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1H, 13C and 15N chemical shift assignment of the stem-loops 5b + c from the 5′-UTR of SARS-CoV-2
(2022)
The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5′- and 3′-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5′-untranslated region (5′-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5′-UUUCGU-3′ hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.
Background: Through the rapid development in DNA sequencing methods and tools, microbiome studies on a various number of species were performed during the last decade. This advance makes it possible to analyze hundreds of samples from different species at the same time in order to obtain a general overview of the microbiota. However, there is still uncertainty on the variability of the microbiota of different animal orders and on whether certain bacteria within a species are subject to greater fluctuations than others. This is largely due to the fact that the analysis in most extensive comparative studies is based on only a few samples per species or per study site. In our study, we aim to close this knowledge gap by analyzing multiple individual samples per species including two carnivore suborders Canoidea and Feloidea as well as the orders of herbivore Perissodactyla and Artiodactyla held in different zoos. To assess microbial diversity, 621 fecal samples from 31 species were characterized by sequencing the V3–V4 region of the 16S rRNA gene using Illumina MiSeq.
Results: We found significant differences in the consistency of microbiota composition and in fecal microbial diversity between carnivore and herbivore species. Whereas the microbiota of Carnivora is highly variable and inconsistent within and between species, Perissodactyla and Ruminantia show fewer differences across species boundaries. Furthermore, low-abundance bacterial families show higher fluctuations in the fecal microbiota than high-abundance ones.
Conclusions: Our data suggest that microbial diversity is significantly higher in herbivores than in carnivores, whereas the microbiota in carnivores, unlike in herbivores, varies widely even within species. This high variability has methodological implications and underlines the need to analyze a minimum amount of about 10 samples per species. In our study, we found considerable differences in the occurrence of different bacterial families when looking at just three and six samples. However, from a sample number of 10 onwards, these within-species fluctuations balanced out in most cases and led to constant and more reliable results.
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.
Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.
Baleen whales (Mysticeti) are a clade of highly adapted carnivorous marine mammals that can reach extremely large body sizes and feature characteristic keratinaceous baleen plates used for obligate filter feeding. From a conservation perspective, nearly all baleen whale species were hunted extensively over a roughly 100 years lasting time period that depleted many of the respective whale stocks with so far unknown consequences for e.g. their molecular viability. From an evolutionary perspective, the lack of fossil records together with conflicting molecular patterns resulted in a still unclear and debated phylogeny of modern baleen whales, particularly in rorquals (Balaenopteridae). In this dissertation, I will demonstrate the application of baleen whale genomes to tackle these open questions by using modern approaches of conservation and evolutionary genomics.
Conservation genomic aspects of baleen whales were addressed in two projects, both using whole genome data of either an Icelandic fin whale (Balaenoptera physalus) population or multiple blue whale (Balaenoptera musculus) populations to evaluate the impact of the industrial whaling era on their molecular viability. The results suggest a substantial drop in effective population size of both species but also a lack of manifestation in genotypes of the fin whale population when compared to the blue whale populations. Especially the rare and short runs of homozygosity (ROH), usually indicative for inbreeding, suggest frequent outcrossing in fin whales while all analyzed blue whale populations featured long and frequent ROH. In addition to these analyses, genome data of blue whale populations was further used to evaluate if northern hemisphere blue whales diverged into different subspecies. Population genetic and gene flow analyses showed clearly separated and well isolated populations in accordance with their assumed geographical distance. In contrast, the genome-wide divergence between all blue whale populations was low compared to other cetacean populations and to the next closely related sei whale species. Because this includes the morphologically different and well recognized pygmy blue whale subspecies, a proposal was made to equally categorize the two northern-hemisphere blue whale populations as subspecies.
Evolutionary aspects were addressed in a third project, by constructing the genome of the pygmy right whale (Caperea marginata) and testing its potential in phylogenetics and cancer research. Phylogenomic analyses using fragments of a whole-genome alignment featuring nearly all extant baleen whales, allowed the revision of the complex evolutionary relationships of rorquals by quantifying and characterizing the amounts of conflicts in early diverging branches. These relationships were further used to identify phylogenetically independent pairs of baleen whales with a maximum of diverging body size differences to compare rates of positive selection between their genomes. The results suggest nearly evenly distributed frequencies of alternative topologies which supports the representation of the early divergence of rorquals as a hard polytomy with high amounts of introgression and incomplete lineage sorting. Within the set of available genomic data, three independent pairs of baleen whales with diverging body sizes were found and comparisons of positive selection rates resulted in many potentially body size and cancer related genes. The lack of conserved selection patterns, however, suggest a more convergent evolution of size and cancer resistance like previously discussed in paleontology.
