related pathogens can be caused both by alteration on the transcriptional level and through functional diversification of the encoded effector proteins.
Global landscapes are changing due to human activities with consequences for both biodiversity and ecosystems. For single species, terrestrial mammal population densities have shown mixed responses to human pressure, with both increasing and decreasing densities reported in the literature. How the impacts of human activities on mammal populations translates into altered global density patterns remains unclear. Here we aim to disentangle the effect of human impacts on large-scale patterns of mammal population densities using a global dataset of 6729 population density estimates for 468 mammal species (representing 59% and 44% of mammalian orders and families). We fitted a mixed effect model to explain the variation in density based on a 1-degree resolution as a function of the human footprint index (HFI), a global proxy of direct and indirect human disturbances, while accounting for body mass, trophic level and primary productivity (normalized vegetation index; NDVI). We found a significant positive relationship between population density and HFI, where population densities were higher in areas with a higher HFI (e.g. agricultural or suburban areas – no populations were located in very high HFI urban areas) compared to areas with a low HFI (e.g. wilderness areas). We also tested the effect of the individual components of the HFI and still found a consistent positive effect. The relationships remained positive even across populations of the same species, although variability among species was high. Our results indicate shifts in mammal population densities in human modified landscapes, which is due to the combined effect of species filtering, increased resources and a possible reduction in competition and predation. Our study provides further evidence that macroecological patterns are being altered by human activities, where some species will benefit from these activities, while others will be negatively impacted or even extirpated.
More than 2 million tons of glycerol are produced during industrial processes each year and, therefore, glycerol is an inexpensive feedstock to produce biocommodities by bacterial fermentation. Acetogenic bacteria are interesting production platforms and there have been few reports in the literature on glycerol utilization by this ecophysiologically important group of strictly anaerobic bacteria. Here, we show that the model acetogen Acetobacterium woodii DSM1030 is able to grow on glycerol, but contrary to expectations, only for 2–3 transfers. Transcriptome analysis revealed the expression of the pdu operon encoding a propanediol dehydratase along with genes encoding bacterial microcompartments. Deletion of pduAB led to a stable growth of A. woodii on glycerol, consistent with the hypothesis that the propanediol dehydratase also acts on glycerol leading to a toxic end-product. Glycerol is oxidized to acetate and the reducing equivalents are reoxidized by reducing CO2 in the Wood–Ljungdahl pathway, leading to an additional acetate. The possible oxidation product of glycerol, dihydroxyacetone (DHA), also served as carbon and energy source for A. woodii and growth was stably maintained on that compound. DHA oxidation was also coupled to CO2 reduction. Based on transcriptome data and enzymatic analysis we present the first metabolic and bioenergetic schemes for glycerol and DHA utilization in A. woodii.
Objectives: The four-dimensional ultrasound (4D-US) enables imaging of the aortic segment and simultaneous determination of the wall expansion. The method shows a high spatial and temporal resolution, but its in vivo reliability is so far unknown for low-measure values. The present study determines the intraobserver repeatability and interobserver reproducibility of 4D-US in the atherosclerotic and non-atherosclerotic infrarenal aorta. Methods: In all, 22 patients with non-aneurysmal aorta were examined by an experienced examiner and a medical student. After registration of 4D images, both the examiners marked the aortic wall manually before the commercially implemented speckle tracking algorithm was applied. The cyclic changes of the aortic diameter and circumferential strain were determined with the help of custom-made software. The reliability of 4D-US was tested by the intraclass correlation coefficient (ICC). Results: The 4D-US measurements showed very good reliability for the maximum aortic diameter and the circumferential strain for all patients and for the non-atherosclerotic aortae (ICC >0.7), but low reliability for circumferential strain in calcified aortae (ICC = 0.29). The observer- and masking-related variances for both maximum diameter and circumferential strain were close to zero. Conclusions: Despite the low-measured values, the high spatial and temporal resolution of the 4D-US enables a reliable evaluation of cyclic diameter changes and circumferential strain in non-aneurysmal aortae independent from the observer experience but with some limitations for calcified aortae. The 4D-US opens up a new perspective with regard to noninvasive, in vivo assessment of kinematic properties of the vessel wall in the abdominal aorta.
Genetic engineering of Saccharomyces cerevisiae for improved cytosolic isobutanol biosynthesis
(2021)
The finite nature of fossil resources and the environmental problems caused by their excessive usage requires alternative approaches. The transformation from a fossil based economy to one based on renewable biomass is called a “bioeconomy”. To substitute fossil resources, various microorganisms have already been modified for the biosynthesis of valuable chemicals from biomass. However, the development of such efficient microorganisms at an industrial scale, remains a major challenge. The most prominent and robust microorganism for industrial production is the yeast Saccharomyces cerevisiae, which is known to produce ethanol that is used as renewable biofuel. However, S. cerevisiae is also naturally able to produce isobutanol in small amounts. Isobutanol is favoured as a biofuel compared to ethanol due to its higher octane number and lower hygroscopicity, which makes it more suitable for application in conventional combustion engines. In S. cerevisiae, the biosynthesis of isobutanol is permitted by the combination of mitochondrial valine synthesis (catalysed by Ilv2, Ilv5 and Ilv3) and its cytosolic degradation (catalysed by Aro10 and Adh2). The different compartmentalisation of the two pathways limit isobutanol biosynthesis. Thus, Brat et al. (2012) were able to increase the isobutanol yield up to 15 mg/gGlc by cytosolic re localisation of the enzymes Ilv2Δ54, Ilv5Δ48 and Ilv3Δ19 (cyt-ILV), with simultaneous deletion of ilv2. This corresponds to approximately 3.7% of the theoretical yield of 410 mg/gGlc, implying existing limitations in isobutanol biosynthesis, which have been investigated in this work.
For yet unknown reasons, isobutanol was only produced by S. cerevisiae in a valine free medium, according to Brat et al. (2012). This work shows that this can be attributed to the catalytic activity of Ilv2Δ54, which acted as growth inhibitor to S. cerevisiae. By this logic, a negative selection on the ILV2∆54 gene was exerted, which made the ilv2 deletion and simultaneous valine exclusion necessary to maintain the functional expression of toxic ILV2∆54. Furthermore, it was shown that valine exclusion is not mandatory due to the feedback regulation of Ilv2, permitted by Ilv6. Rather, increased isobutanol yield was observed when cytosolic Ilv6∆61 was expressed in the valine free medium, which is explained by the enhanced regulation of Ilv2Δ54 by Ilv6∆61 when BCAA are absent. Isobutanol biosynthesis is neither redox nor NAD(P)H co factor balanced. It was seen that co factor imbalance could be mitigated by the expression of an NADH oxidase (NOX), but not by expression of the NADH dependent ilvC6E6, since the latter showed low in vivo activity. Furthermore, it was seen that NAD(H) imbalance did already limit isobutanol biosynthesis, but the NADP(H) imbalance did not. Another limitation of cytosolic isobutanol biosynthesis is the secretion of the intermediate 2‑dihydroxyisovalerate, which then no longer is taken up by S. cerevisiae, causing a reduced isobutanol yield. This is attributed to insufficient Ilv3∆19 activity, due to poor iron sulphur cluster apo protein maturation. Therefore, it was aimed to replace Ilv3∆19 by heterologous dihydroxyacid dehydratases. Even though some of the enzymes were functionally expressed, none showed better in vivo activity than Ilv3∆19. Therefore, the Ilv3∆19 apo protein maturation was improved. This was achieved by the genomic deletion of fra2 or pim1 as well as by the cytosolic expression of Grx5∆29.
In addition to the isobutanol pathway, S. cerevisiae was optimised for isobutanol biosynthesis by rational and evolutionary engineering. For this purpose, the genes which are necessary for isobutanol production were integrated into the ilv2 locus, and the resulting strain was evolved in a medium containing the toxic amino acid analogue norvaline. Evolved single colonies were isolated, which presented improved growth and increased isobutanol yields (0.59 mg/gGlc) in a valine free medium, as compared to the initial strain. This is explained by a gene dosage effect which occurred during the evolutionary engineering experiment. In collaboration with Dr. Wess, the genes ilv2, bdh1/2, leu4/9, ecm31, ilv1, adh1, gpd1/2 and ald6 were cumulatively deleted in CEN.PK113 7D to block competing metabolic pathways. The resulting strain JWY23 achieved isobutanol yields up to 67.3 mg/gGlc, when expressing the cyt ILV enzymes from a multi copy vector. The most promising approaches of this work, namely the deletion of fra2 and the expression of Grx5∆29, Ilv6∆61, and NOX, were confirmed in this JWY23 strain. The highest isobutanol yield from this work was observed at 72 mg/gGlc for Ilv6∆61 and cyt ILV enzymes expressing JWY23, which corresponds to 17.6% of the theoretical isobutanol yield.
