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The biotrophic pathogen Ustilago maydis causes smut disease on maize (Zea mays) and induces the formation of tumours on all aerial parts of the plant. Unlike in other biotrophic interactions, no gene-for-gene interactions have been identified in the maize–U. maydis pathosystem. Thus, maize resistance to U. maydis is considered a polygenic, quantitative trait. Here, we study the molecular mechanisms of quantitative disease resistance (QDR) in maize, and how U. maydis interferes with its components. Based on quantitative scoring of disease symptoms in 26 maize lines, we performed an RNA sequencing (RNA-Seq) analysis of six U. maydis-infected maize lines of highly distinct resistance levels. The different maize lines showed specific responses of diverse cellular processes to U. maydis infection. For U. maydis, our analysis identified 406 genes being differentially expressed between maize lines, of which 102 encode predicted effector proteins. Based on this analysis, we generated U. maydis CRISPR/Cas9 knock-out mutants for selected candidate effector sets. After infections of different maize lines with the fungal mutants, RNA-Seq analysis identified effectors with quantitative, maize line-specific virulence functions, and revealed auxin-related processes as a possible target for one of them. Thus, we show that both transcriptional activity and virulence function of fungal effector genes are modified according to the infected maize line, providing insights into the molecular mechanisms underlying QDR in the maize–U. maydis interaction.
Microglia, the primary immune cells of the central nervous system, hold a multitude of tasks in order to ensure brain homeostasis and are one of the best predictors of biological age on a cellular level. We and others have shown that these long-lived cells undergo an aging process that impedes their ability to perform some of the most vital homeostatic functions such as immune surveillance, acute injury response, and clearance of debris. Microglia have been described as gradually transitioning from a homeostatic state to an activated state in response to various insults, as well as aging. However, microglia show diverse responses to presented stimuli in the form of acute injury or chronic disease. This complexity is potentially further compounded by the distinct alterations that globally occur in the aging process. In this review, we discuss factors that may contribute to microglial aging, as well as transcriptional microglia alterations that occur in old age. We then compare these distinct phenotypic changes with microglial phenotype in neurodegenerative disease.
Cellular communication is a concept that can be explained as the transfer of signals or material (such as cytokines, ions, small molecules) between cells from the same or different type, across either short or long distances. Once this signal or material is received, it will, as a rule, promote a functional effect. Several routes, involved in this transfer, are well described and are of global importance for organ/tissue communication in an organism.
The brain interacts dynamically with the immune system, and the main route known to mediate this communication, is via the release of cytokines (by peripheral blood cells), which can then activate certain brain cell types, such as microglia, directly, or activate the vagus nerve transferring signals to neuronal populations in the brain. The communication between these two systems plays a key role in the pathophysiology of neurodegenerative diseases, and the mechanisms involved in this interaction are of central importance for understanding disease initiation and progression and search for therapeutic models.
The Momma lab previously addressed the mechanisms of interaction between the peripheral immune system and the brain by investigating cellular fusion of haematopoietic cells with neurons after inflammation. They addressed the question of whether this phenomenon also occurs under non-invasive conditions. To approach this problem, a genetic tracing model that relies on the Cre-Lox recombination system was used. Transgenic mice expressing Cre recombinase specifically in the haematopoietic lineage were crossed into a Cre-reporter background, thus all haematopoietic cells irreversibly express the reporter marker-gene EYFP. Using this model, EYFP was detected in non-haematopoietic tissues, suggesting the existence of a communication mechanism never described before. As cells containing two nuclei were never detected, fusion as a mechanism was excluded, suggesting that Cre reaches non-haematopoietic cells via a different signalling pathway. The Momma lab investigated whether the transfer of material through extracellular vesicles (EVs) could be behind this periphery-to-brain communication. Using the genetic mouse model, they were able to trace the transfer of Cre RNA via EVs between cells in vivo, generating the first in vivo evidence of functional RNA transfer by EVs between blood and brain.
The last decade has witnessed a rapid expansion of the field of EVs. Initially considered as waste disposal material, recent evidence has challenged this view. EVs are currently considered as a widespread intercellular communication system that can transport and transfer all types of biomolecules, from nucleic acids to lipids and proteins. However, several important questions are still under investigation. One of them is whether EVs are involved in brain pathophysiology, as inflammation plays an important role in onset and progression of neurodegenerative diseases and is well described in Parkinson Disease (PD). Based on preliminary data in a mouse, peripherally injected with a low dose of Lipopolysaccharide (LPS, an endotoxin found in the outer-membrane of Gram-negative bacteria, which causes an immune response), neurons and other cell population in the brain take up EVs from the periphery. Particularly, dopaminergic neurons from Substantia Nigra and Ventral Tegmental Area have been shown to receive functional RNA, transported through EVs, which can lead up to 20% of recombination. Furthermore, different neuronal populations from Hippocampus, Cortex and Cerebellum exhibit recombination, indicating a widespread signalling from blood to the brain. Therefore, the goal of my PhD thesis was to investigate the mechanisms of this transfer and the triggers that lead to EV uptake by neural cells in vivo both in pathological and physiological conditions.
In this project, the extent and function of EV-mediated signalling from blood to brain is explored in the context of peripheral inflammation and neurodegenerative diseases. Firstly, EVs isolated from WT mice were further characterized using size-exclusion chromatography (SEC), Western Blot (WB) and electron microscopy in order to extend the knowledge from previous work done in the Momma lab. Secondly, to expand on the biological relevance of the fact that inflammation is correlated with an increase in EV uptake, different approaches using the genetic murine tracing model were used. Recombination events from haematopoietic cells to the brain have been followed after peripheral injection of LPS. Peripheral inflammation caused by LPS injection led to widespread recombination events in the brain, specifically in microglia and neurons, including dopaminergic (DA) neurons. In contrast, astrocytes, oligodendrocytes and endothelial cells were never or very rarely recombined. Additionally, peripheral LPS injection in a murine model, where Cre is expressed only in erythrocytes, led to recombination events only in microglia, suggesting that the type of EV-secreting cell plays a role in the targeting of EVs to a specific cell population.
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.
The magnetic field of the Earth provides animals with various kinds of information. Its use as a compass was discovered in the mid-1960s in birds, when it was first met with considerable skepticism, because it initially proved difficult to obtain evidence for magnetic sensitivity by conditioning experiments. Meanwhile, a magnetic compass was found to be widespread. It has now been demonstrated in members of all vertebrate classes, in mollusks and several arthropod species, in crustaceans as well as in insects. The use of the geomagnetic field as a ‘map’ for determining position, although already considered in the nineteenth century, was demonstrated by magnetically simulating displacements only after 2000, namely when animals, tested in the magnetic field of a distant site, responded as if they were physically displaced to that site and compensated for the displacement. Another use of the magnetic field is that as a ‘sign post’ or trigger: specific magnetic conditions elicit spontaneous responses that are helpful when animals reach the regions where these magnetic characteristics occur. Altogether, the geomagnetic field is a widely used valuable source of navigational information for mobile animals.
The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881–1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
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.
Measuring connection to nature - a illustrated extension of the inclusion of nature in self scale
(2021)
The human-nature connection is an important factor that is frequently the subject of environmental education research and environmental psychology. Therefore, over the years, numerous measuring instruments have been established to quantitatively record a person’s connection to nature. However, there is no instrument specifically for children with cognitive limitations. For this reason, in this study, an established scale for connection to nature, the inclusion of nature in self scale (INS), was modified especially for the needs of this group. Study 1 investigated what students understand by the term “nature” in order to create an illustrated version of the INS. In study 2, the new instrument was tested on university students and compared with the original INS and the connectedness to nature scale (CNS). No significant differences between the original INS and the new developed scale were found (p = 0.247), from which it can be concluded that the illustrated INS (IINS) measures the connection to nature with similar accuracy as the original INS. In study 3, the instrument was tested together with other established nature connection instruments on the actual target group, students with disabilities. The correlation between the IINS, the CNS, and nature connectedness scale (NR) were in accordance with the expected literature values (rIINS-CNS = 0.570 & rIINS-NR = 0.605). The results of this study also prove effectiveness of the developed illustrated scale. This research thus provides a suitable measuring instrument for people with learning difficulties and can make a contribution to the investigation of human-nature connections and conservation education.
Beobachten, untersuchen, experimentieren: Wie soll das gehen unter Pandemiebedingungen? Wenn auch die Chancen der digitalen Medien nun gezwungenermaßen zusehends sichtbar wurden, so sind gerade für angehende Biologielehrkräfte Primärerfahrungen mit originalen Organismen und das Einüben naturwissenschaftlicher Arbeitsmethoden sehr wichtig...
Nomadic movements are often a consequence of unpredictable resource dynamics. However, how nomadic ungulates select dynamic resources is still understudied. Here we examined resource selection of nomadic Mongolian gazelles (Procapra gutturosa) in the Eastern Steppe of Mongolia. We used daily GPS locations of 33 gazelles tracked up to 3.5 years. We examined selection for forage during the growing season using the Normalized Difference Vegetation Index (NDVI). In winter we examined selection for snow cover which mediates access to forage and drinking water. We studied selection at the population level using resource selection functions (RSFs) as well as on the individual level using step-selection functions (SSFs) at varying spatio-temporal scales from 1 to 10 days. Results from the population and the individual level analyses differed. At the population level we found selection for higher than average NDVI during the growing season. This may indicate selection for areas with more forage cover within the arid steppe landscape. In winter, gazelles selected for intermediate snow cover, which may indicate preference for areas which offer some snow for hydration but not so much as to hinder movement. At the individual level, in both seasons and across scales, we were not able to detect selection in the majority of individuals, but selection was similar to that seen in the RSFs for those individuals showing selection. Difficulty in finding selection with SSFs may indicate that Mongolian gazelles are using a random search strategy to find forage in a landscape with large, homogeneous areas of vegetation. The combination of random searches and landscape characteristics could therefore obscure results at the fine scale of SSFs. The significant results on the broader scale used for the population level RSF highlight that, although individuals show uncoordinated movement trajectories, they ultimately select for similar vegetation and snow cover.
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.
Downy mildews caused by obligate biotrophic oomycetes result in severe crop losses worldwide. Among these pathogens, Pseudoperonospora cubensis and P. humuli, two closely related oomycetes, adversely affect cucurbits and hop, respectively. Discordant hypotheses concerning their taxonomic relationships have been proposed based on host–pathogen interactions and specificity evidence and gene sequences of a few individuals, but population genetics evidence supporting these scenarios is missing. Furthermore, nuclear and mitochondrial regions of both pathogens have been analyzed using microsatellites and phylogenetically informative molecular markers, but extensive comparative population genetics research has not been done. Here, we genotyped 138 current and historical herbarium specimens of those two taxa using microsatellites (SSRs). Our goals were to assess genetic diversity and spatial distribution, to infer the evolutionary history of P. cubensis and P. humuli, and to visualize genome-scale organizational relationship between both pathogens. High genetic diversity, modest gene flow, and presence of population structure, particularly in P. cubensis, were observed. When tested for cross-amplification, 20 out of 27 P. cubensis-derived gSSRs cross-amplified DNA of P. humuli individuals, but few amplified DNA of downy mildew pathogens from related genera. Collectively, our analyses provided a definite argument for the hypothesis that both pathogens are distinct species, and suggested further speciation in the P. cubensis complex.
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.
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.
Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi–Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.
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.
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.
Der Verzehr von radioaktiv belasteten Pilzfruchtkörpern stellt ein Gesundheitsrisiko für den Menschen dar und auch fast 35 Jahre nach der Reaktorkatastrophe von Tschernobyl im Jahr 1986 sind Pilze aus Waldökosystemen zum Teil noch stark durch das ausgetretene radioaktive 137Cs belastet. Die Einschätzung der Belastung und somit des Gesundheitsrisikos ist aufgrund einer Vielzahl von Einflussfaktoren, wie z. B. der Pilzart, der Tiefe des Myzels, der Bodenkontamination und der Feuchtigkeit des Bodens, schwierig. Ziel dieser Arbeit war es die Variabilität, den Einfluss verschiedener Faktoren sowie die effektive Halbwertszeit der 137Cs-Aktivität in Pilzfruchtkörpern zu ermitteln. Des Weiteren wurde überprüft, ob die Bodenkontamination für eine Abschätzung der 137Cs-Aktivität von Pilzfruchtkörpern herangezogen werden kann. Für die Untersuchungen wurden über mehrere Jahre Proben von Maronenröhrlingen (Imleria badia) und Steinpilzen (Boletus edulis) aus vier Waldgebieten in Mittel- und Süddeutschland mit unterschiedlichem Aktivitätseintrag nach der Reaktorkatastrophe von Tschernobyl im Jahr 1986 analysiert. Die Gebiete waren Eichenzell, Wülfersreuth, Oberschönenfeld und der Nationalpark Bayerischer Wald. Als Ergänzung dienten zugesendete Proben derselben Pilzarten von Mitgliedern aus Pilzvereinen aus ganz Deutschland. Zusätzlich zu den Pilzproben wurden Bodenproben gemessen, um zum einen die aktuelle Bodenkontamination zu bestimmen und zum anderen zu überprüfen, ob der Großteil des 137Cs weiterhin im Bereich des Pilzmyzels zu finden ist.
Für die Untersuchung der örtlichen Variabilität der 137Cs-Aktivität wurden Maronenröhrlinge (Imleria badia) aus dem Waldgebiet Eichenzell in den Jahren 2017 bis 2019 analysiert. Innerhalb eines Sammeltages variierten die Messwerte verschiedener Proben innerhalb des Waldgebietes teilweise um den Faktor sechs. Dabei ist die Variabilität innerhalb eines Teilgebietes größer als zwischen beiden Teilgebieten des Waldgebietes Eichenzell. Für ein repräsentatives Ergebnis eines Gebietes ist es aufgrund der Variabilität erforderlich, eine ausreichende Menge an Fruchtkörpern zu analysieren.