In conclusion, the application of whole genome data using methods of conservation genetics allowed for a comprehensive estimation about the molecular viability of blue and fin whales as well as an assessment of the taxonomic status of northern-hemisphere blue whale populations. The rather different results between blue and fin whales underlines the importance of genomic monitoring of baleen whales because different species show rather different molecular consequences of their potentially varying depletions. Furthermore, as showcased for the northern-hemisphere blue whale, many important isolated populations of baleen whales may still be unknown to conservation management and genome-wide comparisons will most likely contribute to overcome this under-classification problem. The application of whole genome data in evolutionary research allowed the characterization of the complex patterns of molecular conflicts within baleen whales and especially rorquals that will contribute to the still rather unclear understanding of their evolution. The here found molecular support for the idea of convergent evolution of gigantism in whales will further guide the search for molecular patterns responsible for Peto’s paradox.
Compaction and spheroid formation modulates stemness and differentiation of human pancreas organoids
(2023)
The incidence of diabetes type 1 (T1D) in children and young adults is increasing worldwide. T1D is well treated by insulin administration. However, there is currently no long-lasting cure for this ailment. The success rate of pancreatic islet transplantation to treat T1D is limited by the availability of patient-matched islets and the necessity of using life-long immunosuppressive medication. The difficulties caused by transplantation can be overcome by generating bio-engineered pancreatic islets from patient-derived progenitor cells. Aim of this thesis is to establish new strategies for the generation and analysis of pancreatic lineages derived from human progenitor cells. It reports on the optimization of a technique to form human pancreatic spheroids from hollow monolayered human pancreas organoids (hPOs) to investigate how cell-cell and cell-matrix interaction can be leveraged to induce endocrine differentiation of the pancreas progenitor cell organoids. We introduce cell aggregation protocols to generate endocrine pancreas cell lineages from ductal pancreatic cells. Next, we study the effect of co-culture with stromal and endothelial cells to promote cell differentiation toward a pancreatic fate enhancing β cells productivity.
This thesis has focused on identifying the differences in gene expression along with phenotypical transformation during differentiation of human pancreatic organoids (hPOs) towards human β cells to be used in the future of cellular therapeutics in treating T1D patients.
The brains of black 6 mice (Mus musculus) and Seba’s short-tailed bats (Carollia perspicillata) weigh roughly the same and share mammalian neocortical laminar architecture. Bats have highly developed sonar calls and social communication and are an excellent neuroethological animal model for auditory research. Mice are olfactory and somatosensory specialists, used frequently in auditory neuroscience for their advantage of standardization and wide genetic toolkit. This study presents an analytical approach to overcome the challenge of inter-species comparison with existing data. In both data sets, we recorded with linear multichannel electrodes down the depth of the primary auditory cortex (A1) while presenting repetitive stimuli trains at ~5 and ~40 Hz to awake bats and mice. We found that while there are similarities between cortical response profiles in both, there was a better signal to noise ratio in bats under these conditions, which allowed for a clearer following response to stimuli trains. Model fit analysis supported this, illustrating that bats had stronger response amplitude suppression to consecutive stimuli. Additionally, continuous wavelet transform revealed that bats had significantly stronger power and phase coherence during stimulus response and mice had stronger power in the background. Better signal to noise ratio and lower intertrial phase variability in bats could represent specialization for faster and more accurate temporal processing at lower metabolic costs. Our findings demonstrate a potentially different general auditory processing principle; investigating such differences may increase our understanding of how the ecological need of a species shapes the development and function of its nervous system.
Enhanced LTP of population spikes in the dentate gyrus of mice haploinsufficient for neurobeachin
(2020)
Deletion of the autism candidate molecule neurobeachin (Nbea), a large PH-BEACH-domain containing neuronal protein, has been shown to affect synaptic function by interfering with neurotransmitter receptor targeting and dendritic spine formation. Previous analysis of mice lacking one allele of the Nbea gene identified impaired spatial learning and memory in addition to altered autism-related behaviours. However, no functional data from living heterozygous Nbea mice (Nbea+/−) are available to corroborate the behavioural phenotype. Here, we explored the consequences of Nbea haploinsufficiency on excitation/inhibition balance and synaptic plasticity in the intact hippocampal dentate gyrus of Nbea+/− animals in vivo by electrophysiological recordings. Based on field potential recordings, we show that Nbea+/− mice display enhanced LTP of the granule cell population spike, but no differences in basal synaptic transmission, synapse numbers, short-term plasticity, or network inhibition. These data indicate that Nbea haploinsufficiency causes remarkably specific alterations to granule cell excitability in vivo, which may contribute to the behavioural abnormalities in Nbea+/− mice and to related symptoms in patients.