Isobutyric acid (IBA) is a by product of isobutanol biosynthesis, but it is also considered a valuable platform chemical. Therefore, the approaches that improved isobutanol biosynthesis were applied to the biosynthesis of IBA in S. cerevisiae. The highest IBA yield of 9.8 mg/gGlc was observed in a valine free medium by expression of cyt ILV enzymes, NOX and Ald6 in JWY04 (CEN.PK113 7D Δilv2; Δbdh1; Δbdh2; Δleu4; Δleu9; Δecm31; Δilv1). This corresponded to an 8.9 fold increase compared with the control and is, to our best knowledge, the highest IBA yield reported to date for S. cerevisiae.
Climate change causes increased tree mortality leading to canopy loss and thus sun-exposed forest floors. Sun exposure creates extreme temperatures and radiation, with potentially more drastic effects on forest organisms than the current increase in mean temperature. Such conditions might potentially negatively affect the maturation of mushrooms of forest fungi. A failure of reaching maturation would mean no sexual spore release and, thus, entail a loss of genetic diversity. However, we currently have a limited understanding of the quality and quantity of mushroom-specific molecular responses caused by sun exposure. Thus, to understand the short-term responses toward enhanced sun exposure, we exposed mushrooms of the wood-inhabiting forest species Lentinula edodes, while still attached to their mycelium and substrate, to artificial solar light (ca. 30°C and 100,000 lux) for 5, 30, and 60 min. We found significant differentially expressed genes at 30 and 60 min. Eukaryotic Orthologous Groups (KOG) class enrichment pointed to defense mechanisms. The 20 most significant differentially expressed genes showed the expression of heat-shock proteins, an important family of proteins under heat stress. Although preliminary, our results suggest mushroom-specific molecular responses to tolerate enhanced sun exposure as expected under climate change. Whether mushroom-specific molecular responses are able to maintain fungal fitness under opening forest canopies remains to be tested.
1. Plant-fungal interactions are important for plant community assembly, but quantifying these relationships remains challenging. High throughput sequencing of fungal communities allows us to identify plant-fungal associations at a high level of resolution, but often fails to provide information on taxonomic and functional assignment of fungi. 2. We transplanted seeds of Pinus cembra across an elevational gradient (1850–2250 m a.s.l.) and identified environmental factors and known fungal associates important for seedling establishment and survival. We then applied null model tests to identify taxonomically unassigned fungi associated with pine recruitment. 3. Early seedling establishment was determined by abiotic environmental factors, while seedling survival was predominantly affected by biotic environmental factors (i.e., the abundance of a fungal pathogen known from literature and the distance to adult trees). Null model tests identified known mycorrhizal partners and a large number of unknown operational taxonomic units (OTUs) associated with seedling survival, including saprotrophic and pathogenic species. These results highlight that unknown fungal OTUs, which are usually discarded from analyses, could play a crucial role for plant survival. 4. Synthesis. We conclude that high throughput metabarcoding paired with null model tests, is a valuable approach for identifying hidden plant-fungal associations within large and complex DNA metabarcoding datasets. Such an approach can be an important tool in illuminating the black box of plant-microbe interactions, and thus understanding ecosystem dynamics.
The acetogenic model bacterium Acetobacterium woodii is well-known to produce acetate by homoacetogenesis from sugars, but under certain conditions minor amounts of ethanol are produced in addition. Here, we have aimed to identify physiological conditions that increase electron and carbon flow towards ethanol production. Ethanol was only produced from fructose but not from H2 + CO2, formate, pyruvate, lactate or alanine. In the absence of Na+, the Wood–Ljungdahl pathway (WLP) of acetate formation is not functional. Therefore, the ethanol yield increased to 0.42 mol/mol (ethanol/fructose) with an ethanol/acetate ratio of 0.28 mol/mol. The presence of bicarbonate/CO2 stimulated electron and carbon flow through the WLP and led to less ethanol produced. Of the 11 potential alcohol dehydrogenase genes, the most upregulated during ethanologenesis was adh4. A deletion of adh4 led to an increase in ethanol production by 100% to a yield of 0.79 mol/mol (ethanol/fructose); this correlated with an increase in transcript abundance of adh6. In sum, our studies revealed low Na+ and bicarbonate/CO2 as factors that trigger ethanol formation and that a deletion of adh4 drastically increased ethanol formation in A. woodii.
Acinetobacter baumannii is outstanding for its ability to cope with low water activities which significantly contributes to its persistence in hospital environments. The vast majority of bacteria are able to prevent loss of cellular water by amassing osmoactive compatible solutes or their precursors into the cytoplasm. One such precursor of an osmoprotectant is choline that is taken up from the environment and oxidized to the compatible solute glycine betaine. Here, we report the identification of the osmotic stress operon betIBA in A. baumannii. This operon encodes the choline oxidation pathway important for the production of the solute glycine betaine. The salt-sensitive phenotype of a betA deletion strain could not be rescued by addition of choline, which is consistent with the role of BetA in choline oxidation. We found that BetA is a choline dehydrogenase but also mediates in vitro the oxidation of glycine betaine aldehyde to glycine betaine. BetA was found to be associated with the membrane and to contain a flavin, indicative for BetA donating electrons into the respiratory chain. The choline dehydrogenase activity was not salt dependent but was stimulated by the compatible solute glutamate.
An exploration of the relationship between recruitment communication and foraging in stingless bees
(2021)
Social information is widely used in the animal kingdom and can be highly adaptive. In social insects, foragers can use social information to find food, avoid danger, or choose a new nest site. Copying others allows individuals to obtain information without having to sample the environment. When foragers communicate information they will often only advertise high-quality food sources, thereby filtering out less adaptive information. Stingless bees, a large pantropical group of highly eusocial bees, face intense inter- and intra-specific competition for limited resources, yet display disparate foraging strategies. Within the same environment there are species that communicate the location of food resources to nest-mates and species that do not. Our current understanding of why some species communicate foraging sites while others do not is limited. Studying freely foraging colonies of several co-existing stingless bee species in Brazil, we investigated if recruitment to specific food locations is linked to 1) the sugar content of forage, 2) the duration of foraging trips, and 3) the variation in activity of a colony from 1 day to another and the variation in activity in a species over a day. We found that, contrary to our expectations, species with recruitment communication did not return with higher quality forage than species that do not recruit nestmates. Furthermore, foragers from recruiting species did not have shorter foraging trip durations than those from weakly recruiting species. Given the intense inter- and intraspecific competition for resources in these environments, it may be that recruiting species favor food resources that can be monopolized by the colony rather than food sources that offer high-quality rewards.
Obligate endoparasitic oomycetes are known to ubiquitously occur in marine and freshwater diatoms, but their diversity is still largely unexplored. Many of these parasitoids are members of the early-diverging oomycete lineages (Miracula, Diatomophthora), others are within the Leptomitales of the Saprolegniomycetes (Ectrogella, Lagenisma) and some have been described in the Peronosporomycetes (Aphanomycopsis, Lagenidium). Even though some species have been recently described and two new genera were introduced (Miracula and Diatomophthora), the phylogeny and taxonomy of most of these organisms remain unresolved. This is contrasted by the high number of sequences from unclassified species, as recently revealed from environmental sequencing, suggesting the presence of several undiscovered species. In this study, a new species of Miracula is reported from a marine centric diatom (Minidiscus sp.) isolated from Skagaströnd harbor in Northwest Iceland. The morphology and life cycle traits of this novel oomycete parasite are described herein, and its taxonomic placement within the genus Miracula is confirmed by molecular phylogeny. As it cannot be assigned to any previously described species, it is introduced as Miracula islandica in this study. The genus Miracula thus contains three described holocarpic species (M. helgolandica, M. islandica, M. moenusica) to which likely additional species will need to be added in the future, considering the presence of several lineages known only from environmental sequencing that clustered within the Miracula clade.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
Acinetobacter baumannii is an opportunistic pathogen, which has become a rising threat in healthcare facilities worldwide due to increasing antibiotic resistances and optimal adaptation to clinical environments and the human host. We reported in a former publication on the identification of three phopholipases of the phospholipase D (PLD) superfamily in A. baumannii ATCC 19606T acting in concerted manner as virulence factors in Galleria mellonella infection and lung epithelial cell invasion. This study focussed on the function of the three PLDs. A Δpld1-3 mutant was defect in biosynthesis of the phospholipids cardiolipin (CL) and monolysocardiolipin (MLCL), whereas the deletion of pld2 and pld3 abolished the production of MLCL. Complementation of the Δpld1-3 mutant with pld1 restored CL biosynthesis demonstrating that the PLD1 is implicated in CL biosynthesis. Complementation of the Δpld1-3 mutant with either pld2 or pld3 restored MLCL and CL production leading to the conclusion that PLD2 and PLD3 are implicated in CL and MLCL production. Mutant studies revealed that two catalytic motifs are essential for the PLD3-mediated biosynthesis of CL and MLCL. The Δpld1-3 mutant exhibited a decreased colistin and polymyxin B resistance indicating a role of CL in cationic antimicrobial peptides (CAMPs) resistance.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
The extraordinary desiccation resistance of the opportunistic human pathogen Acinetobacter baumannii is a key to its survival and spread in medical care units. The accumulation of compatible solute such as glutamate, mannitol and trehalose contributes to the desiccation resistance. Here, we have used osmolarity as a tool to study the response of cells to low water activities and studied the role of a potential inorganic osmolyte, K+, in osmostress response. Growth of A. baumannii was K+-dependent and the K+-dependence increased with the osmolarity of the medium. After an osmotic upshock, cells accumulated K+ and K+ accumulation increased with the salinity of the medium. K+ uptake was reduced in the presence of glycine betaine. The intracellular pools of compatible solutes were dependent on the K+ concentration: mannitol and glutamate concentrations increased with increasing K+ concentrations whereas trehalose was highest at low K+. After osmotic upshock, cells first accumulated K+ followed by synthesis of glutamate; later, mannitol and trehalose synthesis started, accompanied with a decrease of intracellular K+ and glutamate. These experiments demonstrate K+ uptake as a first response to osmostress in A. baumannii and demonstrate a hierarchy in the time-dependent accumulation of K+ and different organic solutes.