Um die effektive Halbwertszeit der 137Cs-Aktivität in Maronenröhrlingen (Imleria badia) zu ermitteln, wurden Proben aus drei Waldgebieten über fünf bis neun Jahre analysiert. Die Wahl der drei Waldgebiete erfolgte anhand des 137Cs-Aktivitätseintrags nach der Reaktorkatastrophe von Tschernobyl im Jahr 1986. Die Bodenkontaminationswerte variieren von 3.000 Bq/m² in Eichenzell über 12.500 Bq/m² in Wülfersreuth bis 35.000 Bq/m² in Oberschönenfeld. Die effektiven Halbwerts-zeiten liegen in einem engen Bereich von 5,2 bis 5,8 Jahre mit einem Mittelwert von 5,4 ± 0,3 Jahren. Damit reduziert sich die radioaktive Belastung der Pilzfruchtkörper in etwa fünfmal schneller als durch die rein physikalische Halbwertszeit des 137Cs von 30,08 Jahren. Durch die Hinzunahme von bereits im Jahr 1990 veröffentlichten Daten ergab sich eine längere effektive Halbwertszeit von 7,7 ± 0,6 Jahren.
Für die Untersuchung der zwei Einflussfaktoren Exposition des Sammelgebiets (Hangausrichtung nach Ost oder West) und Höhenlage wurden sowohl Maronenröhrlinge (Imleria badia) als auch Steinpilze (Boletus edulis) hinsichtlich der 137Cs-Aktivität gemessen, um die Auswirkung auf Pilzarten mit unterschiedlichem Akkumulationsvermögen zu analysieren. Als Untersuchungsgebiet diente der Nationalpark Bayerischer Wald, da dieser ein großes Gebiet umfasst und verschiedene Ausprägungen der beiden Faktoren abbildet. Zudem wurde das Gebiet in Folge der Reaktorkatastrophe von Tschernobyl stark kontaminiert und der Park ist ein beliebtes Pilzsammelgebiet. Anhand der 137Cs-Aktivität von Bodenproben konnte das Gebiet in zwei Regionen (Cluster) eingeteilt werden: eine Region mit hohem und eine mit niedrigem Aktivitätseintrag. Im Vergleich wiesen Maronenröhrlinge (Imleria badia) durchschnittlich eine um den Faktor fünf höhere 137Cs-Aktivität als Steinpilze (Boletus edulis) auf. Der Faktor Höhenlage zeigte im Gegensatz zur Exposition einen Einfluss auf die Kontamination der Pilzfruchtkörper. In Bezug auf die Höhenlage war der Einfluss nur im Falle eines hohen Aktivitätseintrags signifikant, wobei die Pilzproben aus der niedrigsten Höhenlage am höchsten belastet waren.
Zur Ermittlung der vertikalen Verteilung des 137Cs im Boden wurden in den Waldgebieten Eichenzell und Nationalpark Bayerischer Wald Proben bis zu einer Tiefe von 24 cm entnommen und anschließend in 2 cm Schichten analysiert. Alle Verteilungen konnten mit einem Gauß-Fit oder einem multiplen Gauß-Fit mit 2 bis 3 Maxima abgebildet werden. Das erste Maximum lag in allen Fällen in den organischen Horizonten oder im Übergangsbereich zum Ah-Horizont. Folglich befindet sich der Großteil des 137Cs fast 35 Jahre nach der Reaktorkatastrophe von Tschernobyl immer noch im Bereich des Pilzmyzels und kann somit von den Pilzen aufgenommen und in den Fruchtkörpern angereichert werden.
Der Vergleich der 137Cs-Aktivität der Pilz- und Bodenproben aus dem Nationalpark Bayerischer Wald ergab sowohl für Maronenröhrlinge (Imleria badia) als auch für Steinpilze (Boletus edulis) eine positive Korrelation. Nach Unterteilung der Proben anhand der Höhenlage zeigte sich eine noch stärkere Korrelation. Dies zeigt, dass neben der Bodenkontamination auch die Höhenlage einen Einfluss auf die 137Cs-Aktivität der Fruchtkörper hat.
...
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.
Nature benefits human health. To date, however, little is known whether biodiversity relates to human health. While some local and city level studies show that species diversity, as a measure of biodiversity, can have positive effects, there is a lack of studies about the relationship between different species diversity measures and human health, especially at larger spatial scales. Here, we conduct cross-sectional analyses of the association between species diversity and human health across Germany, while controlling for socio-economic factors and other nature characteristics. As indicators for human health, we use the mental (MCS) and physical health (PCS) component scales of the German Socio-Economic Panel (SOEP, Short Form Health Questionnaire – SF12). For species diversity, we use species richness and abundance estimates of two species groups: plants and birds. We phrase the following hypotheses: plant and bird species are positively associated with mental and physical health (H1 & H3); bird abundance is positively related to mental health (H2). Our results demonstrate a significant positive relationship between plant and bird species richness and mental health across all model variations controlling for a multitude of other factors. These results highlight the importance for species diversity for people’s mental health and well-being. Therefore, policy makers, landscape planners and greenspace managers on the local and national level should consider supporting biodiverse environments to promote mental health and wellbeing. For this purpose, we propose to use species diversity measures as indicators for salutogenic (health promoting) characteristics of nature, landscape and urban green space.
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.
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.
Non-ribosomal peptide synthetases (NRPSs) are large multienzyme machineries. They synthesize numerous important natural products starting from amino acids. For peptide synthesis functionally specialized NRPS modules interact in a defined manner. Individual modules are either located on a single or on multiple different polypeptide chains. The “peptide-antimicrobial-Xenorhabdus” (PAX) peptide producing NRPS PaxS from Xenorhabdus bacteria consists of the three proteins PaxA, PaxB and PaxC. Different docking domains (DDs) located at the N-termini of PaxB and PaxC and at the C-termini of PaxA and BaxB mediate specific non-covalent interactions between them. The N-terminal docking domains precede condensation domains while the C-terminal docking domains follow thiolation domains. The binding specificity of individual DDs is important for the correct assembly of multi-protein NRPS systems. In many multi-protein NRPS systems the docking domains are sufficient to mediate the necessary interactions between individual protein chains. However, it remains unclear if this is a general feature for all types of structurally different docking domains or if the neighboring domains in some cases support the function of the docking domains. Here, we report the 1H, 13C and 15 N NMR resonance assignments for a C-terminal di-domain construct containing a thiolation (T) domain followed by a C-terminal docking domain (CDD) from PaxA and for its binding partner – the N-terminal docking domain (NDD) from PaxB from the Gram-negative entomopathogenic bacterium Xenorhabdus cabanillasii JM26 in their free states and for a 1:1 complex formed by the two proteins. These NMR resonance assignments will facilitate further structural and dynamic studies of this protein complex.
The alpha-proteobacterium Zymomonas mobilis is a promising biofuel producer, based on its native metabolism that efficiently converts sugars to ethanol. Therefore, it has a high potential for industrial-scale biofuel production. Two previous studies suggested that Z. mobilis strain Zm4 might not be monoploid. However, a systematic analysis of the genome copy number is still missing, in spite of the high potential importance of Z. mobilis. To get a deep insight into the ploidy level of Z. mobilis and its regulation, the genome copy numbers of three strains were quantified. The analyses revealed that, during anaerobic growth, the lab strain Zm6, the Zm6 type strain obtained from DSMZ (German Collection of Microorganisms), and the lab strain Zm4, have copy numbers of 18.9, 22.3 and 16.2, respectively, of an origin-adjacent region. The copy numbers of a terminus-adjacent region were somewhat lower with 9.3, 15.8, and 12.9, respectively. The values were similar throughout the growth curves, and they were only slightly downregulated in late stationary phase. During aerobic growth, the copy numbers of the lab strain Zm6 were much higher with around 40 origin-adjacent copies and 17 terminus-adjacent copies. However, the cells were larger during aerobic growth, and the copy numbers per μm3 cell volume were rather similar. Taken together, this first systematic analysis revealed that Z. mobilis is polyploid under regular laboratory growth conditions. The copy number is constant during growth, in contrast to many other polyploid bacteria. This knowledge should be considered in further engineering of the strain for industrial applications.
Research on Podospora anserina unraveled a network of molecular pathways affecting biological aging. In particular, a number of pathways active in the control of mitochondria were identified on different levels. A long-known key process active during aging of P. anserina is the age- related reorganization of the mitochondrial DNA (mtDNA). Mechanisms involved in the stabilization of the mtDNA lead to lifespan extension. Another critical issue is to balance mitochondrial levels of reactive oxygen species (ROS). This is important because ROS are essential signaling molecules, but at increased levels cause molecular damage. At a higher level of the network, mechanisms are active in the repair of damaged compounds. However, if damage passes critical limits, the corresponding pathways are overwhelmed and impaired molecules as well as those present in excess are degraded by specific enzymes or via different forms of autophagy. Subsequently, degraded units need to be replaced by novel functional ones. The corresponding processes are dependent on the availability of intact genetic information. Although a number of different pathways involved in the control of cellular homeostasis were uncovered in the past, certainly many more exist. In addition, the signaling pathways involved in the control and coordination of the underlying pathways are only initially understood. In some cases, like the induction of autophagy, ROS are active. Additionally, sensing and signaling the energetic status of the organism plays a key role. The precise mechanisms involved are elusive and remain to be elucidated.
Bisphenols and phthalates, chemicals frequently used in plastic products, promote obesity in cell and animal models. However, these well-known metabolism disrupting chemicals (MDCs) represent only a minute fraction of all compounds found in plastics. To gain a comprehensive understanding of plastics as a source of exposure to MDCs, we characterized all chemicals present in 34 everyday products using nontarget high-resolution mass spectrometry and analyzed their joint adipogenic activities by high-content imaging. We detected 55,300 chemical features and tentatively identified 629 unique compounds, including 11 known MDCs. Importantly, chemicals that induced proliferation, growth, and triglyceride accumulation in 3T3-L1 adipocytes were found in one third of the products. Since the majority did not target peroxisome proliferator-activated receptor γ, the effects are likely to be caused by unknown MDCs. Our study demonstrates that daily-use plastics contain potent mixtures of MDCs and can, therefore, be a relevant yet underestimated environmental factor contributing to obesity.
Teaser Plastics contain a potent mixture of chemicals promoting adipogenesis, a key process in developing obesity.
Background: Efficient transfer of chemical signals is important for successful mating in many animal species. Multiple evolutionary lineages of animals evolved direct sex pheromone transmission during traumatic mating—the wounding of the partner with specialized devices—which helps to avoid signal loss to the environment. Although such direct transmission modes of so-called allohormone pheromones are well-documented in invertebrates, they are considered rare in vertebrates. Males of several species of the frog genus Plectrohyla (Hylidae, Anura) have elongated teeth and develop swollen lips during the breeding season. Here we investigated the possibility that these structures are used to scratch the females’ skin and apply allohormone pheromones during traumatic mating in several Plectrohyla species.
Results: Our behavioural observations revealed that males press their upper jaw onto the females’ dorsum during amplexus, leaving small skin scratches with their teeth. Histological examinations of the males’ lips identified specialized mucus glands, resembling known amphibian pheromone glands. Whole-transcriptome sequencing of these breeding glands showed high expression of sodefrin precursor-like factor (SPF) proteins, which are known to have a pheromone function in multiple amphibian species.
Conclusions: Our study suggests SPF delivery via traumatic mating in several anuran species: the males have specialized breeding glands in the lips for production and secretion and use their elongated teeth as wounding devices for application. We hypothesize that these SPF proteins end up in the females’ circulatory system, where understanding their exact function will require further molecular, physiological and behavioural testing.
1. During the last century, the practice of fur farming in Europe led to the introduction of two mammal species from opposite ends of the world. With their subsequent unintentional escape from captivity or intentional releases, the process of slow expansion and establishment in Europe began. The raccoon Procyon lotor and the raccoon dog Nyctereutes procyonoides are included on the European Union’s list of invasive alien species.
2. We characterised the current climatic niches of the two species in their native ranges in North America and Asia, and compared them with their non-native-range niches in Europe, where we also projected climatic suitability. The aim was to locate suitable habitats beyond their current ranges and assess where a range expansion can be expected.
3. Niche comparison and the projection of climatic suitability in Europe were based on eight bioclimatic variables and presence records from the Global Biodiversity Information Facility database. For niche modelling, we applied the maximum entropy approach (Maxent) and used the native-range data for training.
4. Minimum temperature of the coldest month (bio06) was identified as the most important bioclimatic variable in the habitat suitability models for both species. Different tolerance levels regarding this variable might explain small differences between the species’ projected ranges, especially in the north and east of Europe. The high niche unfilling for both species in Europe suggests a potential for expansion beyond their present ranges.
5. With only little understanding of their ecological impacts in their new ranges, including the potential risk of Nyctereutes procyonoides as SARS-CoV-2 reservoir hosts, further research and management is required at various spatial scales in Europe.
In der vorliegenden Arbeit wurde das Zinkfinger-µ-Protein HVO_2753 des halophilen Archaeons Haloferax volcanii hinsichtlich seiner biologischen Funktion und seiner Struktur charakterisiert.