he most basic behavioural states of animals can be described as active or passive. While high-resolution observations of activity patterns can provide insights into the ecology of animal species, few methods are able to measure the activity of individuals of small taxa in their natural environment. We present a novel approach in which a combination of automatic radiotracking and machine learning is used to distinguish between active and passive behaviour in small vertebrates fitted with lightweight transmitters (<0.4 g).
We used a dataset containing >3 million signals from very-high-frequency (VHF) telemetry from two forest-dwelling bat species (Myotis bechsteinii [n = 52] and Nyctalus leisleri [n = 20]) to train and test a random forest model in assigning either active or passive behaviour to VHF-tagged individuals. The generalisability of the model was demonstrated by recording and classifying the behaviour of tagged birds and by simulating the effect of different activity levels with the help of humans carrying transmitters. The model successfully classified the activity states of bats as well as those of birds and humans, although the latter were not included in model training (F1 0.96–0.98).
We provide an ecological case-study demonstrating the potential of this automated monitoring tool. We used the trained models to compare differences in the daily activity patterns of two bat species. The analysis showed a pronounced bimodal activity distribution of N. leisleri over the course of the night while the night-time activity of M. bechsteinii was relatively constant. These results show that subtle differences in the timing of species' activity can be distinguished using our method.
Our approach can classify VHF-signal patterns into fundamental behavioural states with high precision and is applicable to different terrestrial and flying vertebrates. To encourage the broader use of our radiotracking method, we provide the trained random forest models together with an R package that includes all necessary data processing functionalities. In combination with state-of-the-art open-source automated radiotracking, this toolset can be used by the scientific community to investigate the activity patterns of small vertebrates with high temporal resolution, even in dense vegetation.
The most basic behavioural states of animals can be described as active or passive. However, while high-resolution observations of activity patterns can provide insights into the ecology of animal species, few methods are able to measure the activity of individuals of small taxa in their natural environment. We present a novel approach in which the automated VHF radio-tracking of small vertebrates fitted with lightweight transmitters (< 0.2 g) is used to distinguish between active and passive behavioural states.
A dataset containing > 3 million VHF signals was used to train and test a random forest model in the assignment of either active or passive behaviour to individuals from two forest-dwelling bat species (Myotis bechsteinii (n = 50) and Nyctalus leisleri (n = 20)). The applicability of the model to other taxonomic groups was demonstrated by recording and classifying the behaviour of a tagged bird and by simulating the effect of different types of vertebrate activity with the help of humans carrying transmitters. The random forest model successfully classified the activity states of bats as well as those of birds and humans, although the latter were not included in model training (F-score 0.96–0.98).
The utility of the model in tackling ecologically relevant questions was demonstrated in a study of the differences in the daily activity patterns of the two bat species. The analysis showed a pronounced bimodal activity distribution of N. leisleri over the course of the night while the night-time activity of M. bechsteinii was relatively constant. These results show that significant differences in the timing of species activity according to ecological preferences or seasonality can be distinguished using our method.
Our approach enables the assignment of VHF signal patterns to fundamental behavioural states with high precision and is applicable to different terrestrial and flying vertebrates. To encourage the broader use of our radio-tracking method, we provide the trained random forest models together with an R-package that includes all necessary data-processing functionalities. In combination with state-of-the-art open-source automated radio-tracking, this toolset can be used by the scientific community to investigate the activity patterns of small vertebrates with high temporal resolution, even in dense vegetation.
One of the earliest consequences of slicing plant storage organs such as potato tubers into thin disks is the formation of polysomes, which in potato slices is complete after 9 hours and is dependent on transcription. Fresh disks do not incorporate 32P, 3H-uridine or 14C-leucine into their ribosomes, whereas ribosomes and polysomes of aged disks use these precursors effectively. This development can be completely blocked by actinomycin D. Among the different RNAs synthesized during aging is 28S- and 16S—rRNA, 5S—RNA, tRNA, and a component sedimenting around 15—18S with a base-composition different from 16S—rRNA, 5S- and 4S—RNA and which supports peptide formation in an in vitro incorporation system.
It is suggested that this compound represents mRNA, which is not available immediately after slicing the tissue. These findings are consistent with the view of a derepression phenomenon in sliced storage tissue.