Acinetobacter baumannii is outstanding for its ability to cope with low water activities and therefore its adaptation mechanism to osmotic stress. Here we report on the identification and characterization of five different secondary active compatible solute transporters, belonging to the betaine-choline-carnitine transporter (BCCT) family. Our studies revealed two choline-specific and three glycine betaine-specific BCCTs. Activity of the BCCTs was differentially dependent to the osmolality: one choline and one betaine transporter were osmostress-independent. Addition of choline to resting cells of Acinetobacter grown in the presence of the co-substrate choline or with phosphatidylcholine as sole carbon source led to ATP synthesis in the wild type but not in the BCCT quadruple mutant. This indicates that the BCCTs are essential to transport the energy substrate choline. The role of the different BCCTs in osmostress resistance and in metabolic adaptation of A. baumannii to the human host is discussed.
A1AO ATP synthases with a V-type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A1AO ATP synthase with a V-type c subunit in a mesophilic organism revealed an A1AO ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 U·mg−1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V-type c subunit with only one ion (Na+)-binding site. Indeed, ATP hydrolysis was strictly Na+-dependent. N,N′-dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na+. Na+ was shown directly to abolish binding of the fluorescence DCCD derivative, NCD-4, to subunit c, demonstrating a competition of Na+ and DCCD/NCD-4 for a common binding site. After incorporation of the A1AO ATP synthase into liposomes, ATP-dependent primary transport of 22Na+ as well as ΔµNa+-driven ATP synthesis could be demonstrated. The Na+ A1AO ATP synthase from E. limosum is the first ATP synthase with a V-type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases.
Understanding hominin expansions requires the comprehension of movement processes at different scales. In many models of hominin expansion these processes are viewed as being determined by large-scale effects, such as changes in climate and vegetation spanning continents and thousands or even millions of years. However, these large-scale patterns of expansions also need to be considered as possibly resulting from the accumulation of small-scale decisions of individual hominins. Moving on a continental scale may for instance involve crossing a water barrier. We present a generalized agent-based model for simulating the crossing of a water barrier where the agents represent the hominin individuals. The model can be configured to represent a variety of movement modes across water. Here, we compare four different behavioral scenarios in conjunction with a set of water barrier configurations, in which agents move in water by either paddling, drifting, swimming or rafting. We introduce the crossing-success-rate (CSR) to quantify the performance in water crossing. Our study suggests that more focus should be directed towards the exploration of behavioral models for hominins, as directionality may be a more powerful factor for crossing a barrier than environmental opportunities alone. A prerequisite for this is to perceive the opposite shore. Furthermore, to provide a comprehensive understanding of hominin expansions, the CSR allows for the integration of results obtained from small-scale simulations into large-scale models for hominin expansion.
Young trees of deciduous Quercus robur and Q. petraea and evergreen Q. ilex were grown together in a competition lysimeter experiment to assess i) – whether the observed growth differences between evergreen Q. ilex and the deciduous Q. robur and Q. petraea on sandy soil in the field and ii) – whether the different natural distribution of Q. robur and Q. petraea could be attributed to physiological differences between the species under experimental drought stress (DS). Half of the plants were subjected to long-term DS in two consecutive years and monitored for physiological and growth parameters. In the first year, water withholding for more than three months did not lead to significant drought stress, probably because of a sufficient residual water volume in the lysimeter for the relatively small plants. However, in the second year, 2018, which was warmer, the bigger plants now competed for the residual water and clear drought stress symptoms developed for more than two months in all trees in the DS lysimeter basin. Growth was only moderately (and mostly not significantly) affected by the DS in the second year, except for a smaller total leaf area in DS Q. ilex as compared to DS Q. robur and Q. petraea and smaller root collar diameter in DS Q. ilex compared to DS Q. robur. Under DS, the deciduous species revealed significant decreases in ΔVIP, indicating a negative effect on electron transport through PS I. Pn, PIabs and water relations parameters (ΨPD and LWC) all decreased to various extents under DS in all three species, leading to clear separation of the deciduous from the evergreen species by PCA. However, PCA did not separate the two deciduous species from each other. It is concluded that longer root growth in the two deciduous species as compared to Q. ilex ameliorates DS effects in Q. robur and Q. petraea and may be the key to understand the better performance of deciduous oaks on sandy soil in the field.
The Southern Ocean (SO) continental shelf and deep sea are environments characterised by different benthic communities. Their structure and composition are driven and shaped by different variables: whilst on the continental shelf physical environmental variables are the main drivers shaping faunal abundance, structure and composition, the deep-sea fauna is most problably driven by biological variables such as predation and competition. Among shelf and deep-sea benthic communities, peracarids (e.g. amphipods and isopods) are one of the most dominant groups, showing high levels of abundance and diversity in both environments. Knowledge on their assemblage structure and composition in the SO remains limited, as well as the knowledge of the environmental variables that influence them. Therefore, the aim of our study was to investigate peracarid assemblages from the SO continental shelf and deep sea and to assess the main drivers shaping their assemblage structure along a wide bathymetric gradient (from 160 m to about 6000 m depth) and at a large geographic scale. We analysed the spatial distribution of 183,606 peracarids sampled using an epibenthic sledge (EBS) during nine different expeditions in the SO, covering a latitudinal range of 77° to 41° South. Depth was identified as the main driver shaping peracarid abundance pattern, their assemblage structure from the continental shelf (<1499 m) was dissimilar to that from the deep sea (>1500 m). Also, depth was differently correlated with different peracarid orders: while isopod abundances increased with depth, amphipods and mysids were negatively correlated; no correlation was found with cumaceans and tanaidaceans. The dissimilar peracarid assemblage structure between the SO continental shelf and the SO deep sea can be due to the assumption that there are different driving forces shaping benthic assemblages from these two environments (physical variables on the continental shelf, biological interactions in the deep sea). As a result, we also suggest that environmental changes due to climate change (e.g. temperature, ice coverage, productivity) would have different consequences depending on the bathymetric range considered.
Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, partial and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and their severe discomfort.
FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae, the gene encoding for FAD synthase is FAD1, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms—that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3′ region of FAD1 mRNA was analyzed by 3′RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3′UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3′UTRs allowed us to predict the existence of a cis-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3′UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform.
In recent years, several neuronal differentiation protocols were published that circumvent the requirement of embryoid body (EB) formation under serum-deprivation and simplified medium conditions. But a neuronal default model to establish an approach that works efficiently for all pluripotent cells and neuronal precursors is still lacking. Whether such a default neural mechanism exist and how this is implemented across a broad spectrum of cell source, is addressed in several studies and still controversially discussed. It was proposed that the default neuronal fate is initiated in the absence of extrinsic signals and is achieved by eliminating extracellular inhibitors of neuroectodermal fate and suppressing cell-cell signalling through limited cell density. Previous studies reported that ESC and ECC grown at low density and in absence of exogenous factors or feeder layers die within 24 h but acquire a neural identity as indicated by expression of the neural marker Nestin. Thus, this application is not suitable for generating neural cultures. Furthermore, it was reported that P19 cells survive and express neuroectodermal marker genes in serum-free DMEM/F12 medium containing transferrin, insulin, and selenite, although no neurites were identified.