Zinkfinger-µ-Proteine wurden bisher nur sehr wenig untersucht, während ihnen jedoch in den letzten Jahren steigendes Interesse entgegengebracht wird. Im Genom von H. volcanii sind mehr als 40 solcher Zinkfinger-µ-Proteine codiert. Von diesen besitzt mit HVO_2753 lediglich eines nicht nur zwei, sondern vier der charakteristischen C(P)XCG-Muster, was für die Anwesenheit von zwei Zinkfinger-Motiven spricht. Während Homologe von HVO_2753 in vielen Euryachaeota vorkommen und manche davon als Zink-Ribbon RNA-Bindeproteine annotiert sind, ist über ihre Funktion jedoch nichts bekannt. Zur Charakterisierung des Proteins wurde zunächst eine in frame-Deletionsmutante seines Gens erstellt und diese einer phänotypischen Charakterisierung unterzogen. Die Mutante wies, verglichen mit dem Wildtyp, keine Unterschiede im Wachstum in Komplexmedium oder in synthetischem Medium mit Glukose als Kohlenstoffquelle auf. Ein schweres Defizit konnte jedoch sowohl bei der Adhäsion und Biofilmbildung als auch der Schwärmfähigkeit der Deletionsmutante festgestellt werden. Während die Schwärmfähigkeit des Wildtyps durch plasmidische Expression von HVO_2753 in der Deletionsmutante teilweise wiederhergestellt werden konnte, war eine solche Komplementation bei der Biofilmbildung nicht möglich. Die Analyse der Relevanz ausgewählter Aminosäuren, wie beispielsweise das jeweils erste Cystein in jedem C(P)XCG-Muster zeigte, dass die Substitution jeder einzelnen der getesteten Aminosäuren einen Funktionsverlust des Proteins nach sich zieht. Die Untersuchung des HVO_2753-Transkripts mittels Northern Blot-Analyse bestätigte erste Hinweise aus vorangegangenen dRNA- und RNA-Seq-Studien, die eine Co-Transkription von HVO_2753 mit dem Nachbargen HVO_2752, das für den Translations-Elongationsfaktor aEF-1 beta codiert, aufzeigten. Daraufhin erfolgte eine Untersuchung des Ribosomenprofils, bei der keine Unterschiede zwischen der Deletionsmutante und der Überexpressionsmutante von HVO_2753 festgestellt werden konnten.
Eine Variante von HVO_2753 mit N-terminalem Hexahistidin-Tag wurde homolog überproduziert und aufgereinigt. Die Überproduktion und Aufreinigung wurden im Zuge dieser Arbeit weiter, speziell für HVO_2753, optimiert. So konnten große Mengen von HVO_2753n überproduziert und bei nativen Salzbedingungen mittels Nickel-Affinitätschromatographie und anschließender Größenausschlusschromatographie aufgereinigt werden. Eine massenspektrometrische Analyse bestätigte sowohl das Molekulargewicht als auch die Abwesenheit posttranslationaler Modifikationen. Die Untersuchung der Menge an gebundenem Zink im Protein erfolgte beim Zink-Assay mit Hilfe des hochsensitiven und hochspezifischen Fluorophors ZnAF-2F. Dabei konnte gezeigt werden, dass überraschenderweise lediglich ein Zink-Ion in HVO_2753 gebunden vorliegt.
Zur weiteren Funktionsaufklärung erfolgte eine Interaktionspartnersuche. Hierfür wurde HVO_2753 überproduziert, ein in vivo-Crosslink und anschließend eine native Aufreinung durchgeführt. Die massenspektrometrische Analyse ausgewählter Fraktionen nach der Größenausschlusschromatographie ergaben eine Vielzahl an möglichen Bindepartnern. Besonders häufig wurde hier die GalE family Epimerase/Dehydratase gefunden. Eine weitere Methode zur Suche nach Interaktionspartnern richtete sich auf RNAs. Hier konnten mittels eines eigens entwickelten Protokolls neben RNAs des Translationsapparates auch mehrfach die tRNA(Glu) gefunden werden.
Zusätzlich sollte die Transkriptomanalyse mittels RNA-Sequenzierung Unterschiede zwischen Wildtyp, Deletionsmutante und Komplementationsmutante aufzeigen. Hier wurden weitreichende Auswirkungen der Deletion von HVO_2753 gefunden. Zahlreiche Gene in mehreren Operons zur Motilität und Chemotaxis lagen in der Deletionsmutante stark herunterreguliert vor, während die Gene einiger Metallionen-Transporter und der Eisen(III)-Siderophor-Biosynthese hochreguliert vorlagen. In der Komplementationsmutante konnten nur von den letzteren Genen Transkriptlevel vergleichbar mit denen des Wildtyps wiedergefunden werden.
In dieser Arbeit konnte gezeigt werden, dass das kleine Zinkfinger-Protein HVO_2753 eine essenzielle Rolle in der positiven Regulation der Motilität, Chemotaxis und der Adhäsion bzw. Biofilmbildung spielt. Gleichzeitig übt HVO_2753 eine negative Regulation auf den Metallionen-Transport und die Biosynthese des Eisen(III)-Siderophors aus.
Gene conversion is defined as the non-reciprocal transfer of genetic information from one site to a homologous, but not identical site of the genome. In prokaryotes, gene conversion can increase the variance of sequences, like in antigenic variation, but can also lead to a homogenization of sequences, like in the concerted evolution of multigene families. In contrast to these intramolecular mechanisms, the intermolecular gene conversion in polyploid prokaryotes, which leads to the equalization of the multiple genome copies, has hardly been studied. We have previously shown the intermolecular gene conversion in halophilic and methanogenic archaea is so efficient that it can be studied without selecting for conversion events. Here, we have established an approach to characterize unselected intermolecular gene conversion in Haloferax volcanii making use of two genes that encode enzymes involved in carotenoid biosynthesis. Heterozygous strains were generated by protoplast fusion, and gene conversion was quantified by phenotype analysis or/and PCR. It was verified that unselected gene conversion is extremely efficient and it was shown that gene conversion tracts are much longer than in antigenic variation or concerted evolution in bacteria. Two sites were nearly always co-converted when they were 600 bp apart, and more than 30% co-conversion even occurred when two sites were 5 kbp apart. The gene conversion frequency was independent from the extent of genome differences, and even a one nucleotide difference triggered conversion.
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.
Mitochondrial F1Fo-ATP-synthase dimers play a critical role in shaping and maintenance of mitochondrial ultrastructure. Previous studies have revealed that ablation of the F1Fo-ATP-synthase assembly factor PaATPE of the ascomycete Podospora anserina strongly affects cristae formation, increases hydrogen peroxide levels, impairs mitochondrial function and leads to premature cell death. In the present study, we investigated the underlying mechanistic basis. Compared to the wild type, we observed a slight increase in non-selective and a pronounced increase in mitophagy, the selective vacuolar degradation of mitochondria. This effect depends on the availability of functional cyclophilin D (PaCYPD), the regulator of the mitochondrial permeability transition pore (mPTP). Simultaneous deletion of PaAtpe and PaAtg1, encoding a key component of the autophagy machinery or of PaCypD, led to a reduction of mitophagy and a partial restoration of the wild-type specific lifespan. The same effect was observed in the PaAtpe deletion strain after inhibition of PaCYPD by its specific inhibitor, cyclosporin A. Overall, our data identify autophagy-dependent cell death (ADCD) as part of the cellular response to impaired F1Fo-ATP-synthase dimerization, and emphasize the crucial role of functional mitochondria in aging.
The factors that vary the aroma of Tuber magnatum fruiting bodies are poorly understood. The study determined the headspace aroma composition, sensory aroma profiles, maturity and bacterial communities from T. magnatum originating from Italy, Croatia, Hungary, and Serbia, and tested if truffle aroma is dependent on provenance and if fruiting body volatiles are explained by maturity and/or bacterial communities.
Headspace volatile profiles were determined using gas chromatography–mass spectrometry–olfactometry (GC-MS-O) and aroma of fruiting body extracts were sensorially assessed. Fruiting body maturity was estimated through spore melanisation. Bacterial community was determined using 16S rRNA amplicon sequencing.
Main odour active compounds were present in all truffles but varied in concentration. Aroma of truffle extracts were sensorially discriminated by sites. However, volatile profiles of individual fruiting bodies varied more within sites than across geographic area, while maturity level did not play a role. Bacterial communities varied highly and were partially explained by provenance. A few rare bacterial operational taxonomical units associated with a select few nonodour active volatile compounds.
Specificities of the aroma of T. magnatum truffles are more likely to be linked to individual properties than provenance. Some constituents of bacteria may provide biomarkers of provenance and be linked to nonodour active volatiles.
Clonal hematopoiesis of indeterminate potential (CHIP) is caused by recurrent somatic mutations leading to clonal blood cell expansion. However, direct evidence of the fitness of CHIP-mutated human hematopoietic stem cells (HSCs) in blood reconstitution is lacking. Because myeloablative treatment and transplantation enforce stress on HSCs, we followed 81 patients with solid tumors or lymphoid diseases undergoing autologous stem cell transplantation (ASCT) for the development of CHIP. We found a high incidence of CHIP (22%) after ASCT with a high mean variant allele frequency (VAF) of 10.7%. Most mutations were already present in the graft, albeit at lower VAFs, demonstrating a selective reconstitution advantage of mutated HSCs after ASCT. Thus, CHIP-mutated stem and progenitor cells largely gain on clone size upon ASCT-related blood reconstitution, leading to an increased future risk of CHIP-associated complications. CHIP increase with age and is also associated with atherosclerosis and inflammation. Age and inflammation are the major risk factors for heart failure, yet the association of CHIP with chronic ischemic heart failure (CHF) in humans is unknown. Therefore, we analyzed bone marrow-derived mononuclear cells from 200 patients with CHF by NGS to detect the presence of CHIP and associated such with long-term prognosis in patients with CHF. Forty-seven mutations with a VAF of at least 2% were found in 18.5% of 200 patients with CHF. The mutations most commonly occurred in the genes DNMT3A and TET2. During a median follow-up of 4.4 years, a significantly worse clinical outcome for patients with either DNMT3A or TET2 mutations compared with non-CHIP carriers was notable. Importantly, there was a significant dose-response association between VAF and clinical outcome. Our data suggest that somatic mutations in hematopoietic cells, may be significantly associated with the progression and poor prognosis of CHF.
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.
Fatty acids in oomycetes
(2021)
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.
Thermoanaerobacter kivui is a thermophilic acetogen that can grow on carbon monoxide as sole carbon and energy source. To identify the gene(s) involved in CO oxidation, the genome sequence was analyzed. Two genes potentially encoding CO dehydrogenases were identified. One, cooS, potentially encodes a monofunctional CO dehydrogenase, whereas another, acsA, potentially encodes the CODH component of the CODH/ACS complex. Both genes were cloned, a His-tag encoding sequence was added, and the proteins were produced from a plasmid in T. kivui. His-AcsA copurified by affinity chromatography with AcsB, the acetyl-CoA synthase of the CO dehydrogenase/acetyl CoA synthase complex. His-CooS copurified with CooF1, a small iron-sulfur center containing protein likely involved in electron transport. Both protein complexes had CO:ferredoxin oxidoreductase as well as CO:methyl viologen oxidoreductase activity, but the activity of CooSF1 was 15-times and 231-times lower, respectively. To underline the importance of CooS, the gene was deleted in the CO-adapted strain. Interestingly, the ∆cooS deletion mutant did not grow on CO anymore. These experiments clearly demonstrated that CooS is essential for growth of T. kivui on CO. This is in line with the hypothesis that CooS is the CO-oxidizing enzyme in cells growing on CO.
Camellia sinensis is one of the major crops grown in Taiwan and has been widely cultivated around the island. Tea leaves are prone to various fungal infections, and leaf spot is considered one of the major diseases in Taiwan tea fields. As part of a survey on fungal species causing leaf spots on tea leaves in Taiwan, 19 fungal strains morphologically similar to the genus Diaporthe were collected. ITS (internal transcribed spacer), tef1-α (translation elongation factor 1-α), tub2 (beta-tubulin), and cal (calmodulin) gene regions were used to construct phylogenetic trees and determine the evolutionary relationships among the collected strains. In total, six Diaporthe species, including one new species, Diaporthe hsinchuensis, were identified as linked with leaf spot of C. sinensis in Taiwan based on both phenotypic characters and phylogeny. These species were further characterized in terms of their pathogenicity, temperature, and pH requirements under laboratory conditions. Diaporthe tulliensis, D. passiflorae, and D. perseae were isolated from C. sinensis for the first time. Furthermore, pathogenicity tests revealed that, with wound inoculation, only D. hongkongensis was pathogenic on tea leaves. This investigation delivers the first assessment of Diaporthe taxa related to leaf spots on tea in Taiwan.
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.
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.
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.
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 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.