Whereas ribosome preparations of freshly sliced potato disks do not show appreciable activity in an in-vitro amino acid incorporation system, aging of the tissue leads to a greatly enhanced incorporation activity which reaches its maximum 24 hours after slicing. If ribosomes from freshly excised disks are provided with polyuridylic acid, their activity in the incorporation of phenylalanine is increased about 8 fold.
Moreover, an RNA-fraction can be dissociated by EDTA from ribosomes of aged potato tuber slices, which sediments at 15 —18S, has a base composition different from that of 16S — rRNA, 5S-and 4S —RNA, and is not present on ribosomes of fresh slices. Its appearance is inhibited by actinomycin D and therefore most probably dependent on transcription. This compound, purified from sucrose gradients, enhances in vitro leucine incorporation into peptide material by ribosomes of fresh potato slices.
The possibility is discussed that this fraction-among other factors-is responsible for the enhanced protein synthesis after slicing plant storage organs, and is indicative of a general derepression phenomenon in these tissues.
Macrophage infectivity potentiator (MIP) proteins are widespread in human pathogens including Legionella pneumophila, the causative agent of Legionnaires’ disease and protozoans such as Trypanosoma cruzi. All MIP proteins contain a FKBP (FK506 binding protein)-like prolyl-cis/trans-isomerase domain that hence presents an attractive drug target. Some MIPs such as the Legionella protein (LpMIP) have additional appendage domains of mostly unknown function. In full-length, homodimeric LpMIP, the N-terminal dimerization domain is linked to the FKBP-like domain via a long, free-standing stalk helix. Combining X-ray crystallography, NMR and EPR spectroscopy and SAXS, we elucidated the importance of the stalk helix for protein dynamics and inhibitor binding to the FKBP-like domain and bidirectional crosstalk between the different protein regions. The first comparison of a microbial MIP and a human FKBP in complex with the same synthetic inhibitor was made possible by high-resolution structures of LpMIP with a [4.3.1]-aza-bicyclic sulfonamide and provides a basis for designing pathogen-selective inhibitors. Through stereospecific methylation, the affinity of inhibitors to to L. pneumophila and T. cruzi MIP was greatly improved. The resulting X-ray inhibitor-complex structures of LpMIP and TcMIP at 1.49 and 1.34 Å, respectively, provide a starting point for developing potent inhibitors against MIPs from multiple pathogenic microorganisms.
Fluorescense spectra of lactate dehydrogenase * (E.C. 1.1.1.27) were investigated in the presence of the coenzyme fragments dihydronicotinamide mononucleotide and dihydronicotinamide-ribose-5'-pyrophospho- (P2) -5“-ribose. The reduced mononucleotide is enzymatically less active as a hydrogen donor. However, formation of a complex with the enzyme was not observed under the conditions used. All the other substances: dihydronicotinamide-ribose-5'-pyrophospho- (P2) -5“-ribose, dihydronicotinamide- benzimidazole-dinucleotide, dihydronicotinamide-3-desazapurine-dinucleotide and dihydronicotinamide-6-mercaptopurine-dinucleotide form more or less stable complexes with lactate dehydrogenase. The complexes do not markedly differ from the complex formed with the natural cofactor. In all cases spectra indicate change in conformation of the coenzyme by forming the coenzyme-enzyme-complex which has been proposed by VELICK 1 too. The cysteine residues of the lactate dehydrogenase are not essential for binding the coenzyme to the active center; this was shown with mercury blocked enzyme.
The production of ribosomes is a complicated multistep, that is susceptible to changes occurring within the cell and its environment. The process itself requires many proteins, known as ribosome biogenesis factors (RBFs) and many non-coding RNAs like the small nucleolar RNAs (snoRNAs). While RBFs are required for the accurate processing of the pre-rRNA into mature rRNAs, the snoRNAs act to coordinate and guide enzymes for post-transcriptional modifications, chiefly 2´-O-ribose methylation and pseudouridylation. While ribosome biogenesis is mostly described in human and yeast model eucaryotes, similar detailed studies in the model plant Arabidopsis thaliana are far less explored and understood. Furthermore, for many experimentally confirmed modification sites the according snoRNAs and for many pre-rRNA processing steps the responsible RBFs are missing. Therefore, it is expected that a high number of snoRNAs and RBFs are not identified till yet. For this reason, RNA-deep sequencing was performed in order to identify novel snoRNAs and MS analysis data of nucleoli and nuclei of A. thaliana from a former PhD student were used in order to find new proteins involved in pre-rRNA processing.