Based on this background, in this study, a novel approach to induce neuronal differentiation in vitro was developed that implements a nutrient-poor environment, which, in contrast to previous studies, ensures the survival of neuronally differentiated cells over a long period of time and allows normal formation of neurites. Neither the formation of free-floating aggregates nor supplementation of growth factors or known inducers was required to establish a reliable neuronal differentiation protocol. A simple medium, consisting of DMEM/F12+N2 that was highly diluted in salt solution, was sufficient to drive a fast neuronal differentiation in monolayer cultures. Serum deprivation and strong dilution of DMEM/F12+N2 medium cause a nutrient-poor environment in which the influence of growth factors and inducers is minimized. This medium creates a metabolically defined environment that is presumably free of extrinsic signals that prevent the decision of neuronal fate. Analysis of the medium components discovered no actual inducer. Hence, it was suggested that the metabolic composition of the medium exclusively covers specific cell requirements of neurons, therefore ensures their survival, and drives the switch from pluripotent cells to neurons. The self-developed method was established by usage of the murine embryonal carcinoma cell line P19 and could be transferred to murine ESC. Consequently, the method could provide a feasible protocol for a generally valid neuronal default model.
The established protocol provides several advantages such as the possibility to generate stable pure neuronal cultures by a fast, simple, and highly reproducible one-step induction under defined medium conditions with a minimum of exogen effectors. The method is characterised by clear and steady medium conditions that makes the investigation of specific cell requirements during differentiation accessible. It is therefore expected to be a useful tool to investigate the molecular basis of neuronal differentiation as well as for high throughput screenings. The phenotype of mature postmitotic neurons was arising within one week and cultures were shown to stay stable at least for three weeks. The neuronal identity was confirmed by expression of neuronal markers through immunofluorescence staining and mass spectrometry analysis. Furthermore, increased levels of axon markers were detected in early neuronal differentiation and functionality of the synapses of the P19-derived neurons was ascertained by detection of calcium activity. Axonal laser ablation, immediately followed by fast regrowth of connections in the neuronal network, revealed a strong regeneration potential under the given conditions. Furthermore, the generated neurons showed a morphologically distinct phenotype and the formation of neural rosettes. Immunofluorescence staining demonstrated the generation of pure and homogeneous neuronal cultures, free of glial cells.
Retinoic acid (RA) plays an essential role in cell signalling during embryogenesis and efficiently induces neuronal differentiation in vitro in a concentration dependent manner. Neither retinol nor retinoic acid was included in any of the components of the self-prepared medium in this work. However, I observed, dependence on RARβ- and/or RARγ-regulated RA signalling in serum-free monolayer cultures. Nevertheless, neuronal differentiation in serum-free monolayer cultures was assumed to be RARα-independent because (i) RARα was slightly downregulated after neuronal induction, (ii) the truncated RARα of the RAC65 mutant had no effect on induction efficiency, and (iii) a pan-RAR inhibitor suppressed neuronal differentiation. In contrast to serum-free monolayer cultures, the truncated RARα prevented neuronal differentiation by application of the conventional protocol where cells are grown in free floating cell aggregates in serum-containing medium. Proteome analysis of P19 cells, treated by the self-developed differentiation protocol over five days showed increased levels of cellular RA binding proteins that mediate the cellular RA transport and are involved in canonical as well as non-canonical RA signalling.
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Acetogenic bacteria are a polyphyletic group of organisms that fix carbon dioxide under anaerobic, non-phototrophic conditions by reduction of two mol of CO2 to acetyl-CoA via the Wood–Ljungdahl pathway. This pathway also allows for lithotrophic growth with H2 as electron donor and this pathway is considered to be one of the oldest, if not the oldest metabolic pathway on Earth for CO2 reduction, since it is coupled to the synthesis of ATP. How ATP is synthesized has been an enigma for decades, but in the last decade two ferredoxin-dependent respiratory chains were discovered. Those respiratory chains comprise of a cytochrome-free, ferredoxin-dependent respiratory enzyme complex, which is either the Rnf or Ech complex. However, it was discovered already 50 years ago that some acetogens contain cytochromes and quinones, but their role had only a shadowy existence. Here, we review the literature on the characterization of cytochromes and quinones in acetogens and present a hypothesis that they may function in electron transport chains in addition to Rnf and Ech.
Plastic products leach chemicals that induce in vitro toxicity under realistic use conditions
(2021)
Plastic products contain complex mixtures of extractable chemicals that can be toxic. However, humans and wildlife will only be exposed to plastic chemicals that are released under realistic conditions. Thus, we investigated the toxicological and chemical profiles leaching into water from 24 everyday plastic products covering eight polymer types. We performed migration experiments over 10 days at 40 °C and analyzed the migrates using four in vitro bioassays and nontarget high-resolution mass spectrometry (UPLC-QTOF-MSE). All migrates induced baseline toxicity, 22 an oxidative stress response, 13 antiandrogenicity, and one estrogenicity. Overall, between 17 and 8681 relevant chemical features were present in the migrates. In other words, between 1 and 88% of the plastic chemicals associated with one product were migrating. Further, we tentatively identified ∼8% of all detected features implying that most plastic chemicals remain unknown. While low-density polyethylene, polyvinyl chloride, and polyurethane induced most toxicological endpoints, a generalization for other materials is not possible. Our results demonstrate that plastic products readily leach many more chemicals than previously known, some of which are toxic in vitro. This highlights that humans are exposed to many more plastic chemicals than currently considered in public health science and policies.
Nature's non-material contributions to people are difficult to quantify and one aspect in particular, nature's contributions to communication (NCC), has so far been neglected. Recent advances in automated language processing tools enable us to quantify diversity patterns underlying the distribution of plant and animal taxon labels in creative literature, which we term BiL (biodiversity in literature). We assume BiL to provide a proxy for people's openness to nature's non-material contributions enhancing our understanding of NCC. We assembled a comprehensive list of 240,000 English biological taxon labels. We pre-processed and searched a subcorpus of digitised literature on Project Gutenberg for these labels. We quantified changes in biodiversity indices commonly used in ecological studies for 16,000 books, encompassing 4,000 authors, as proxies for BiL between 1705 and 1969. We observed hump-shape patterns for taxon label richness, abundance and Shannon diversity indicating a peak of BiL in the middle of the 19th century. This is also true for the ratio of biological to general lexical richness. The variation in label use between different sections within books, quantified as β-diversity, declined until the 1830s and recovered little, indicating a less specialised use of taxon labels over time. This pattern corroborates our hypothesis that before the onset of industrialisation BiL may have increased, reflecting several concomitant influences such as the general broadening of literary content, improved education and possibly an intensified awareness of the starting loss of biodiversity during the period of romanticism. Given that these positive trends continued and that we do not find support for alternative processes reducing BiL, such as language streamlining, we suggest that this pronounced trend reversal and subsequent decline of BiL over more than 100 years may be the consequence of humans’ increasing alienation from nature owing to major societal changes in the wake of industrialisation. We conclude that our computational approach of analysing literary communication using biodiversity indices has a high potential for understanding aspects of non-material contributions of biodiversity to people. Our approach can be applied to other corpora and would benefit from additional metadata on taxa, works and authors.
This work comprises the investigation of four different biosynthesis gene clusters from Xenorhabdus. Xenorhabdus is an entomopathogenic bacterium that lives in mutualistic symbiosis with its Steinernema nematode host and together they infect and kill insect larvae. Xenorhabdus is well known for the production of so-called specialised metabolites and many of these compounds are synthesised by non-ribosomal peptide synthetases (NRPSs) or NRPS-polyketide synthase (PKS)-hybrids. These enzymes are organised in a modular manner and produce structurally very diverse molecules, often with the help of modifying domains and tailoring enzymes. In general, the genes involved in the biosynthesis are organised in so-called biosynthetic gene clusters (BGCs) in the genome of the producing strain. Exchanging the native promoter with an inducible promoter, e.g. PBAD, allows the targeted activation of the BGC and in turn the analysis of the biosynthesis product via LC-MS analysis.
The first BGC investigated in this work is responsible for the biosynthesis of xenofuranones. Based on gene deletions, this work shows that the NRPS-like enzyme XfsA produces a carboxylated furanone intermediate which is subsequently decarboxylated by XfsB to yield xenofuranone B. The next step in xenofuranone biosynthesis is the O-methylation of xenofuranone B to yield xenofuranone A. A comparative proteomics approach allowed the identification of four methyltransferase candidates and subsequent gene deletions confirmed one of the candidates to be responsible for methylation of xenofuranone B. The proteome analysis was based on the comparison of X. szentirmaii WT and X. szentirmaii Δhfq because distinct levels of the methylated xenofuranone A were observed when the xfs BGC was activated in either WT or Δhfq strain. Hfq is a global transcriptional regulator whose deletion is associated with the down regulation of natural product biosynthesis in Xenorhabdus. The strong PBAD activation of the xfs BGC also allowed the detection of two novel xenofuranone derivatives which arise from incorporation of one 4-hydroxyphenylpyruvic acid as first or second building block, respectively.