Get3 in Arabidopsis
(2021)
Der guided entry of tail-anchored proteins (GET) Biogenese-Weg vermittelt den Transport und die Insertion von tail-anchor (TA) Proteinen in die Doppellipidschicht des Endoplasmatischen Retikulums (ER). TA Proteine sind dadurch gekennzeichnet, dass sie eine Transmembran Domäne (TMD) in den letzten 50 Aminosäuren ihrer Sequenz beherbergen. Diese TMD enthält die notwendigen Informationen, mit denen die Proteine an ihren jeweiligen subzellulären Zielort transportiert werden können. TA Proteine erfüllen eine Vielzahl von essentiellen biologischen Prozessen, sie fungieren zum Beispiel als Rezeptoren, sind maßgeblich an der Fusion von Vesikeln beteiligt sowie an der Initiation von Apoptose. Durch ihren modularen Aufbau können TA Proteine nicht mit dem Signalerkennungspartikel interagieren und müssen deshalb posttranslational zum ER geleitet werden. Im Modellorganismus Bäckerhefe (Saccharomyces cerevisiae) ist der GET Biogenese-Weg am besten beschrieben und läuft wie folgt ab: Nach der Termination der Translation bindet das Protein SgtA das TA Protein und händigt es über den Adapter-Komplex, bestehend aus Get4 und Get5, an die zytosolische ATPase Get3 aus. Get3 ist der zentrale Zielsteuerungsfaktor des GET Biogenese-Weges. Sobald sich ein Komplex aus Zeilsteuerungsfaktor und TA Protein gebildet hat, wird dieses zur Membran des ERs überführt. Dort wird das TA Protein an den Rezeptorkomplex bestehend aus Get1 und Get2 übergeben, welcher anschließend die Insertion des TA Proteins in die Doppellipidschicht des ERs initiiert.
Get3 hat im zellulären Kontext noch eine weitere Funktion. Unter oxidativem Stress oder Energie depletierenden Bedingungen wird Get3 zu spezifischen Foci rekrutiert, an denen sich noch weitere durch Stress -induzierbare Proteine, wie z.B. die der Familie der Hitze Stress Proteine (HSPs) versammeln. Analysen haben gezeigt, dass Get3 unter den oben genannten Bedingungen, Konformationsänderungen durchläuft und dann als ATP unabhängige Holdase fungiert. Diese kann die exponierten, hydrophoben Anteile von Proteinen binden, um dadurch die Proteostasis aufrechtzuhalten.
Durch die Bedeutsamkeit der TA Proteinen ist die zentrale ATPase Get3 in allen Domänen des Lebens hochgradig konserviert. Phylogenetische Analysen ergaben, dass sich Get3 im Allgemeinen in eine „A“ Gruppe sowie eine „BC“ Gruppe aufspaltet. Im Modellorganismus Arabidopsis thaliana (Ackerschmalwand) wurden drei Orthologe zu Get3 identifiziert. Eins davon gehört zu der „A“ Gruppe und befindet sich im Zytoplasma. Die anderen zwei Orthologe befinden sich in den Organellen endo-symbiotischen Ursprungs und gehören der „BC“ Gruppe an. Untersuchungen an verschiedenen Deletionsmutanten in A. thaliana haben gezeigt, dass die Mutationen einzelner GET Komponenten zu einer signifikanten Verkürzung der Haarwurzeln führen, obwohl der restliche Habitus der Pflanze unverändert bleibt. Diesbezüglich wurde SYP123 als einziges TA Proteine identifiziert, dessen Abundanz durch die Deletion von GET Komponenten beeinflusst werden kann. Von den anderen beiden Orthologen organellären Ursprungs ist, abgesehen von ihrer Lokalisation nichts weiter bekannt
Vier Orthologe Gruppen in Pflanzen
Da bislang nicht mehr als zehn Pflanzenarten für phylogenetische Analysen herangezogen wurden, wurden in dieser Arbeit die taxonomischen Beziehungen von Get3 zu einander in 50 Spezies der Viridiplantae auf Basis der Orthologie sowie Homologie untersucht. Dies führte zur Identifizierung einer zytolischen (AtGet3a), einer plastidären (AtGet3b), einer mitochondriellen (AtGet3c) sowie einer Monokotyledone spezifischen Gruppe (SBGet3). Die Lokalisation der ersten drei Gruppen wurde in selektierten Pflanzen, sowohl homolog als auch heterolog, der unterschiedlichen Spezies mittels saGFP untersucht, und es konnte gezeigt werden, dass mehrere Get3 Orthologe mit unterschiedlichen subzellulären Lokalisationen eine unter Pflanze häufig auftretende Eigenschaft ist. Das Weitern konnte gezeigt werden, dass manche Komponenten des Präzielsteuerungskomplexes (SgtA und Get4) sowie des Rezeptorkomplexes (Get1) in fast allen der 50 untersuchten Pflanzenarten vorhanden sind. Dies weist auf eine Konservierung des gesamten GET Biogenese-Weges in Pflanzen hin.
Get3a in Arabidopsis thaliana
Da die molekulare Zusammensetzung des Präzielsteuerungskomplexes für AtGet3a in A. thaliana nicht bekannt ist, habe ich Co-Immunpräzipitationen mit Zellextrakten aus weißer Zellkultur und einen von mir selbst aufgereinigten Antikörper gegen AtGet3a durchgeführt. Nach anschließender Gelelektrophorese und einer Anfärbung mit Coomassie Brilliant Blue ließ sich ein reproduzierbares Muster aus Proteinbanden erkennen, welche ausgeschnitten und mittels LC-MS/MS analysiert wurden. Dadurch wurde ein putativer Kandidat für Get5 identifiziert sowie eine Assoziation mit Chaperonen und proteasomalen Untereinheiten.
Um die Zielsteuerungseffizienz und Topologie von ER-Membranproteinen zu analysieren habe ich (i) die rekombinante Synthese eines Modell-TA Proteins mit glykosylierbarem opsin bovine glycosylation Tag (OPG) etabliert sowie (ii) eine Methode etabliert um in isolierten Protoplasten die Richtigkeit der Insertion zu überprüfen. Mit Hilfe dieser Methoden können nun verschiedene Mutanten auf ihre Insertions-Wirksamkeit untersucht werden. Desweitern können durch Mutationsanalysen die notwendigen physikochemischen Eigenschaften für die Erkennung des Substrates ermittelt werden.
Eine weit verbreitete Methode im GET Feld ist die tail-anchor translocation (TAT). Bei dieser Methode werden isolierte mikrosomale Fraktionen des rauen ERs mit rekombinanten Komplexen bestehend aus Zielsteuerungsfaktor und TA Protein inkubiert. Durch einen rekombinanten OPG, der im Lumen des ERs post-translational modifiziert werden kann, ist die Beobachtung einer zeitabhängigen Kinetik der Glykosylierung möglich. Dieses System wurde bislang nur für Komponenten aus Säugern oder Hefen benutzt, aber noch nie mit einem System auf pflanzlicher Basis. Um dies zu verwirklichen, habe ich die rekombinante Proteinexpression soweit optimiert, dass der Großteil des synthetisierten Proteins sich im löslichen Anteil des Lysats statt in den Inclusion Bodies befand. Mittels dieser Optimierung konnte ich die Ko-Expression von Zielsteuerungsfaktor mit TA Protein als löslichen Komplex etablieren. Ergänzend zu den löslichen Komplexen habe ich eine geeignete Methode etabliert um mittels Saccharosegradienten mikrosomale Fraktionen aufzutrennen in denen AtGet3a angereichert ist. Leider müssen noch die Parameter der Reaktion optimiert werden, aber die Akquirierung alle nötigen Bestandteile ist etabliert.
The central dogma of biology is based on the concatenated transfer of information from DNA, via transcribed mRNA, to the translated protein. In eukaryotes, transcription and translation are separated locally as well as temporally by cellular compartmentalization. Prior to active export factor-dependent transport from the nucleus to the cytosol, the newly formed pre-mRNA must mature. This involves 5'capping, splicing, and endonucleolytic cleavage and polyadenylation (CPA).
Transcription of a new pre-mRNA is terminated by hydrolytic cleavage in the 3'-UTR, and the newly formed 3'-end is protected from premature degradation by synthesis of a poly(A) tail. These processes are catalyzed by four multi-protein complexes (CFIm, CFIIm, CPSF, and CsTF) and poly(A) polymerase (PAP). CPA is sequence-specific and dependent on RNA-binding proteins (RBPs). APA-specific sequences include the poly(A) motif ('AAUAAA' and certain motif variants), the UGUA motif, and U/GU-rich sequences upstream and downstream of the poly(A) signal, respectively. About 70% of mammalian genes have more than one polyadenylation site (PAS) and express transcripts of different lengths by a mechanism called alternative polyadenylation (APA). This can affect the length of the 3'UTR (3'UTR-APA) or the coding sequence of the transcript (CDS-APA) if the alternative PAS is upstream of the STOP codon. The length of the 3'UTR affects the stability, export efficiency, subcellular localization, translation rate, and local translation of the nascent transcript. 3'UTR-APA is regulated in the interplay of the cis-elements (poly(A) motif, UGUA and U/GU) and trans-elements (expression of CPA factors). In this context, the functions of the individual cis and trans elements have been extensively studied, yet the regulation of alternative polyadenylation-the decision whether to use the proximal or distal PAS-is less deciphered and requires additional study.
In murine P19 cells, we were able to demonstrate for the first time a direct link between 3'UTR-APA and nuclear export of mature mRNA by the splicing factors SRSF3 and SRSF7 and decipher the mechanism. At the core here is the direct recruitment of the export factor NXF1 by SRSF3 and SRSF7 to transcripts with 3'UTRs of different lengths.
The primary goal of the thesis presented here was to decipher the function of SRSF3 and SRSF7 in the regulation of 3'UTR-APA and to determine the basic mechanism. For this purpose, various genome-wide methods, such as RNA-Seq, MACE-Seq, and iCLIP-Seq, were integrated and the findings were supported by reporter gene and mutation studies.
Initial determination of the poly(A)-tome in P19 cells by MACE-Seq yielded approximately 16,000 PAS and showed that slightly less than 50% of all genes used two or more PAS and expressed alternative 3'UTR isoforms. Further DaPARS analyses after knockdown of Srsf3 or Srsf7 confirmed that SRSF3 affected more transcripts than SRSF7 and led primarily to the expression of long 3'UTRs, whereas SRSF7 promoted the expression of short 3'UTRs. Integration of SRSF3- and SRSF7-specific iCLIP data suggested a possible competition between SRSF3 and SRSF7 at the proximal PAS (pPAS), which could thus act as a hotspot of 3'UTR regulation.
Experiments with intron-free reporter genes revealed that SRSF3- and SRSF7-dependent regulation of 3'UTR-APA is independent of splicing. With respect to SRSF7, a concentration dependence was demonstrated. Mutation experiments involving the SRSF3- and SRSF7-specific binding motifs in the 3'UTR also confirmed the hypothesis of competition between the two SR proteins.
Extensive Co-IP experiments clearly demonstrated that only SRSF7, but not SRSF3, can interact with CFIm and FIP1 (a subunit from the CPSF complex) in an RNA-independent manner. In addition, we showed that these interactions exhibited some phosphorylation dependence, such that the interaction to FIP1 arose primarily in the semi- to hypophosphorylated state of SRSF7. Whereas the interaction to CFIm was mainly detected in the hyperphosphorylated state. The differential affinity between SRSF3 and SRSF7 for polyadenylation factors could be attributed to two SRSF7-specific domains in subsequent mutation experiments: A CCHC-type Zn finger between the RRM and the RS domain, and a hydrophobic 27 amino acid long region in the middle of the RS domain. Together, this suggested that SRSF3 could block the utilization of pPAS, whereas SRSF7 could activate it by directly recruiting polyadenylation factors.
Interestingly, we showed that knockdown of Srsf3 also negatively regulates the expression of Cpsf6 (a subunit of CFIm) through alternative splicing, which subsequently leads to decreased expression of CPSF6 and of CFIm. Reduction of CFIm led to increased expression of transcripts with short 3'UTR, analogous to knockdown of Srsf3. This mirrors the results of previous studies. A direct comparison between SRSF3- and CPSF6-specific transcripts revealed that not all targets were congruent. In addition, we found preliminary evidence for CFIm-related masking of essential cis-pPAS elements by bimodal UGUA motifs at the pPAS. In summary, we present a novel mechanism of indirect 3'UTR-APA regulation through SRSF3-conditional expression of the CFIm subunit CPSF6.
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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.
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.
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.
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.
Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions worldwide. The success of A. baumannii is based on the rise of multiple antibiotic resistances and its outstanding potential to persist in the human host and under conditions of low water activity in hospital environments. Combating low water activities involves osmoprotective measures such as uptake of compatible solutes and K+. To address the role of K+ uptake in the physiology of A. baumannii we have identified K+ transporter encoding genes in the genome of A. baumannii ATCC 19606. The corresponding genes (kup, trk, kdp) were deleted and the phenotype of the mutants was studied. The triple mutant was defective in K+ uptake which resulted in a pronounced growth defect at high osmolarities (300 mM NaCl). Additionally, mannitol and glutamate synthesis were strongly reduced in the mutant. To mimic host conditions and to study its role as an uropathogen, we performed growth studies with the K+ transporter deletion mutants in human urine. Both, the double (ΔkupΔtrk) and the triple mutant were significantly impaired in growth. This could be explained by the inability of ΔkupΔtrkΔkdp to metabolize various amino acids properly. Moreover, the reactive oxygen species resistance of the triple mutant was significantly reduced in comparison to the wild type, making it susceptible to one essential part of the innate immune response. Finally, the triple and the double mutant were strongly impaired in Galleria mellonella killing giving first insights in the importance of K+ uptake in virulence.
Morbus Parkinson (abgekürzt als PD vom Englischen Parkinson’s disease) ist nach Alzheimer die zweithäufigste neurodegenerative Erkrankung. Die Hauptmerkmale sind Rigidität und Bradykinesie, sowie Tremor und posturale Instabilität. Im Gehirn lässt sich bei Parkinsonpatienten post mortem ein Verlust an Neuronen in der Substantia nigra feststellen, was zu den ersten beiden Anzeichen führt. Zudem gibt es intrazelluläre Einschlüsse in den betroffenen Nervenzellen – Lewy-Körperchen genannt – die aus Alpha-Synuklein und anderen Proteinen wie Ubiquitin zusammengesetzt sind. Außerdem ist der Eisenmetabolismus in Gehirnen von Parkinsonpatienten gestört und man findet Eisen-Ablagerungen, vor allem im Mittelhirn. Die Ursachen für PD sind bislang nicht abschließend geklärt. Der Großteil der Fälle ist sporadischer Natur mit unbekannter Ursache und nur bei einem geringen Anteil liegt eine Mutation in einem einzelnen Gen zugrunde. Die häufigsten Mutationen tritt in den Genen für Alpha-Synuklein (SNCA), PINK1 und PARKIN auf.
Die Serin-Threonin-Kinase PINK1 und die E3-Ubiquitin-Protein-Ligase PARKIN sind zwei Proteine, die in Stresssituationen an der Mitochondrien-Außenmembran am Abbau von alten oder nicht richtig funktionierenden Mitochondrien beteiligt sind. Dieser Vorgang nennt sich Mitophagie.
Die dieser Arbeit zugrunde liegenden Publikationen gehen den Zusammenhängen zwischen mitochondrialen Fehlfunktionen und der Pathogenese von PD nach. Da die Krankheit meist erst im hohen Alter auftritt, davon größtenteils ohne direkte Ursache, liegt der Schluss nahe, dass neben genetischen Ursachen auch Umweltfaktoren eine größere Rolle spielen könnten. Um dies näher zu analysieren, wurden experimentell verschiedene Stressoren eingesetzt.
Insgesamt wurden folgende Aspekte untersucht:
I. Welche Auswirkungen hat das Fehlen von PINK1 auf die Zelle? Gibt es einen Biomarker, der mit höherem Alter immer stärker verändert ist?
II. Welchen Einfluss haben Umweltfaktoren wie veränderte Eisen-Exposition auf die Zelle und was verändert sich beim Fehlen von PINK1?
III. Wie können mitochondriale Fehlfunktionen präferentiell das Nervensystem betreffen, wenn es nicht um respiratorische Insuffizienz geht?
Die einzelnen Studien zeigten folgende Ergebnisse:
Torres-Odio/Key et al. 2017 widmete sich der Suche nach molekularen Biomarkern, wodurch PD präsymptomatisch erkannt und die Progression der Erkrankung eingeschätzt werden kann. Die Transkriptom-Analyse der Kleinhirne von Mäusen mit Pink1-/--Mutation in drei verschiedenen Altersstufen zeigte eindrücklich, dass nicht ein einzelner Faktor immer stärker verändert war, sondern, dass immer mehr Faktoren und daher auch eine steigende Zahl an
Signalwegen mit höherem Alter beteiligt waren. Diese Veränderungen betrafen inflammatorische Signalwege, insbesondere Faktoren, die mit der Erkennung und Verarbeitung von zellfremden Nukleinsäuren assoziiert sind. Aufgrund der evolutionären Herkunft von Mitochondrien als frühere Protobakterien haben mitochondriale Nukleinsäuren und Proteine zum Teil bakterielle Ähnlichkeiten, und könnten bei Fehlfunktionen ins Zytosol gelangen. Vor diesem Hintergrund lassen die Ergebnisse der Studie den Schluss zu, dass das angeborene Immunsystem in Neuronen durch eine PINK1-assoziierte mitochondriale Störung aktiviert wird.
In der Publikation Key et al. 2020 wurde Eisen als ein im täglichen Leben vorkommender Stressor eingesetzt und es wurden systematisch Faktoren des Eisenstoffwechsels bei hohen und niedrigen Eisenspiegeln im Zusammenhang mit Parkinson-Mutationen untersucht. Da Eisen für die Gesundheit von Mitochondrien eine große Rolle spielt und Eisen-Chelatoren als Therapie bei PD Patienten bereits diskutiert werden, haben die molekularen Befunde große Relevanz. Die Ergebnisse zeigen, dass unter niedrigen Eisenspiegeln Proteine reduziert waren, die am Nukleotid-Stoffwechsel beteiligt sind, sowie Faktoren, die Eisen-Schwefel-Cluster als Cofaktoren haben und wichtig für die Nukleotid-Qualitätskontrolle sind. Das Fehlen von Eisen führte zu einer Induktion von Pink1 und Prkn, was auf verstärkte Mitophagie hindeutet. Insgesamt konnte gezeigt werden, dass die mitochondriale Eisen-Schwefel-Cluster Biogenese und die post-transkriptionelle Eisenregulation entscheidend für die Pathogenese von PD, bzw. das gesunde Fortbestehen einer Zelle und letztlich auch eines Organismus sind.
In Key et al. 2019 wurde erstmalig das Gesamt-Ubiquitylom aus Gehirnen von gealterten Parkin-knockout (KO) Mäusen erhoben und analysiert, um Ubiquitylierungs-Substrate von PARKIN zu identifizieren. Hierbei zeigte sich eine veränderte Ubiquitylierung von mehreren Faktoren, die an der zellulären Calcium-Homöostase beteiligt sind. Weitere elektrophysiologische Experimente in Gehirnen von gealterten Parkin-/--KO Mäusen ergaben, dass in Nervenzellen im Locus coeruleus die Geschwindigkeit der spontanen Taktgeber erhöht, dass die langsame Nachhyperpolarisation reduziert und, dass die Dauer der Aktionspotentiale erniedrigt war, ohne Veränderung der Kaliumkanal-Ströme.
Insgesamt geht aus den drei Studien hervor, dass mitochondriale Fehlfunktionen bei dauerhaftem Bestehen weitreichende Folgen für die Gesundheit des Nervensystems haben können, denn auch kleine Veränderungen, seien es durch Mutationen oder Umweltfaktoren wie Eisen, können in einer so großen Lebensspanne wie der des Menschen über Krankheit oder Gesundheit entscheiden!
Background: Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3′ untranslated regions (3′UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown.
Results: Here we combine iCLIP and 3′-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3′UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3′UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3′UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3′UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3′UTRs.
Conclusions; We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.
Identification and regulation of tomato Serine/Arginine-rich proteins under high temperatures
(2021)
Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.
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.
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.
Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg2+ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg2+ and ligand binding.
Land use change has led to large-scale insect decline, threatening ecosystem resilience through reduced functional diversity. Even in nature reserves, losses in insect diversity have been detected. Hereby, changes in local habitat quality and landscape-scale habitat quantity can play a role driving functional diversity toward erosion. Our aim was to analyze how local and landscape-scale factors simultaneously affect functional insect diversity. Therefore, we sampled moths in two Italian coastal forest reserves at 60 sites. Our focus was on functional richness, redundancy and niche occupation, being important for ecosystem resilience, following the insurance framework. Ecological information about 387 species and 14 traits was used to analyze functional diversity. Twenty-five functional groups were recognized and used to estimate niche occupation and redundancy. Fourteen local and 12 landscape-scale factors were measured and condensed by using Principal Components Analysis. The resulting PC-axes served as predictors in linear mixed effects models. Functional richness, redundancy and niche occupation of moths were lower at sites with low habitat quality and quantity, indicating reduced ecosystem resilience. Especially landscape diversity and habitat structure, viz. a humidity-nutrient gradient, but also plant diversity, were promoting functional richness. Landscape fragmentation, indicating increased impermeability for insects, reduced local functional richness, redundancy and niche occupation. Local habitat quality and landscape-wide habitat quantity are both important for maintaining functional insect diversity inside reserves. Therefore, small and isolated nature reserves might fail in preserving biodiversity and ecosystem functions through adverse effects acting from the surrounding landscape structure and configuration.
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.
Seit den 1950er Jahren hat sich Plastik als unverzichtbare Ressource im menschlichen Alltag etabliert. Als negative Folge dieses Booms wird seit einigen Jahrzehnten jedoch eine zunehmende Belastung aquatischer Ökosysteme mit Plastikmüll bzw. dessen Degradationsprodukten, sogenanntes „Mikroplastik“ (MP, < 5 mm) bzw. „Nanoplastik“ (NP, < 1 µm), beobachtet. Ziel dieser Arbeit war die Untersuchung des aktuellen Vorkommens von MP in limnischen Gewässern, die Analyse der Interaktion zwischen MP und limnischen Wirbellosenarten und der daraus resultierenden Toxizität sowie eine erste Risikoabschätzung.
Das Vorkommen von Mikroplastik in limnischen Gewässern wurde exemplarisch anhand der Elbe als großes Fließgewässer in Deutschland untersucht. Durch die Auswertung von elf Probestellen entlang des Verlaufs der Mittel- und Unterelbe konnte gezeigt werden, dass die MP-Konzentrationen im Sediment (2,26x10^4 – 2,27x10^7 P m^-3) im Mittel fast 150.000-fach höher sind als in der Wasserphase (0,88–13,24 P m^-3). Sedimente sind somit eine Senke für MP. Die Zusammensetzung der Polymerarten sowie MP-Formen deuten zudem an, dass die Partikel sowohl aus diffusen wie auch aus Punktquellen (z.B. Industrieabwässer) stammen. Im globalen Vergleich können die MP-Konzentrationen in deutschen Fließgewässern z. Z. als durchschnittlich betrachtet werden. Allerdings muss insgesamt davon ausgegangen werden, dass die bisher bestimmten MP-Umweltkonzentrationen die realen Konzentrationen möglicherweise unterschätzen. So zeigte die Elbestudie, dass die Sedimentfeinfraktion < 100 µm einen bedeutenden Polymeranteil enthielt. Die meisten bisher durchgeführten Studien zur Bestimmung von MP-Partikeln in Flüssen haben Partikel < 100 µm jedoch nicht in ihrer Analyse berücksichtigt.
Die Interaktion von MP mit limnischer Biota wurde anhand der Artgruppen der Muscheln (Bivalvia), Schnecken (Gastropoda) sowie Krebstiere (Crustacea) näher untersucht. Die Intensität der Interaktion ist maßgeblich von der Aufnahme von MP durch die verschiedenen Arten abhängig. Anhand von zahlreichen Aufnahmestudien mit verschiedenen limnischen Arten, darunter den Muschelarten Dreissena polymorpha, Sinanodonta woodiana und Anodonta anatina, der Lungenschnecke Lymnaea stagnalis sowie der Amphipodenart Gammarus pulex, wurde nachgewiesen, dass die MP-Aufnahme von den Eigenschaften der exponierten Arten bzw. Individuen, den MP-Charakteristika sowie den Expositionsbedingungen abhängt. Die Experimente mit Muscheln verdeutlichten die rasche Aufnahme, aber auch Exkretion von MP-Partikeln innerhalb weniger Stunden. In allen drei Artgruppen war die Aufnahme konzentrationsabhängig mit zunehmender Aufnahme bei steigenden MP-Konzentrationen. Die Muschelexperimente zeigten jedoch auch, dass eine gleichzeitige Exposition mit anderen Partikeln (z.B. Nahrung) zu einer reduzierten Aufnahme führt. Auch die Größe der Testorganismen beeinflusste die Aufnahme: So nahmen im Fall der Muscheln und Krebse kleinere Individuen (bzw. im Fall der Muscheln auch Arten) relativ pro Körpermasse mehr MP-Partikel auf als größere Individuen bzw. Arten. Für alle untersuchten Arten wurde darüber hinaus gezeigt, dass die MP-Größe relevanten Einfluss auf die Menge an aufgenommenen Partikeln hat.
Ein Vergleich zwischen den Artgruppen zeigte, dass Muscheln als filtrierende Organismen in den Laboruntersuchungen bei gleicher Expositionskonzentration mehr MP aufnahmen als Krebse (Zerkleinerer) und Schnecken (Weidegänger). Im Gegensatz zu Muscheln nutzen Krebstiere und Schnecken allerdings die Grenzschicht zwischen Wasser- und Sedimentphase als Suchraum für ihre Nahrung und sind in der Umwelt (auf Grund des höheren MP-Vorkommens in Sedimenten) somit möglicherweise gegenüber höheren MP-Konzentrationen exponiert als Muscheln. Die Extrapolation der gewonnenen Labordaten sowie der Vergleich mit publizierten Umweltdaten legen allerdings nahe, dass das MP-Vorkommen in Individuen aller drei Artgruppen bisher auf einige wenige MP-Partikel begrenzt ist. Ausgeprägte Unterschiede zwischen den Artgruppen sind bisher nicht erkennbar.
MP-Toxizitätsstudien mit G. pulex, L. stagnalis sowie D. polymorpha konnten trotz der Berücksichtigung einer Vielzahl an Endpunkten (Mortalität, Reproduktion, Nahrungsaufnahme, oxidativer Stress, Energiereserven, Immunzellaktivität) und trotz des Einsatzes zum Teil sehr hoher MP-Konzentrationen weit oberhalb aktueller Umweltkonzentrationen nur sehr wenige MP-induzierte Effekte nachweisen, darunter eine Steigerung der Filtrationsaktivität (D. polymorpha) bzw. Veränderung der Immunfunktion von Hämolymphzellen (L. stagnalis).
Zur weiteren Risikoabschätzung wurden diese Studienergebnisse mit publizierten Daten für marine und limnische Muschel- und Krebsarten in Artenempfindlichkeitsverteilungen (Species Sensitivity Distributions, SSD) zusammengeführt und jeweils eine SSD für Muscheln und Krebstiere erstellt. Die Erstellung einer SSD nur für limnische Arten ist zum jetzigen Zeitpunkt auf Grund der geringen Datenlage noch nicht möglich.
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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.
Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen–surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin–ligand interaction, supported by present high-throughput “omics” technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.
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.
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.
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 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).
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.
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.
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.