PBAD based activation of the second BGC addressed in this work lead to the detection of a novel metabolite and compound purification allowed NMR-based structure elucidation. The molecule exhibits two pyrrolizidine moieties and was named pyrrolizwilline (pyrrolizidine + twin (German: “Zwilling”)). The BGC comprises seven genes and single gene deletions as well as heterologous expression in E. coli and NRPS engineering were conducted to investigate the biosynthesis. The first two genes xhpA and xhpB encode a bimodular NRPS and a monooxygenase which synthesise a pyrrolizixenamide-like structure, similar to PxaA and PxaB in pyrrolizixenamide biosynthesis. It is suggested that the acyl side chain incorporated by XhpA is removed by the α,β-hydrolase XhpG. The keto function is then reduced by two subsequent two electron reductions catalysed by XhpC and XhpD. One of these two reduced pyrrolizidine units most likely is extended with glyoxalate prior to non-enzymatic dimerisation with the second pyrrolizidine moiety. To finally yield pyrrolizwilline, L-valine is incorporated, probably by the free-standing condensation domain XhpF.
The third BGC investigated is responsible for the production of a tripeptide composed of β-D-homoserine, α-hydroxyglycine and L-valine and is referred to as glyoxpeptide. This work demonstrates that the previously observed glyoxpeptide derivative is derived from glycerol present in the culture medium. Furthermore, this work shows that the monooxygenase domain, which is found in an unusual position between motifs A8 and A9 within the adenylation domain, is responsible for the α-hydroxylation of glycine. It is suggested that the α-hydroxylation of glycine renders the tripeptide prone to hydrolysis via hemiacetal formation. Hence, the XgsC_MonoOx domain might be an interesting candidate for further NRPS engineering.
The fourth BGC addressed is responsible for the production of xildivalines and this work describes two additional derivatives which are detected only when the promoter is exchanged and activated in the X. hominickii WT strain but not in X. hominickii Δhfq. Deletion of the methyltransferase encoding gene xisE results in the production of non-methylated xildivalines. It remains to be determined when the N-methylation of L-valine takes place. It is discussed that the methyltransferase could act on the NRPS released product but also during the assembly. The peptide deformylase is not involved in the proposed biosynthesis as xildivaline production is detected in a ΔxisD strain. The PKS XisB features two adjacent, so-called tandem T domains. The inactivation of the first or the second T domain by point mutation causes decreased production titres of detected xildivalines in the respective mutant strain when compared to the wild type.
Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
Acetogenic bacteria are a group of strictly anaerobic bacteria that may have been first life forms on Earth since they employ an ancient pathway for CO2 fixation into acetyl-CoA that is coupled to the synthesis of ATP, the Wood–Ljungdahl pathway. Electrons for CO2 reduction are derived from oxidation of H2 or CO and thus, these bacteria can grow lithotrophically on gases present on early Earth. Among the organic molecules present on early Earth is acetaldehyde, a highly volatile C2 compound. Here, we demonstrate that the acetogenic model bacterium Acetobacterium woodii grows on acetaldehyde. Acetaldehyde is dismutated to ethanol and acetyl-CoA, most likely by the bifunctional alcohol dehydrogenase AdhE. Acetyl-CoA is converted to acetate by two subsequent enzymes, phosphotransacetylase and acetate kinase, accompanied by the synthesis of ATP by substrate-level phosphorylation. Apparently, growth on acetaldehyde does not employ the Wood–Ljungdahl pathway. Our finding opens the possibility of a simple and ancient metabolic pathway with only three enzymes that allows for biomass (acetyl-CoA) and ATP formation on early Earth.
Frontal areas of the mammalian cortex are thought to be important for cognitive control and complex behaviour. These areas have been studied mostly in humans, non-human primates and rodents. In this article, we present a quantitative characterization of response properties of a frontal auditory area responsive to sound in the brain of Carollia perspicillata, the frontal auditory field (FAF). Bats are highly vocal animals, and they constitute an important experimental model for studying the auditory system. We combined electrophysiology experiments and computational simulations to compare the response properties of auditory neurons found in the bat FAF and auditory cortex (AC) to simple sounds (pure tones). Anatomical studies have shown that the latter provides feedforward inputs to the former. Our results show that bat FAF neurons are responsive to sounds, and however, when compared to AC neurons, they presented sparser, less precise spiking and longer-lasting responses. Based on the results of an integrate-and-fire neuronal model, we suggest that slow, subthreshold, synaptic dynamics can account for the activity pattern of neurons in the FAF. These properties reflect the general function of the frontal cortex and likely result from its connections with multiple brain regions, including cortico-cortical projections from the AC to the FAF.
Clean water is fundamental to human health and ecosystem integrity. However, water quality deteriorates due to novel anthropogenic pollutants present at microgram per liter concentrations in urban water cycles (termed micropollutants). Wastewater treatment plants (WWTP) have been identified as major point sources for aquatic (micro-)pollutants. Chemical and ecotoxicological analyses have shown that conventional biological WWTPs do not fully remove micropollutants and associated toxicities, which is often because of mobile, polar and/or recalcitrant compounds and transformation products (TPs). To minimize possible environmental risks, advanced wastewater treatment (AWWT) technologies could be a promising mitigation measure. Multiple processes are therefore being developed and evaluated such as ozonation and ozonation followed by granulated activated carbon (GAC) or biological filtration. Assessing the performance of these combined AWWTs was the focus the TransRisk project. Within this project, this thesis accomplished four major goals.
Firstly, the preparation of (waste)water samples was optimised for in vitro bioassays. Acidification, filtration and solid phase extraction (SPE) were tested for their impact on environmentally relevant in vitro endocrine activities, mutagenicity, genotoxicity and cytotoxicity. Significantly different outcomes of these assays were detected comparing neutral and acidified samples. Sample filtration had a lesser impact, but in some cases retention of particle-bound compounds could have caused significant toxicity losses. Out of three SPE sorbents the Telos C18/ENV at sample pH 2.5 extracted highest toxicity, some undetected in aqueous samples. These results indicate that sample preparation needs to be optimised for specific sample matrices and bioassays to avoid false-positive or -negative detects in effect-based analyses.
Secondly, the above listed in vitro toxicities were monitored in a protected region for drinking water production in South-West Germany (2012-2015). Out of 30 sampling sites surface water and groundwater were the least polluted. Nonetheless, a few groundwater samples induced high anti-estrogenic activity that prompted further monitoring. The latter included a waterworks in which no toxicity was detected. Hospital wastewater also had elevated in vitro toxicities and hospitals are, thus, relevant intervention points for source control. The biological WWTPs were effective in removing most of the detected toxicity, and the selected bioassays proved to be pertinent tools for water quality assessment and prioritisation of pollution hotspots.
Thirdly, the in vivo bioassay ISO10872 based on Caenorhabditis elegans (C. elegans) was adapted for this thesis. Using this model, a median effect concentration (EC50) for reproductive toxicity of the polycyclic aromatic hydrocarbon β-naphthoflavone (β- NF) of 114 µg/L was computed which is slightly lower than reported in the scientific literature. β-NF induced cyp-35A3::GFP (a biomarker in transgenic animals) in a time and concentration dependent manner (≤ 21.3–24 fold above controls). β-NF spiked wastewater samples supported earlier hypotheses on particle-bound pollutants. Reproductive toxicity (96 h) and cyp-35A3 induction (24 h) of biologically treated and/or ozonated wastewater extracts and growth promoting effects of GAC/biologically filtered ozonated wastewater extracts were observed. This suggested the presence of residual bioactive/toxic chemicals not included in the targeted chemical analysis. It also highlighted the importance of integrating multiple (apical and molecular) endpoints in wastewater assessments.
Fourthly, five in vitro and the adapted C. elegans bioassay were integrated into a wastewater quality evaluation (developed within TransRisk). Out of the five AWWT options, ozonation (at 1 g O3,applied/g DOC, HRT ~ 18 min) combined with nonaerated GAC filtration was rated most effective for toxicity removal. All five AWWTs largely removed estrogenic and (anti-)androgenic activities, but not anti-estrogenic activity and mutagenicity, which even increased during ozonation. This has been observed in related studies and points towards toxic TPs. These results also emphasized the need for implementing an effective post-treatment for ozonation. The results from a parallel in vivo study with Lumbriculus variegatus and Potamopyrgus antipodarum conducted on site at the WWTP (using flow through systems) were in accordance with the C. elegans results. In this context, it is suggested to further implement C. elegans as sensitive, feasible and ecologically relevant model.
In conclusion, this thesis shows how optimised sample preparation, long-term (in vitro) environmental monitoring, sensitive and ecologically relevant (in vivo) bioassays as well as innovative evaluation concepts, are pivotal in improving the removal of micropollutants and their toxicities with AWWTs. Future research should further develop and evaluate measures at sewer systems, conventional biological, tertiary and other advanced treatment technologies, as well as sociopolitical strategies (e.g., source control or natural conservation) and restoration projects. The effect-based tools optimised in this thesis will support assessing their success.