Die Bildung von Blutgefäßen ist essentiell für die Entwicklung und Homöostase von Wirbeltieren und die Endothelzellspezifikation ist ein wichtiger erster Schritt in diesem Prozess. Das früheste bekannte Ereignis bei der Endothelzellspezifikation im Zebrafisch ist die Expression des bHLH-PAS-Transkriptionsfaktor-Gens npas4l. Ich habe eine transgene V5-Linie zum Nachweis des markierten Npas4l auf Proteinebene und eine Gal4-VP16-Reporterlinie zur Visualisierung und Verfolgung von npas4l exprimierenden Zellen in vivo generiert. Beide Linien können bereits in frühen Entwicklungsstadien nachgewiesen werden und komplementieren auch starke npas4l-Mutanten Allele. Um npas4l Reporter exprimierende Zellen in npas4l Mutanten zu verfolgen, habe ich anschließend eine mutierte Variante der Gal4-Reporterlinie erzeugt. Diese Mutante trägt eine Insertion in der Region, die die DNA-Bindedomäne kodiert. Dadurch stört sie die Npas4l-Funktion, aber nicht die Reporterexpression. Dieses mutierte Reporterallel komplementiert nicht die npas4l-Mutanten und zeigt einen starken Phänotyp, was darauf hindeutet, dass es sich um ein funktionelles Nullallel handelt. Phänotypische Analysen zeigten, dass npas4l-Reporter positive Zellen in npas4l-Mutanten nicht spezifizieren oder zur Mittelachse wandern. Stattdessen tragen sie zu den vom intermediären Mesoderm abgeleiteten pronephrischen Tubuli und dem vom paraxialen Mesoderm abgeleiteten Skelettmuskel bei. Ich habe diese Phänotypen durch Einzelzell-RNAseq an den npas4l-Reporter positiven Zellen in npas4l+/- und npas4l-/- Embryonen bestätigt. Zusammen erklären diese beiden alternativen Zellschicksale den Großteil der beobachteten Veränderungen zwischen den Genotypen. Npas4l ist dafür bekannt die Expression der drei Transkriptionsfaktorgene etsrp, tal1 und lmo2 zu fördern. Ich stellte die Hypothese auf, dass das Fehlen jedes dieser Transkriptionsfaktoren in npas4l-Mutanten verschiedene Aspekte des npas4l-Phänotyps verursacht. Daher habe ich Mutantenlinien für alle drei Gene generiert und sie sowohl in vaskulären Reporterlinien als auch im npas4l-Reporterhintergrund analysiert. Die Daten legen nahe, dass verschiedene Gene unterschiedliche Prozesse während der frühen Endothelentwicklung regulieren. In npas4l-/- und etsrp-/- Embryonen differenzieren npas4l-Reporter exprimierende Zellen nicht zu Endothelzellen und tragen stattdessen zur Skelettmuskelzellpopulation bei. In npas4l-/- und tal1-/- Embryonen können npas4l-Reporter exprimierende Zellen nicht migrieren und tragen stattdessen zu der Bildung der pronephrischen Tubuli bei. Um die Beziehung zwischen diesen Faktoren besser zu verstehen, habe ich getestet, ob die Injektion von etsrp-, tal1- oder lmo2-mRNA verschiedene Aspekte des npas4l-Phänotyps retten würde. npas4l-, etsrp- und tal1-Mutanten zeigen alle schwere vaskuläre Phänotypen. Einige Endothelzellen und vaskuläre Strukturen bleiben jedoch in jeder Mutante erhalten. Der Phänotyp ist am stärksten in npas4l-/- Embryonen, aber selbst in diesen Embryonen können einige fli1a-positive Endothelzellen in der Schwanzregion beobachtet werden. Es war unklar, ob sich diese Population von Endothelzellen unabhängig von der Npas4l-, Tal1- und Etsrp-Funktion entwickelt oder als Folge einer restlichen tal1- oder etsrp-Expression unabhängig von Npas4l. Um diese Frage zu untersuchen, habe ich Doppelmutanten generiert und nach dem Vorhandensein von fli1a-positiven Endothelzellen in diesen Mutanten gesucht. Während fli1a-positive Endothelzellen in npas4l-/- und npas4l-/-;tal1-/- Embryonen deutlich vorhanden sind, können keine solchen Zellen in npas4l-/-;etsrp-/- oder etsrp-/-;tal1-/- Embryonen beobachtet werden. Diese Daten deuten darauf hin, dass sich im Zebrafisch keine Endothelzellen entwickeln können, wenn zugleich npas4l und etsrp oder etsrp und tal1 gestört sind. Während der Verlust von etsrp zu stärkeren Defekten in npas4l-Mutanten führt, gibt es keinen zusätzlichen Phänotyp, der durch den Verlust von tal1verursacht wird, was darauf hindeutet, dass die Expression von etsrp, aber nicht die von tal1, unabhängig von Npas4l auftreten kann. Diese Idee wird durch die Beobachtung unterstützt, dass etsrp, aber nicht tal1-Expression in den meisten fli1a-exprimierenden Zellen in npas4l-/- Embryonen beobachtet wird. Dennoch wird der Großteil -Expression durch Npas4l reguliert. tal1-mRNA-Injektionen reichten aus, um eine Wildtyp-ähnliche vaskuläre Musterbildung im Bauchbereich der npas4l-/- Embryonen wiederherzustellen, einschließlich der Rettung sowohl der Zellmigration als auch der Differenzierung. Da Npas4l mehrere unterschiedliche transkriptionelle Effektoren hat, war eine so starke Rettung durch nur einen dieser Effektoren unerwartet. In den geretteten Mutanten wurde die bilaterale Population von npas4l-Reporter-positiven pronephrischen Tubuluszellen nicht entdeckt, aber die Anzahl der ektopischen npas4l-Reporter exprimierenden Muskelzellen war im Vergleich zu nicht injizierten npas4l-Mutanten gleichbleibend.
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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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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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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.
Background: The industrial production of various alcohols from organic carbon compounds may be performed at high rates and with a low risk of contamination using thermophilic microorganisms as whole-cell catalysts. Thermoanaerobacter species that thrive around 50–75 °C not only perform fermentation of sugars to alcohols, but some also utilize different organic acids as electron acceptors, reducing them to their corresponding alcohols. Results: We purified AdhE as the major NADH- and AdhB as the major NADPH-dependent alcohol dehydrogenase (ADH) from the cell extract of the organic acid-reducing Thermoanaerobacter sp. strain X514. Both enzymes were present in high amounts during growth on glucose with and without isobutyrate, had broad substrate spectra including different aldehydes, with high affinities (< 1 mM) for acetaldehyde and for NADH (AdhE) or NADPH (AdhB). Both enzymes were highly thermostable at the physiological temperature of alcohol production. In addition to AdhE and AdhB, we identified two abundant AdhA-type ADHs based on their genes, which were recombinantly produced and biochemically characterized. The other five ADHs encoded in the genome were only expressed at low levels. Conclusions: According to their biochemical and kinetic properties, AdhE and AdhB are most important for ethanol formation from sugar and reduction of organic acids to alcohols, while the role of the two AdhA-type enzymes is less clear. AdhE is the only abundant aldehyde dehydrogenase for the acetyl-CoA reduction to aldehydes, however, acid reduction may also proceed directly by aldehyde:ferredoxin oxidoreductase. The role of the latter in bio-alcohol formation from sugar and in organic acid reduction needs to be elucidated in future studies.
The Global South is facing severe challenges in ensuring livelihood security due to climate change impacts, environmental degradation and population growth as well as changing lifestyles. These complex problems cannot be solely solved by single scientific disciplines – they require transdisciplinary research (TDR). Stakeholders from civil society, the corporate sector, government and science need to pool their knowledge to find solutions for sustainable transformations. In Namibia, we have been involved in TDR projects on water supply, and sanitation services as well as livestock management in rangeland systems. In this paper, we review two TDR projects that differ in multiple ways and hence allow us to carve out structural differences and critically discuss research outcomes, lessons learned and the challenge of North–South collaborations. Our review builds upon published and unpublished project documents as well as expert interviews with Namibian and German researchers who were involved in the projects. Our results show that TDR can be put into practice in different ways, depending on the research focus and the period available. The TDR phases of problem framing, inter- and transdisciplinary integration were implemented with different tools and foci points. We discuss the role of project length and funding conditions for project success and outcome generation. In addition, we critically consider the role of Namibian and German researchers in these international collaborations. The conclusions we draw touch upon the points of preparatory research funding, the equal acknowledgement of Global South contributions to joint research projects and the explicit handling of TDR components in project work. Significance: • The current social-ecological challenges are complex and require TDR as a mode of knowledge coproduction, particularly in a development context. • Inter- and transdisciplinary integration are critical processes for a project to be successful and require the allocation of adequate time and monetary resources. • Longer-term projects with a funded preparatory research phase constitute a structural model for TDR as project outcomes can evolve over time. • Global South researchers carry a hidden burden in international collaborations that has to be adequately acknowledged upfront in project planning and final products.
Highlights
• Protocol for extracting and analyzing pollen grains from fossil insects
• Individual fossil grains can be analyzed using a combined approach
• Simple and fast TEM embedding and sectioning protocol
• Protocol enables a taxonomic assignment of pollen
Summary
This protocol explains how to extract pollen from fossil insects with subsequent descriptions of pollen treatment. We also describe how to document morphological and ultrastructural features with light-microscopy and electron microscopy. It enables a taxonomic assignment of pollen that can be used to interpret flower-insect interactions, foraging and feeding behavior of insects, and the paleoenvironment. The protocol is limited by the state of the fossil, the presence/absence of pollen on fossil specimens, and the availability of extant pollen for comparison.
Mitochondria are ubiquitous organelles of eukaryotic organisms with a number of essential functions, including synthesis of iron-sulfur clusters, amino acids, lipids, and adenosine triphosphate (ATP). During aging of the fungal aging model Podospora anserina, the inner mitochondrial membrane (IMM) undergoes prominent morphological alterations, ultimately resulting in functional impairments. Since phospholipids (PLs) are key components of biological membranes, maintenance of membrane plasticity and integrity via regulation of PL biosynthesis is indispensable. Here, we report results from a lipidomic analysis of isolated mitochondria from P. anserina that revealed an age-related reorganization of the mitochondrial PL profile and the involvement of the i-AAA protease PaIAP in proteolytic regulation of PL metabolism. The absence of PaIAP enhances biosynthesis of characteristic mitochondrial PLs, leads to significant alterations in the acyl composition of the mitochondrial signature PL cardiolipin (CL), and induces mitophagy. These alterations presumably cause the lifespan increase of the PaIap deletion mutant under standard growth conditions. However, PaIAP is required at elevated temperatures and for degradation of superfluous CL synthase PaCRD1 during glycolytic growth. Overall, our study uncovers a prominent role of PaIAP in the regulation of PL homeostasis in order to adapt membrane plasticity to fluctuating environmental conditions as they occur in nature.
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.
Eukaryotic ribosome assembly starts in the nucleolus, where the ribosomal DNA (rDNA) is transcribed into the 35S pre-ribosomal RNA (pre-rRNA). More than two-hundred ribosome biogenesis factors (RBFs) and more than two-hundred small nucleolar RNAs (snoRNA) catalyze the processing, folding and modification of the rRNA in Arabidopsis thaliana. The initial pre-ribosomal 90S complex is formed already during transcription by association of ribosomal proteins (RPs) and RBFs. In addition, small nucleolar ribonucleoprotein particles (snoRNPs) composed of snoRNAs and RBFs catalyze the two major rRNA modification types, 2′-O-ribose-methylation and pseudouridylation. Besides these two modifications, rRNAs can also undergo base methylations and acetylation. However, the latter two modifications have not yet been systematically explored in plants. The snoRNAs of these snoRNPs serve as targeting factors to direct modifications to specific rRNA regions by antisense elements. Today, hundreds of different sites of modifications in the rRNA have been described for eukaryotic ribosomes in general. While our understanding of the general process of ribosome biogenesis has advanced rapidly, the diversities appearing during plant ribosome biogenesis is beginning to emerge. Today, more than two-hundred RBFs were identified by bioinformatics or biochemical approaches, including several plant specific factors. Similarly, more than two hundred snoRNA were predicted based on RNA sequencing experiments. Here, we discuss the predicted and verified rRNA modification sites and the corresponding identified snoRNAs on the example of the model plant Arabidopsis thaliana. Our summary uncovers the plant modification sites in comparison to the human and yeast modification sites.
Climate change imposes severe stress on European forests, with forest degradation already visible in several parts of Europe. Thus adaptation of forestry applications in Mediterranean areas and central Europe is necessary. Proactive forestry management may include the planting of Mediter- ranean oak species in oak-bearing Central European regions. Five replicate common gardens of Greek and Italian provenances of Quercus ilex, Q. pubescens and Q. frainetto seedlings (210 each per plantation) were established in Central Italy, NE Greece (two) and Southern Germany (two, including Q. robur) to assess their performance under different climate conditions. Climate and soil data of the plantation sites are given and seedling establishment was monitored for survival and morphological parameters. After 3 years (2019) survival rates were satisfactory in the German and Italian sites, whereas the Greek sites exerted extremely harsh conditions for the seedlings, including extreme frost and drought events. In Germany, seedlings suffered extreme heat and drought periods in 2018 and 2019 but responded well. Provenances were ranked for each country for their performance after plan- tation. In Greece and Italy, Q. pubescens was the best performing species. In Germany, Q. pubescens and Q. robur performed best. We suggest that Greek or Italian provenances of Q. pubescens may be effectively used for future forestation purposes in Central Europe. For the establishment of Quercus plantations in Northern Greece, irrigation appears to be a crucial factor in seedling establishment.
Most cellular processes are regulated by RNA-binding proteins (RBPs). These RBPs usually use defined binding sites to recognize and directly interact with their target RNA molecule. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments are an important tool to de- scribe such interactions in cell cultures in-vivo. This experimental protocol yields millions of individual sequencing reads from which the binding spec- trum of the RBP under study can be deduced. In this PhD thesis I studied how RNA processing is driven from RBP binding by analyzing iCLIP-derived sequencing datasets.
First, I described a complete data analysis pipeline to detect RBP binding sites from iCLIP sequencing reads. This workflow covers all essential process- ing steps, from the first quality control to the final annotation of binding sites. I described the accurate integration of biological iCLIP replicates to boost the initial peak calling step while ensuring high specificity through replicate re- producibility analysis. Further I proposed a routine to level binding site width to streamline downstream analysis processes. This was exemplified in the re- analysis of the binding spectrum of the U2 small nuclear RNA auxiliary factor 2 (U2AF2, U2AF65). I recaptured the known dominance of U2AF65 to bind to intronic sequences of protein-coding genes, where it likely recognizes the polypyrimidine tract as part of the core spliceosome machinery.
In the second part of my thesis, I analyzed the binding spectrum of the serine and arginine rich splicing factor 6 (SRSF6) in the context of diabetes. In pancreatic beta-cells, the expression of SRSF6 is regulated by the transcription factor GLIS3, which encodes for a diabetes susceptibility gene. It is known that SRSF6 promotes beta-cell death through the splicing dysregulation of genes essential to beta-cell function and survival. However, the exact mechanism of how these RNAs are targeted by SRSF6 remains poorly understood. Here, I applied the defined iCLIP processing pipeline to describe the binding landscape of the splicing factor SRSF6 in the human pancreatic beta-cell line EndoC-H1. The initial binding sites definition revealed a predominant binding to coding sequences (CDS) of protein-coding genes. This was followed up by extensive motif analysis which revealed a so far, in human, unknown purine-rich binding motif. SRSF6 seemed to specifically recognize repetitions of the triplet GAA. I also showed that the number of contiguous triplets correlated with increasing binding site strength. I further integrated RNA-sequencing data from the same cell type, with SRSF6 in KD and in basal conditions, to analyze SRSF6- related splicing changes. I showed that the exact positioning of SRSF6 on alternatively spliced exons regulates the produced transcript isoforms. This mechanism seemed to control exons in several known susceptibility genes for diabetes.
In summary, in my PhD thesis, I presented a comprehensive workflow for the processing of iCLIP-derived sequencing data. I applied this pipeline on a dataset from pancreatic beta-cells to unveil the impact of SRSF6-mediated splicing changes. Thus, my analysis provides novel insights into the regulation of diabetes susceptibility genes.
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.
As abundant carbohydrates in renewable feedstocks, such as pectin-rich and lignocellulosic hydrolysates, the pentoses arabinose and xylose are regarded as important substrates for production of biofuels and chemicals by engineered microbial hosts. Their efficient transport across the cellular membrane is a prerequisite for economically viable fermentation processes. Thus, there is a need for transporter variants exhibiting a high transport rate of pentoses, especially in the presence of glucose, another major constituent of biomass-based feedstocks. Here, we describe a variant of the galactose permease Gal2 from Saccharomyces cerevisiae (Gal2N376Y/M435I), which is fully insensitive to competitive inhibition by glucose, but, at the same time, exhibits an improved transport capacity for xylose compared to the wildtype protein. Due to this unique property, it significantly reduces the fermentation time of a diploid industrial yeast strain engineered for efficient xylose consumption in mixed glucose/xylose media. When the N376Y/M435I mutations are introduced into a Gal2 variant resistant to glucose-induced degradation, the time necessary for the complete consumption of xylose is reduced by approximately 40%. Moreover, Gal2N376Y/M435I confers improved growth of engineered yeast on arabinose. Therefore, it is a valuable addition to the toolbox necessary for valorization of complex carbohydrate mixtures.
Natural products can contribute to abiotic stress tolerance in plants and fungi. We hypothesize that biosynthetic gene clusters (BGCs), the genomic elements that underlie natural product biosynthesis, display structured differences along elevation gradients. We analysed biosynthetic gene variation in natural populations of the lichen-forming fungus Umbilicaria pustulata. We collected a total of 600 individuals from the Mediterranean and cold-temperate climates. Population genomic analyses indicate that U. pustulata contains three clusters that are highly differentiated between the Mediterranean and cold-temperate populations. One entire cluster is exclusively present in cold-temperate populations, and a second cluster is putatively dysfunctional in all cold-temperate populations. In the third cluster variation is fixed in all cold-temperate populations due to hitchhiking. In these two clusters the presence of consistent allele frequency differences among replicate populations/gradients suggests that selection rather than drift is driving the pattern. We advocate that the landscape of fungal biosynthetic genes is shaped by both positive and hitchhiking selection. We demonstrate, for the first time, the presence of climate-associated BGCs and BGC variations in lichen-forming fungi. While the associated secondary metabolites of the candidate clusters are presently unknown, our study paves the way for targeted discovery of natural products with ecological significance.
White stork (Ciconia ciconia) nestlings can provide quantitative information on the quality of the surrounding environment by indicating the presence of pollutants, as they depend on locally foraged food. This study represents the first comparison of biomarkers in two fractions of white stork nestling blood: plasma and S9 (the post-mitochondrial fraction). The aim of this study was to evaluate acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as to establish a novel fluorescence-based method for glutathione (GSH) and reactive oxygen species (ROS) detection in plasma and S9. Considering the enzymatic biomarkers, lower variability in plasma was detected only for AChE, as CES, GST, and GR had lower variability in S9. Enzyme activity was higher in plasma for AChE, CES, and GST, while GR had higher activity in S9. Regarding the fluorescence-based method, lower variability was detected in plasma for GSH and ROS, although higher GSH detection was reported in S9, and higher ROS was detected in plasma. The present study indicated valuable differences by successfully establishing protocols for biomarker measurement in plasma and S9 based on variability, enzyme activity, and fluorescence. For a better understanding of the environmental effects on nestlings’ physiological condition, biomarkers can be measured in plasma and S9.
The geomagnetic field provides directional information for birds. The avian magnetic compass is an inclination compass that uses not the polarity of the magnetic field but the axial course of the field lines and their inclination in space. It works in a flexible functional window, and it requires short-wavelength light. These characteristics result from the underlying sensory mechanism based on radical pair processes in the eyes, with cryptochrome suggested as the receptor molecule. The chromophore of cryptochrome, flavin adenine dinucleotide (FAD), undergoes a photocycle, where radical pairs are formed during photo-reduction as well as during re-oxidation; behavioral data indicate that the latter is crucial for detecting magnetic directions. Five types of cryptochromes are found in the retina of birds: cryptochrome 1a (Cry1a), cryptochrome 1b, cryptochrome 2, cryptochrome 4a, and cryptochrome 4b. Because of its location in the outer segments of the ultraviolet cones with their clear oil droplets, Cry1a appears to be the most likely receptor molecule for magnetic compass information.
Although macroecology is a well-established field, much remains to be learned about the large-scale variation of fungal traits. We conducted a global analysis of mean fruit body size of 59 geographical regions worldwide, comprising 5340 fungal species exploring the response of fruit body size to latitude, resource availability and temperature. The results showed a hump-shaped relationship between mean fruit body size and distance to the equator. Areas with large fruit bodies were characterised by a high seasonality and an intermediate mean temperature. The responses of mutualistic species and saprotrophs were similar. These findings support the resource availability hypothesis, predicting large fruit bodies due to a seasonal resource surplus, and the thermoregulation hypothesis, according to which small fruit bodies offer a strategy to avoid heat and cold stress and therefore occur at temperature extremes. Fruit body size may thus be an adaptive trait driving the large-scale distribution of fungal species.
In recent years, the popularity of rock-climbing has grown tremendously, setting an increasing pressure on cliff habitats. Climbing may be particularly harmful in the Mediterranean biome due to its appropriate environmental conditions for climbing. A few studies have identified the effect of climbing on plant diversity at a small-scale (namely locally or even just in specific climbing areas). However, no studies exist assessing the potential risk of rock-climbing on a broad-scale (e.g., regional or national). The study aims to identify the priority locations and priority cliff plant species in Spain to focus future study efforts. Spain was selected because it is a plant biodiversity hotspot, with a great diversity of endemic and endangered species, and one of the most popular destinations for climbers. We used a geographic information system-based approach to model the spatial concurrence among Spanish climbing areas (and climbing intensity), natural protected areas (NPAs), and distribution of threatened cliff plants (and their IUCN threat category). We found that 53.5% of climbing areas in Spain are located within a NPA, most of them falling into NPAs of medium protection level. We mapped 151 threatened cliff plants, identifying four medium priority Mediterranean locations and eight priority species in which future research efforts should be focused. High-priority study locations are absent in Spain according to our spatial modeling. For the first time on a national scale, this study identifies areas in which climbing represents a potential threat for cliff habitats and threatened plants. These findings contribute to designing field studies on the effects of rock-climbing on Mediterranean cliffs, laying the groundwork for a sustainable, yet challenging, balance between the protection of these unique habitats and rock-climbing.
Until quite recently, stem cell technology mainly focused on pure populations of embryonic stem cells (ES) derived from the inner cell mass of the blastocyst and induced pluripotent stem cells (iPS). Using organoids, a newly established culture technique, it is now possible to culture also organ and patient-specific adult stem (AS) and induced pluripotent stem (IPS) cells in vitro. Furthermore, it has been shown that adult stem cells, grown as organoids, are genetically stable, proliferate and maintain their multi-potency (often a bi-potency) for months. This is possible by providing conditions that recapitulate the stem cell niche of the corresponding organ. Particularly, defined growth factors and a physiological scaffold, which is provided by an extracellular matrix (ECM). Because of increasing research activities, organoids became influential in the recent years. Wide-ranging interest also led to a clearer definition: organoids must contain multiple organ-specific cell types, must be able to recapitulate some organ specific functions, and the cells must be spatially organized in a way similar to the organ they are derived from. The excitement about organoids is based on their high potential as a model to understand wound healing, cellular behaviour and differentiation processes in organogenesis. Furthermore, high potential in the drug development and in personalized stem cell therapeutic approaches has been shown. Specifically, for personalized stem cell therapy, one potential application is for chronic autoimmune diseases such as Diabetes type 1 (T1D). T1D is characterized by the immune-mediated destruction of ß-cells in the Pancreas that leads to absolute insulin deficiency. In T1D the first-line therapeutic approach is exogenous insulin replacement therapy, which always implicates the risk of high fluctuations in blood-sugar levels and therefore the risk of hypoglycaemia. Another therapeutic approach is the xenotransplantation of islets from human donors. A successful islet transplantation allows patients a years-long insulin independence. However, the therapeutic value of islet transplantation is highly limited by the availability of organ donors and by the need for chronic administration of immune suppressive medication. The use of pancreas organoids offers a promising alternative as a personalized cell therapeutic approach to treat T1D without the hypoglycaemia risks of the established therapies. In 2013 Meritxell Huch and colleagues established for the first-time organoids from the exocrine, ductal part of the pancreas. These pancreas organoids are characterized by a monolayered, spherical cell epithelium which comprises a liquid filled lumen. In addition, they showed that after transplantation of these cells into immunodeficient mice, they differentiate into ß-cells and cure T1D. However, basic knowledge of the culture growth behaviour is still lacking: to date, no growth parameters are defined and reliable and robust investigation approaches are still missing. Furthermore, basic knowledge about the organoid development and biochemical/biophysical mechanisms that generate the phenotypic structure are not identified. For a clinical approach these parameters are fundamental and therefore must be defined pre-clinically.
The aim of this study is the preclinical characterization of the hPOs...
Coupling between epidermis and amphid morphogenesis during embryonic development of C. elegans
(2021)
Sensory organs are fundamental for survival of animal populations, since the detection of environmental stimuli is crucial for localization of nourishment, predators or mating partners. In nematodes, the amphid (AM) sensilla are the largest sensory organs for detection of chemical compounds.
This study investigates how the AM sensilla acquire their special elongated shape during lima-bean to 1.5-fold embryonic stages of C. elegans head development. The dissertation also examines events facilitating the morphogenesis of other head sensilla (IL/OL/CEP) and addresses aspects of general embryonic head morphogenesis. Using high resolution live-cell imaging techniques with different combinations of markers highlighting specific tissues, this study shows that epidermal head enclosure, migration of AM socket cells (pores) and translocation of AM dendrite tips are coupled processes, facilitating the elongation of AM dendrites. Importantly, during AM dendrite elongation the AM neural cell bodies are staying stationary. Manipulation through conducting UV-Laser ablation (epidermis close to pore/pore) and RPN-6.1 dsRNA interference resulted in compromised AM pore migration and impaired dendrite elongation. This leads to the conclusion that AM pores need to be physically attached (through C. elegans apical junctions, CeAJ) to the migrating epidermal sheet and to AM dendrite tips for successful AM morphogenesis. This study infers that RPN-6.1 plays an important role for correct AM pore morphogenesis and AM pore to AM dendrite tip attachment. Our results lead to the conclusion that head enclosure drives AM pore migration and AM dendrite elongation with AM neural cell bodies staying stationary. Thereby, CeAJ are interconnecting AM dendrite tips to AM pores and CeAJ link the sensillar ending to the migrating epidermis. Thus, migration of attached target tissue (pore), with neural cell bodies staying stationary (constituting an abutment), creates a pulling force facilitating AM dendrite elongation. This passive neurite elongation procedure is coined dendrite towing in this study.