Extremophilic prokaryotes live under harsh environmental conditions which require far-reaching cellular adaptations. The acquisition of novel genetic information via natural transformation plays an important role in bacterial adaptation. This mode of DNA transfer permits the transfer of genetic information between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal gene transfer (HGT), significantly contributes to genome plasticity over evolutionary history and is a driving force for the spread of fitness-enhancing functions including virulence genes and antibiotic resistances. In particular, HGT has played an important role for adaptation of bacteria to extreme environments. Here, we present a survey of the natural transformation systems in bacteria that live under extreme conditions: the thermophile Thermus thermophilus and two desiccation-resistant members of the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter baumannii. The latter is an opportunistic pathogen and has become a world-wide threat in health-care institutions. We highlight conserved and unique features of the DNA transporter in Thermus and Acinetobacter and present tentative models of both systems. The structure and function of both DNA transporter are described and the mechanism of DNA uptake is discussed.
Extremophilic prokaryotes live under harsh environmental conditions which require far-reaching cellular adaptations. The acquisition of novel genetic information via natural transformation plays an important role in bacterial adaptation. This mode of DNA transfer permits the transfer of genetic information between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal gene transfer (HGT), significantly contributes to genome plasticity over evolutionary history and is a driving force for the spread of fitness-enhancing functions including virulence genes and antibiotic resistances. In particular, HGT has played an important role for adaptation of bacteria to extreme environments. Here, we present a survey of the natural transformation systems in bacteria that live under extreme conditions: the thermophile Thermus thermophilus and two desiccation-resistant members of the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter baumannii. The latter is an opportunistic pathogen and has become a world-wide threat in health-care institutions. We highlight conserved and unique features of the DNA transporter in Thermus and Acinetobacter and present tentative models of both systems. The structure and function of both DNA transporter are described and the mechanism of DNA uptake is discussed.
Human GLUTs represent a family of specialized transporters that facilitate the diffusion of hexoses through membranes along a concentration gradient. The 14 isoforms share high sequence identity but differ in substrate specificity and affinity, and tissue distribution. According to their structure similarity, GLUTs are divided into three classes, with class 1 comprising the most intensively studied isoforms GLUTs1 4. An abnormal function of different GLUT members has been related to the pathogenesis of various diseases, including cancer and diabetes. Hence, GLUTs are the subject of intensive research, and efforts concentrate on identifying GLUT-selective ligands for putative medical purposes and their application in studies aiming to further unravel the metabolic roles of these transporters.
The hexose transporter deficient (hxt0) yeast strain EBY.VW4000 is devoid of all its endogenous hexose transporters and unable to grow on glucose or related hexoses. This strain has proven to be a valuable platform to investigate heterologous transporters due to its easy handling, increased robustness, and versatile applications. However, the functional expression of GLUTs in yeast requires certain modifications. Single point mutations of GLUT1 and GLUT5 led to their functional expression in EBY.VW4000, whereas the native GLUT1 was actively expressed in EBY.S7, a hxt0 strain carrying the fgy1 mutation that putatively reduces the phosphatidylinositol-4-phosphate (PI4P) content in the plasma membrane. GLUT4 was only actively expressed in the hxt0 strain SDY.022, which also contains the fgy1 mutation and in which ERG4 is additionally deleted. Erg4 is one of the late enzymes in the ergosterol pathway, and therefore SDY.022 probably has an altered sterol composition in its membrane.
The goal of this thesis was to actively express GLUT2 and GLUT3 in a hxt0 yeast strain, providing a convenient system for their ligand screening. A PCR-derived amino acid exchange in the sequence of GLUT3 enabled its functional expression in EBY.VW4000 and the unmodified GLUT3 protein was active in EBY.S7. Functional expression of GLUT2 was achieved by rational design. The extracellular loop between the transmembrane regions 1 and 2 is significantly larger in GLUT2 than in other class 1 GLUTs. By truncating this loop by 34 amino acids and exchanging an alanine for a serine, a GLUT3-like loop was implemented. The resulting construct GLUT2∆loopS was functional in EBY.S7. With an additional point mutation in the transmembrane region 11, GLUT2∆loopS_Q455R was also actively expressed in EBY.VW4000. Inhibition studies with the known GLUT inhibitors phloretin and quercetin showed a reduced transporter activity for GLUT2 and GLUT3 in uptake assays and growth tests when inhibitors were present, demonstrating that both systems are amenable for ligand screening experiments.
The newly established GLUT2 yeast system was then used to screen a library of compounds pre-selected by in silico screening. Thereby, eleven identified GLUT2 inhibitors exhibited strong potencies with IC50 values ranging from 0.61 to 19.3 µM. By employing the other yeast systems, these compounds were tested for their effects on GLUT1, and GLUTs3-5, revealing that nine of the identified ligands were GLUT2-selective. In contrast, one was a pan-class 1 inhibitor (inhibiting GLUTs1-4), and one affected GLUT2 and GLUT5, the two fructose transporting isoforms. These compounds will serve as useful tools for investigations on the role of GLUT2 in metabolic diseases and might even evolve into pharmaceutical agents targeting GLUT2-associated diseases.
Due to the beneficial effect of the putatively changed sterol composition in SDY.022 (by ERG4 deletion) on the functional expression of GLUT4, it was hypothesized that the presence of the human sterol cholesterol, or cholesterol-like sterols, might have a beneficial effect on GLUT expression, too. Thus, it was attempted to generate hxt0 strains that synthesize these sterols by genetic modifications targeting the ergosterol pathway. In the scope of these experiments, several strains with different sterol compositions were generated. Drop tests on glucose medium with the different strains expressing GLUT1 or GLUT4 revealed that the deletion of ERG6 is clearly advantageous for a functional expression of GLUT1 (but not GLUT4). This indicates that the methyl group at the ergosterol side chain (introduced by Erg6 and reduced by Erg4) negatively influences GLUT1 activity. However, this effect on GLUT1 activity was less pronounced than the putative altered PI4P content in EBY.S7.
Additionally, in this thesis, a new tool to measure glucose transport rates of transporters expressed in the hxt0 yeast system was developed to facilitate their kinetic characterization. For this, the pH-sensitive GFP variant pHluorin was employed as a biosensor for the cytosolic pH (pHcyt) by measuring the ratio (R390/470) of emission intensities at 512 nm from two different excitation wavelengths (390 and 470 nm). Sugar-starved cells exhibit a slightly acidic pHcyt because ATP production is depleted, reducing the activity of ATP-dependent proton pumps.
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The development of photosynthesis was a highlight in the progression of bacteria. In addition to the photosystems with their structural proteins, the photosynthesis apparatus consists of different cofactors including essential carotenoids. Thus, the evolution of the carotenoid pathways in relation to the functionality of the resulting structures in photosynthesis is the focus of this review. Analysis of carotenoid pathway genes indicates early evolutionary roots in prokaryotes. The pathway complexity leading to a multitude of structures is a result of gene acquisition, including their functional modifications, emergence of novel genes and gene exchange between species. Along with the progression of photosynthesis, carotenoid pathways coevolved with photosynthesis according to their advancing functionality. Cyanobacteria, with their oxygenic photosynthesis, became a landmark for evolutionary events including carotenogenesis. Concurrent with endosymbiosis, the cyanobacterial carotenoid pathways were inherited into algal plastids. In the lineage leading to Chlorophyta and plants, carotenoids evolved to their prominent role in protection and regulation of light energy input as constituents of a highly efficient light-harvesting complex.
My PhD work employed genetic and pharmacological manipulations, coupled with highresolution live imaging, to understand intercellular communications during zebrafish cardiovascular development. The heart is the first organ to form, and it is composed of several tissues, among which interactions are crucial. I identified two important interactions between muscular and non-muscular tissues in poorly characterized contexts, and the molecules required for the signalling. First, I discovered an important cellular and molecular crosstalk orchestrating the development of the cardiac outflow tract (i.e., the aortic root in mammals).
Endothelial-derived TGF-beta signalling controls the generation of the local extracellular matrix (ECM). The ECM in turn affects endothelial proliferation as well as smooth muscle cell organization (Boezio et al, 2020; Bensimon-Brito*, Boezio* et al, 2020). In my second project, I investigated the crosstalk between the epicardial layer and the myocardial wall. By generating epicardial-impairment models, I identified a novel role for the epicardium in regulating cardiomyocyte volume during heart development (Boezio et al, 2021). Ultimately, this research contributed to our understanding of how paracrine signalling controls the multicellular interactions integral to organogenesis.
The UN 2030 Agenda for Sustainable Development stresses the fundamental role science should play in implementing the 17 Sustainable Development Goals endorsed by the global community. But how can and should researchers respond to this societal demand on science? We argue that answering this question requires systematic engagement with the fundamental normative dimensions of the 2030 Agenda and those of the scientific community—and with the implications these dimensions have for research and practice. We suggest that the production of knowledge relevant to sustainable development entails analytic engagement with norms and values through four tasks. First, to unravel and critically reflect on the ethical values involved in sustainability, values should increasingly become an empirical and theoretical object of sustainability research. Second, to ensure that research on social–ecological systems is related to sustainability values, researchers should reflect on and spell out what sustainability values guide their research, taking into account possible interdependencies, synergies, and trade-offs. Third, to find common ground on what sustainability means for specific situations, scientists should engage in deliberative learning processes with societal actors, with a view to jointly reflecting on existing development visions and creating new, contextualized ones. Fourth, this implies that researchers and scientific disciplines must clarify their own ethical and epistemic values, as this defines accountability and shapes identification of problems, research questions, and results. We believe that ignoring these tasks, whether one is in favor or critical of the 2030 Agenda, will undermine the credibility and relevance of scientific contributions for sustainable development.