Additionally, this study discovers that translocation of IL, OL and CEP head sensilla pores is influenced by apical constriction. This conclusion was made based on the findings that IL/OL/CEP pores migrate towards the prospective mouth anterior to the epidermal leading edge, separated from AM pores and irrespective of highly impaired AM sensilla morphogenesis after strong RPN-6.1 depletion. Also, concurrent with translocation of IL/OL/CEP pores, bottle-shaped cells occur and non-muscle-myosin and apical polarity factors are getting enriched at the anterior most part of the head, indicating de-novo manifestation of apical constriction. It is furthermore assumed that apical constriction in arcade cells might contribute to early pharynx development. All in all, this study reveals two force-generating events: Head enclosure-driven AM sensilla morphogenesis via dendrite towing and, otherwise, apical constriction-facilitated translocation of IL/OL/CEP sensilla pores. These events can get separated by graded depletion of the proteasome activator RPN-6.1.
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.
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.
Climatic niches describe the climatic conditions in which species can persist. Shifts in climatic niches have been observed to coincide with major climatic change, suggesting that species adapt to new conditions. We test the relationship between rates of climatic niche evolution and paleoclimatic conditions through time for 65 Old-World flycatcher species (Aves: Muscicapidae). We combine niche quantification for all species with dated phylogenies to infer past changes in the rates of niche evolution for temperature and precipitation niches. Paleoclimatic conditions were inferred independently using two datasets: a paleoelevation reconstruction and the mammal fossil record. We find changes in climatic niches through time, but no or weak support for a relationship between niche evolution rates and rates of paleoclimatic change for both temperature and precipitation niche and for both reconstruction methods. In contrast, the inferred relationship between climatic conditions and niche evolution rates depends on paleoclimatic reconstruction method: rates of temperature niche evolution are significantly negatively related to absolute temperatures inferred using the paleoelevation model but not those reconstructed from the fossil record. We suggest that paleoclimatic change might be a weak driver of climatic niche evolution in birds and highlight the need for greater integration of different paleoclimate reconstructions.
Thermoanaerobacter kivui ist ein thermophiles acetogenes Bakterium, das chemolithoautotroph auf CO2 unter Verwendung von molekularem H2 als Elektronendonor wächst und Acetat als Produkt über den Wood-Ljungdahl-Weg (WLP) bildet. Im WLP werden 2 Mol CO2 reduziert, um ein Mol Acetyl-CoA zu bilden. Erste Studien wurden durchgeführt, um die Physiologie von T. kivui zu verstehen. T. kivui wächst autotroph auf H2 + CO2 und nach Adaptation auch auf CO oder Syngas. T. kivui wächst ebenfalls auch in Minimalmedium ohne weitere Zugabe von Vitaminen, was es zu einem Biokatalysator mit hohem Potenzial für die Produktion von Chemikalien mit hohem Mehrwert macht. Heterotroph wächst T. kivui auf Glucose, Fructose, Mannose, Pyruvat oder Formiat. Kürzlich wurde beschrieben, dass T. kivui in der Lage ist, auf dem Zuckeralkohol Mannitol in Gegenwart und Abwesenheit von HCO3- (oder externem CO2) zu wachsen. Allerdings war das Wachstum in Abwesenheit von externem CO2 deutlich verlangsamt. Daher wurde in dieser Studie getestet, ob eine Zugabe von externem Formiat das "fehlende" CO2 kompensieren kann. In Kombination mit Formiat wurde das Wachstum auf Mannitol in CO2 und HCO3- freien definierten Medien bis zu einer maximalen OD600 von 2,34 und mit einer Verdopplungszeit von 2,0 ± 0,0 stimuliert, was dem Wachstumsverhalten auf Mannitol in Anwesenheit von CO2/HCO3- entsprach. In Abwesenheit von Formiat (oder CO2) erreichte T. kivui nur eine endgültige optische Dichte von bis zu 0,7 mit einer verlängerten Verdoppelungszeit von 5,2 ± 0,2 Stunden. Dieses Experiment zeigte die höhe metabolische Flexibilität von T. kivui durch die Nutzung von Formiat als Elektronenakzeptor, wenn kein oder nur wenig CO2 vorhanden ist.
Genomanalysen ergaben, dass T. kivui ein Trehalose- und Maltose-Transportsystem-Permeaseprotein (MalF) besitzt. Darüber hinaus verfügt T. kivui über Trehalose- und Maltosehydrolase-Gene, die als Kojibiose-Phosphorylase annotiert sind. Obwohl in der Originalveröffentlichung beschrieben wurde, dass der Organismus nicht auf Maltose oder Trehalose wachsen kann, konnte T. kivui im Laufe dieser Arbeit an das Wachstum auf Maltose und Trehalose adaptiert werden. Nach dem Transfer von einer Glukose-Vorkultur auf ein Medium mit 25 mM Maltose oder 25 mM Trehalose als alleinige C-Quelle wurde kein Wachstum erzielt. Bei Verwendung der gleichen Vorkultur in einem Medium mit höherer Konzentration (50 mM) Maltose oder Trehalose, begannen die Zellen zu wachsen. Bei Verwendung dieser adaptierten kulturen als Vorkultur wuchsen die Zellen in Gegenwart von in 25 mM Maltose oder Trehalose bis zu einer maximalen OD600 von 1,12 bzw. 0,73. Die Adaptation hing mit der Tatsache zusammen, dass der Organismus eine höhere Konzentration benötigt, um sich an diese Kohlenstoffquellen zu gewöhnen. Durch diese Daten wird das heterotrophe Potenzial von T. kivui erhöht.
Um die Bedeutung der wasserstoffabhängigen Kohlendioxidreduktase (HDCR) während des Wachstums auf Formiat oder auf H2 + CO2 im Stoffwechsel von T. kivui zu verstehen, wurden Studien auf molekularer Ebene durchgeführt. Die HDCR nutzt H2 direkt für die Reduktion von CO2 zu Formiat im ersten Schritt des Wood-Ljungdahl-Wegs (WLP). Um die Rolle der HDCR in dieser Reaktion zu untersuchen, wurde das hdcr-Gencluster mit Hilfe des kürzlich entwickelten Mutagenesytems für T. kivui deletiert. In Wachstumstudien konnte anschliessend gezeigt werden, dass die ߡhdcr-Deletionsmutante nicht mehr auf Formiat oder H2 + CO2 als alleiniger Kohlenstoffquelle wachsen konnte. Nach Komplementation der Mutante mit dem hdcr-Gene in cis wuchsen die Kulture wieder auf Formiat oder H2 + CO2. Diese Experimente zeigten, dass die HDCR für das Wachstum auf H2 + CO2 oder Formiat essentiell ist. Interessanterweise konnte in der ߡhdcr-Mutante ebenfalls ein verändertes Wachstum auf Glukose als alleiniger C-Quelle festgestellt werden. Die T. kivui ߡhdcr-Mutante wuchs nur bis zu einer OD600 von 0,2, während der Wildtyp und der hdcr-komplementierte Stamm bis zu einer OD600 von 2,64 bzw. 2,4 wuchsen. Damit wurde bewiesen, dass die HDCR auch für die vollständige Glukoseoxidation in T. kivui erforderlich ist. Durch die Zugabe von Formiat wurde das Wachstum vollständig wiederhergestellt, ähnlich wie beim Wildtyp. Dies belegt wieder die Nutzung Formiat als terminalen Elektronenakzeptor. Auch auf Mannitol oder Pyruvat konnte die Mutanten nur in Gegenwart von Formiat wachsen. Der Substratverbrauch und die Produktbildung der T. kivui ߡhdcr-Mutante wurden in einem Zellsuspensionsexperiment untersucht. Die Zellen verbrauchten Formiat nur in Gegenwart von Glukose und produzierten Acetat mit einem Acetat/Substrat-Verhältnis von etwas mehr als 3,0, während die Acetatproduktion nur 12 mM betrug, wenn Glukose als alleiniges Substrat verwendet wurde. Diese Ergebnisse zeigen eine enge Kopplung der Oxidation von Multikohlenstoffsubstraten an den WLP.
T. kivui ist eines der wenigen Acetogenen, die CO als einzige Kohlenstoff- und Energiequelle nutzen können. ...
In times of global warming caused by the extensive use of fossil fuels, the need to capture gaseous carbon compounds is growing bigger. Several groups of microorganisms can fix the greenhouse gas CO2. Out of these, acetogenic bacteria are role models in their ability to reduce CO2 with hydrogen to acetate, which makes acetogens prime candidates for genetic modification towards biotechnological production of value-added compounds from CO2, such as biofuels. However, growth of acetogens on gaseous substrates is strongly energy-limited, and successful metabolic engineering requires a detailed knowledge of the bioenergetics. In 1939, Clostridium aceticum was the first acetogen to be described. A recent genomic study revealed that this organism contains cytochromes and therefore may use a proton gradient in its respiratory chain. We have followed up these studies and will present data that C. aceticum does not use a H+ but a Na+ gradient for ATP synthesis, established by a Na+-Rnf. Experimental data and in silico analyses enabled us to propose the biochemistry and bioenergetics of acetogenesis from H2 + CO2 in C. aceticum.
Vocal communication is essential to coordinate social interactions in mammals and it requires a fine discrimination of communication sounds. Auditory neurons can exhibit selectivity for specific calls, but how it is affected by preceding sounds is still debated. We tackled this using ethologically relevant vocalizations in a highly vocal mammalian species: Seba’s short-tailed bat. We show that cortical neurons present several degrees of selectivity for echolocation and distress calls. Embedding vocalizations within natural acoustic streams leads to stimulus-specific suppression of neuronal responses that changes sound selectivity in disparate manners: increases in neurons with poor discriminability in silence and decreases in neurons selective in silent settings. A computational model indicates that the observed effects arise from two forms of adaptation: presynaptic frequency specific adaptation acting in cortical inputs and stimulus unspecific postsynaptic adaptation. These results shed light into how acoustic context modulates natural sound discriminability in the mammalian cortex.
Pectin-rich residues are considered as promising feedstocks for sustainable production of platform chemicals. Enzymatic hydrolysis of extracted sugar beet press pulp (SBPP) releases the main constituent of pectin, d-galacturonic acid (d-GalA). Using engineered Saccharomyces cerevisiae, d-GalA is then reduced to l-galactonate (l-GalOA) with sorbitol as co-substrate. The current work addresses the combination of enzymatic hydrolysis of pectin in SBPP with a consecutive optimized biotransformation of the released d-GalA to l-GalOA in simple batch processes in stirred-tank bioreactors. Process conditions were first identified with synthetic media, where a product concentration of 9.9 g L-1 L-GalOA was obtained with a product selectivity of 99% (L-GalOA D-GalA-1) at pH 5 with 4% (w/v) sorbitol within 48 h. A very similar batch process performance with a product selectivity of 97% was achieved with potassium citrate buffered SBPP hydrolysate, demonstrating for the first time direct production of L-GalOA from hydrolyzed biomass using engineered S. cerevisiae. Combining the hydrolysis process of extracted SBPP and the biotransformation process with engineered S. cerevisiae paves the way towards repurposing pectin-rich residues as substrates for value-added chemicals.
High-temperature tolerant enzymes offer multiple advantages over enzymes from mesophilic organisms for the industrial production of sustainable chemicals due to high specific activities and stabilities towards fluctuations in pH, heat, and organic solvents. The production of molecular hydrogen (H2) is of particular interest because of the multiple uses of hydrogen in energy and chemicals applications, and the ability of hydrogenase enzymes to reduce protons to H2 at a cathode. We examined the activity of Hydrogen-Dependent CO2 Reductase (HDCR) from the thermophilic bacterium Thermoanaerobacter kivui when immobilized in a redox polymer, cobaltocene-functionalized polyallylamine (Cc-PAA), on a cathode for enzyme-mediated H2 formation from electricity. The presence of Cc-PAA increased reductive current density 340-fold when used on an electrode with HDCR at 40 °C, reaching unprecedented current densities of up to 3 mA·cm−2 with minimal overpotential and high faradaic efficiency. In contrast to other hydrogenases, T. kivui HDCR showed substantial reversibility of CO-dependent inactivation, revealing an opportunity for usage in gas mixtures containing CO, such as syngas. This study highlights the important potential of combining redox polymers with novel enzymes from thermophiles for enhanced electrosynthesis.
Biological and environmental factors as sources of variation in nocturnal behavior of giraffe
(2021)
Upon a drastic decline of the giraffe population in the wild, conservation efforts and therefore the role of zoos have become more important than ever. With their unique opportunities, zoos provide excellent conditions to study animal behavior, expanding the knowledge about the giraffe's behavior repertoire and their ability to adapt. This study therefore examined the nocturnal behavior of 63 giraffe living in 13 different EAZA zoos across Germany and the Netherlands. Giraffe were observed and videos recorded via infrared sensitive cameras during the winter seasons 2015–2018. The observation period spanned nightly from 17:00 to 7:00. Thus, 198 nights, with a total of 2772 h were recorded and analyzed. Linear mixed models were then used to assess potential biological and environmental factors influencing behavior during the dark phase. Results show that individual variables such as age, subspecies and motherhood determined nocturnal activity and sleep behavior most. Among the variables studied, husbandry conditions and environmental factors complying with EAZA standards had no influence on the giraffe's nocturnal behavior. By combining nocturnal activity analyses and an assessment of potential influencing factors, our findings present a holistic approach to a better understanding of captive giraffe behavior and allow for management implications.