Tissue translocation, multigenerational and population effects of microplastics in Daphnia magna
(2021)
The last century saw the widespread adoption of plastic materials throughout nearly every aspect of our lives. Plastics are synthetic polymers that are made up of monomer chains. The properties of the monomer in conjunction with chemical additives allow plastics to have a sheer endless variety of features and use cases. They are cheap, lightweight, and extremely durable. Plastic materials are often engineered for single-use and in conjunction with high production volumes and insufficient waste management and recycling across the globe, this leads to a large number of plastics entering the environment. Marine ecosystems are considered sinks. However, freshwater ecosystems as entry pathways are highly affected by plastic waste as well. Throughout the past decade, the impact of plastic waste on human and environmental health has received a lot of attention from the ecotoxicological community as well as the public. Small plastic fragments (< 1 mm called microplastics) are a large part of this emerging field of research. Within this, the water flea Daphnia magna is probably the most common organism that is used to assess microplastics toxicity. As a filter-feeding organism, it indiscriminately ingests particles from the water column and is thus highly susceptible to microplastics. For this thesis, we identified some gaps in the available data on the ecotoxicity of microplastics to daphnids. To illuminate some of those gaps the present thesis was aimed at five main aspects:
(1) Tissue translocation of spherical microplastics in Daphnia magna
(2) Investigation of the toxicity of irregularly shaped microplastics
(3) Multigenerational and population effects of microplastics
(4) Comparison of the toxicity of microplastics and natural particles
(5) Effects of particle-aging on microplastics toxicity
The thesis is comprised of three peer-reviewed articles and one so-far unpublished study as “additional results”. The first study was aimed at understanding tissue translocation of spherical microplastics to lipid storage droplets of daphnids. The crossing of biological membranes is discussed as a prerequisite to eliciting tissue damage and an inflammatory response. Previously, researchers reported the translocation of fluorescently labeled spherical microplastics to lipid storage droplets of daphnids, even though no plausible biological mechanism to explain this occurrence. Therefore, in order to learn more about this process and potentially illuminate the mechanism we replicated the study. We were able to observe a fluorescence signal inside the lipid droplets only after increasing the exposure concentrations. Nonetheless, it appeared to be independent of particles. This led to the hypothesis, that the lipophilic fluorescent dye uncoupled from the particles and subsequently accumulated in lipid storage droplets. The hypothesis was further confirmed through an additional experiment with a silicone-based passive sampling device showing that the fluorescence occurred both independent of particles and digestive processes. Accordingly, we concluded that the reported findings were a microscopic artifact caused by the uncoupling of the dye from the particles. Therefore, a fluorescence signal alone is not a sufficient proxy to assume that particles have translocated. It needs to be coupled with additional methods to ensure that the observation is indeed caused by the translocation of particles.
It is still unclear whether the toxicity profile of microplastics is different from that of naturally occurring particles or if they are “just another particle”, as there are innumerable amounts in the natural environment surrounding an organism. The goal of the second study was to compare the toxicity of irregularly shaped polystyrene microplastics to that of the natural particle kaolin. The environment is full of natural non-food particles that daphnids ingest more or less indiscriminately and therefore are well adapted to deal with. Daphnids have a short generation time and usually experience food limitation in nature. Therefore, short-term studies only looking at acute toxicity with ad libitum food availability are not representative of the exposure scenario in nature. For a more realistic scenario, we, therefore, used a four-generation multigenerational design under food limitation to investigate how effects translate from one generation to the next. We observed concentration-dependent effects of microplastics but not of natural particles on mortality, reproduction, and growth. Some of the effects increased from generation to generation, leading to the extinction of two treatment groups. Here, microplastics were more toxic than natural particles. At least part of this difference can be explained by physical properties leading to the quick sedimentation of the kaolin, while microplastics remained in the water column. Nonetheless, buoyancy and sedimentation would also affect exposure in the environment and are likely different for most microplastics than for most naturally occurring particle types.
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Translation is an important step in gene expression. Initiation of translation is rate-limiting, and it is phylogenetically more diverse than elongation or termination. Bacteria contain only three initiation factors. In stark contrast, eukaryotes contain more than 10 (subunits of) initiation factors (eIFs). The genomes of archaea contain many genes that are annotated to encode archaeal homologs of eukaryotic initiation factors (aIFs). However, experimental characterization of aIFs is scarce and mostly restricted to very few species. To broaden the view, the protein–protein interaction network of aIFs in the halophilic archaeon Haloferax volcanii has been characterized. To this end, tagged versions of 14 aIFs were overproduced, affinity isolated, and the co-isolated binding partners were identified by peptide mass fingerprinting and MS/MS analyses. The aIF–aIF interaction network was resolved, and it was found to contain two interaction hubs, (1) the universally conserved factor aIF5B, and (2) a protein that has been annotated as the enzyme ribose-1,5-bisphosphate isomerase, which we propose to rename to aIF2Bα. Affinity isolation of aIFs also led to the co-isolation of many ribosomal proteins, but also transcription factors and subunits of the RNA polymerase (Rpo). To analyze a possible coupling of transcription and translation, seven tagged Rpo subunits were overproduced, affinity isolated, and co-isolated proteins were identified. The Rpo interaction network contained many transcription factors, but also many ribosomal proteins as well as the initiation factors aIF5B and aIF2Bα. These results showed that transcription and translation are coupled in haloarchaea, like in Escherichia coli. It seems that aIF5B and aIF2Bα are not only interaction hubs in the translation initiation network, but also key players in the transcription-translation coupling.
In order to form an organ, cells need to take up specialized functions and tasks. Cellular specialization is guided by an interplay of chemical signals and physical forces, where one influences the other. One aspect in cellular identity is its shape, which e.g. defines how susceptible the cell may be to intercellular signaling or in which section of the cell cycle it is and therefore can tell us about its current state. Shape changes are introduced by motor proteins that are controlled and activated in a locally confined manner. For my thesis, I was interested to understand better how cellular shape and geometry impacts downstream cell and organ development. What happens if a cell cant transition to a specific shape? How does it affect tissue structure? How does it affect further development?
One regulator of motor proteins like non-muscle myosin is Shroom3, which recently has been been shown to be expressed and involved in the development of the zebrafish lateral line organ (1 ). Development of the lateral line occurs through a migrating cluster of initially about 150 cells, the posterior lateral line primordium (pLLP), which migrates from the anterior (head) to the posterior (tail) while depositing cell clusters in a regular pattern. Literature on development of the lateral line suggests that in order for a cell cluster to be deposited from the pLLP, rosette formation is a key requirement. Therefore our expectation from the shroom3 mutant was that the number of clusters deposited was significantly reduced. To our surprise, when we first inspected the end of migration lateral line phenotype we found many individuals with a significant increase in cell clusters deposited.
This made us re-think the role of Shroom3 during rosette assembly and the processes its involved in.
To study the effects of Shroom3 on lateral line development, a mutant line was generated and crossed with various transgenic lines which express fluorescently labeled proteins that locate to organelles such as the plasmamembrane or the nucleus. Following, the mutant with its fluorescent labels was microscopically imaged under different conditions to quantify and analyze various cell-morphometric features. Even though the zebrafish is a popular model organism and its perfectly suited for developmental biology and advanced microscopy, there were no methods that would allow for a standardized and more automated pipeline of data acquisition and processing.
Therefore, in order to accurately quantify the morphogenic processes Shroom3 is involved in, I developed a new toolset that significantly improved and facilitated my research. The toolset consists of (1) a new sample mounting method that is based on a 3D agarose gel that increases the number of embryos that can be mounted and imaged at once and speeds up the imaging process significantly (2) for subseqent image analysis I developed four programs that automate the process and therefore make the results much more reproducible and the analysis much more efficient. The first program is used for end of migration analyses, to deduce the pattern, count and size of Lateral Line cell clusters. The second is used not for end of migration, but for migration analyses (on timelapse recordings). Besides this it also prepares the images for more advanced downstream migration analyses and allows to analyse fluorescence signal on a second channel. The third program is used to analyse the pLLP only at high spatial resolution and to deduce the cell count, 3D cell morphometrics (like the volume) and cell orientation. The fourth program finally is used downstream of the second and third program and is capable of detecting and comparing them with the look of wildtype rosettes.
Here I show that in absence of Shroom3 rosette formation in the migrating pLLP is destabilized leading to facilitated cell cluster deposition and I show how this might be related to traction forces due to a possible interdependence of pLLP acceleration and speed of migration. Furthermore I show that apical constriction and rosette formation is not blocked in Shroom3 deficient embryos, but that larger rosettes are fragmented into many smaller ones. Finally, I give an outlook on how the absense of Shroom3 and hence the absense of morphological changes may deregulate gene transcription by elevating the levels Atoh1a, a transcription factor necessary for hair cell development.
My results and methodology demonstrate the importance of morphology in guiding developmental processes and how rather small morphological changes on the cellular level can impact further development significantly. My work also shows how powerful modern genetics, imaging and image analysis are and how diverse they are in terms of range of questions they are capable of answering. The methods and tools I developed prepare the ground for at least three quarters of the analyses I carried out and together with the documentation and data I provide, they are highly reproducible. In that regard I am especially happy that one of my developments, an improved sample preparation method, is already used by many different labs all over the world helping them to make their results more reproducible.
Background: The application of chemical dispersants is a common remediation strategy when accidental oil spills occur in aquatic environments. Breaking down the oil slick into small droplets, dispersants facilitate the increase of particulate and dissolved oil compounds, enhancing the bioavailability of toxic oil constituents. The aim of the present work was to explore the effects of water accommodated fractions (WAF) of a naphthenic North Sea crude oil produced with and without the addition of the chemical dispersant FINASOL OSR 52 to adult zebrafish exposed for 3 and 21 d. Fish were exposed to environmentally relevant concentrations of 5% and 25% WAFOIL (1:200) and to 5% WAFOIL+D (dispersant–oil ratio 1:10) in a semi-static exposure setup. Results: The chemically dispersed WAF presented a 20-fold increase of target polycyclic aromatic hydrocarbons (PAHs) in the water phase compared to the corresponding treatment without dispersant and was the only treatment resulting in markedly bioaccumulation of PAHs in carcass after 21 d compared to the control. Furthermore, only 5% WAFOIL+D caused fish mortality. In general, the undispersed oil treatments did not lead to significant effects compared to control, while the dispersed oil induced significant alterations at gene transcription and enzyme activity levels. Significant up-regulation of biotransformation and oxidative stress response genes (cyp1a, gstp1, sod1 and gpx1a) was recorded in the livers. For the same group, a significant increment in EROD activity was detected in liver along with significant increased GST and CAT activities in gills. The addition of the chemical dispersant also reduced brain AChE activity and showed a potential genotoxic effect as indicated by the increased frequency of micronuclei in erythrocytes after 21 d of exposure. Conclusions: The results demonstrate that the addition of chemical dispersants accentuates the effect of toxic compounds present in oil as it increases PAH bioavailability resulting in diverse alterations on different levels of biological organization in zebrafish. Furthermore, the study emphasizes the importance to combine multilevel endpoints for a reliable risk assessment due to high variable biomarker responses. The present results of dispersant impact on oil toxicity can support decision making for oil spill response strategies.
Across the entire animal kingdom, sociality, i.e. the tendency of individual animals to form a group with conspecifics, is a common trait. Environmental changes have to be met with corresponding, quick adaptations. For social species, the presence of conspecifics is important for survival and if social animals are deprived of access to conspecifics, this can lead to strong and lasting changes on a physiological level as well as behaviour. Gene expression changes responsible for these adaptations have so far not been understood in detail. As social isolation leads to changes on a neuronal level, it is important to investigate the gene expression changes that are induced in the brain. In this thesis, next-generation RNA-sequencing was applied to zebrafish, a well-established model organism characterized by its high degree of companionship. Within the entire brain, gene expression was analysed in zebrafish that were raised either with conspecifis or in isolation, ranging from 5 to 21 days post fertilization. Using this approach, several genes were identified that were downregulated by social isolation. In this thesis, I focused on one of these consistently downregulated genes, parathyroid hormone 2 (pth2). The expression of pth2 was demonstrated to be bidirectionally regulated by the number of conspecifics present and to be responsive to changes in the social environment within 30 minutes. Regulation of pth2 does not occur by visual or chemosensory access to conspecifcs, but is mediated by mechanosensory perception of other fish via the lateral line. In an experiment using an artificial mechanical stimulation paradigm, it was shown that the features necessary to elicit pth2 transcription closely mimick the locomotion of actual zebrafish. Other, similar stimulation paradigms are not capable to induce this transcriptional response.
Terpenes are one of the largest and most diverse class of natural products, produced by organisms from all kingdoms of life and with important applications in the pharma, flavor and fragrance industries. Well-known examples of terpenes are the pharmaceuticals artemisinin and taxol, the flavor and fragrance compounds menthol, santalol and sclareol, the structural material polyisoprene and the biofuel precursor farnesene. The methods and results presented in this work offer a variety of ways to modify terpene precursors for the creation of new terpene molecules. The application of these methodologies in well-established production systems could lead to the production of new substances, with applications in the industrial fields of pharmaceuticals, flavors and fragrances, and biofuels.
Global biodiversity is changing rapidly and contemporary climate change is an important driver of this change. As climate change continues, the challenge is to understand how it may affect the future of biodiversity. This is relevant to informing policy and conservation, but it requires reliable future projections of biodiversity. Biodiversity is the variety of life on Earth which includes the diversity of species. The species on Earth are linked in diverse networks of biotic interactions. Interacting species can respond differently to climate change. This can cause spatial or temporal mismatches between interacting species and result in secondary extinctions of species that lose obligate interaction partners. Yet, accounting for biotic interactions in biodiversity projections remains challenging. One way to address this challenge is the use of trait-based approaches because the impact of climate change on interacting species is influenced by species’ functional traits, i.e., measurable characteristics of the species that influence their abiotic and biotic interactions. First, species’ functional traits influence how species respond to climate change. Second, they influence whether the species find compatible interaction partners in reshuffled species assemblages under climate change. Thus, the overarching aim of this dissertation was to explore how trait-based approaches can increase our understanding of how climate change might affect interacting species. For this, I focussed on interactions between fleshy-fruited plants and avian frugivores along a tropical elevational gradient.
I investigated three principal research questions. First, I investigated how traits related to the sensitivity of avian frugivores to climate change and their adaptive capacity vary along elevation and covary across species. I combined estimates of species’ climatic niche breadth (approximating species’ sensitivity) with traits influencing species’ dispersal ability, dietary niche breadth and habitat niche breadth (aspects of species’ adaptive capacity). Species’ climatic niche breadth increased with increasing elevation, while their dispersal ability and dietary niche breadth decreased with increasing elevation. Across species, there was no significant relationship of the sensitivity of the avian frugivores to climate change and their adaptive capacity. The opposing patterns of species’ sensitivity to climate change and their adaptive capacity along elevation imply that species from assemblages at different elevations may respond differently to climate change. The independence between species’ sensitivity and adaptive capacity suggests that it is important to account for both sensitivity and adaptive capacity to fully understand how climate change might affect biodiversity.
Second, I assessed how climate change might influence the co-occurrence of interaction partners with compatible traits, i.e., the functional correspondence of interacting species. I integrated future projections of species’ elevational ranges considering different vertical dispersal scenarios with analyses of the functional diversity of interacting species assemblages. The functional correspondence of fleshy-fruited plants and avian frugivores was lowest if plant and bird species were projected to contract their ranges towards higher elevations in response to increasing temperatures. Contrastingly, if species were projected to expand their ranges upslope, the functional correspondence remained close. The low functional correspondence under a scenario of range contraction indicates that plant species with specific traits might miss compatible interaction partners in future assemblages. This could negatively affect their seed dispersal ability. These results suggest that ensuring the integrity of biotic interactions under climate change requires that species can shift their ranges upslope unlimitedly.
Third, I examined whether avian seed dispersal is sufficient for plants to track future temperature change along the elevational gradient. With a trait-based modelling approach, I simulated seed-dispersal distances avian frugivores can provide to fleshy-fruited woody plant species and quantified the number of long-distance dispersal events the plant species would require to fully track projected temperature shifts along elevation. Most plant species were projected to require several long-distance dispersal events to fully track the projected temperature shifts in time. However, the number of required long-distance dispersal events varied with the degree of trait matching and plant species’ traits. These findings suggest that avian seed dispersal is insufficient for plants to track future temperature change along the elevational gradient as woody plant species might not be able to undergo several consecutive long-distance dispersal events within a short time window, due to their long maturation times. These results also imply that the ability of bird-dispersed plant species to track climate change is associated with the specialization of the seed dispersal system and with plant species’ traits.
Trait-based approaches are promising tools to study impacts of climate change on interacting species. The trait-based approaches that I have developed in this thesis are applicable more widely, e.g., to other types of biotic interactions, or to assess the effects of other drivers of global change. Moreover, these approaches may be further developed to model changes in biotic interactions under global change more dynamically. Taken together, I have shown how a trait-based perspective could help to account for biotic interactions in biodiversity projections. The development of such approaches and the gained knowledge are urgently needed to facilitate the conservation of biodiversity in a rapidly changing world.