In here, it is shown that with RNA deep-sequencing still new snoRNAs and snRNAs can be identified and that detection of predicted snoRNAs can be fulfilled with a) antisense oligonucleotides tagged with fluorescence dyes and b) with radioactive labeled antisense probes. Furthermore, a secondary structure map of the 60S and 40S subunit highlighting the predicted and moreover verified modification sites in 5.8S, 25S and 18S rRNA was created. Especially, the correlation between the modification sites and the guiding snoRNA is highlighted further shedding light on overview about current pre-rRNA modification sites and corresponding guiding snoRNAs. The next chapter reveals the complex and multi-layered existence of the 5.8S rRNA and its numerous precursors. The mutant prp24 (also known as seap1) encoding AtPRP24, is recognized as factor being important for splicing as it is promoting the recruitment of the U4 and U6 snRNAs to the spliceosome. In here, it was found that AtPRP24 is involved in processing of 5.8S rRNA precursors, recognizable by precursors that are over accumulating in the mutant. Moreover, it could be shown for the first time that the plant-specific precursor 5´-5.8S is exported to the cytoplasm, where final cleavage steps of 5.8S rRNA takes place. In the prp24.2 mutant, this precursor is exported at an increased rate to the cytoplasm, where it can be detected in the actively translating ribosomes (polysomes). A lower sensitivity of the mutant seeds to cycloheximide (CHX) suggests that due to the extension at the 5´-end of 5.8S, the structure of the 60S subunit has altered CHX binding. In conclusion, this work highlights the importance and complexity of 5.8S rRNA and its precursors for ribosome biogenesis and displays new insights into pre-rRNA processing in A. thaliana.
Xylose, an abundant sugar fraction of lignocellulosic biomass, is a five-carbon skeleton molecule. Since decades, utilization of this sugar has gained much attention and has been in particular focus as a substrate for production of biofuels like ethanol by microbial hosts, including Saccharomyces cerevisiae. In this yeast, xylose is naturally not used as a carbon source, but its utilization could be achieved by metabolic engineering either via the oxidoreductive route or through the isomerase pathway. Both pathways share xylulose as a common intermediate that must be phosphorylated before entering the endogenous metabolism via the non-oxidative pentose phosphate pathway (noxPPP). Besides this, in some bacteria a non-phosphorylating oxidative pathway for xylose degradation exists, known as Weimberg pathway, where a molecule of xylose is converted by a series of enzymes - xylose dehydrogenase (XylB), xylonate dehydratase (XylD), 3-keto-2-deoxy-xylonate dehydratase (XylX) and α-ketoglutarate semialdehyde dehydrogenase (KsaD) - to form α-ketoglutarate (AKG). Besides having several useful properties as a product, AKG could also be used for cell growth as an intermediate of the tricarboxylic acid (TCA) cycle. One target of the present study is to establish a functional Weimberg pathway in S. cerevisiae. Previous studies have shown that this task is not trivial, for instance due to the toxicity of xylonate (the first metabolite of the pathway) and the involvement of an iron-sulfur cluster dependent enzyme, the D-xylonate dehydratase. The assembly of iron-sulfur clusters on a heterologous protein in yeast is known to be challenging.
To establish the Weimberg pathway in yeast, the genes xylB, xylD, and xylX were obtained from Caulobacter cresentus and ksaD was from Corynebacterium glutamicum. In a variant, the dehydratase xylD was replaced with orf41 from Arthrobacter nicotinovorans, which is believed to be independent of iron-sulfur clusters. Growth of yeast cells on xylose as a sole carbon source was expected as an indicator of a functional Weimberg pathway. However, the heterologous expression of the codon optimized genes was not sufficient to reach this goal. Due to the complexity of the interactions of the heterologous pathway with the endogenous cellular processes, it was assumed that potential limitations could be overcome by adaptive laboratory evolution, using xylose as a sole source of carbon. Increasing selection pressure was applied on a strain with Weimberg pathway genes integrated into the genome over several generations. As a variant of the evolutionary engineering approach, mutator strains were generated. For this, RAD27 and MSH2 genes were deleted, which are involved in nucleotide excision and mismatch repair mechanisms, respectively. Some of the resulting strains PRY24, PRY25, PRY27 and PRY28 were able grow in xylose as a sole carbon source after evolutionary engineering. As a control, a non-mutator strain PRY19 was also included. Strikingly, only the mutator strains were able to consume xylose as a sole carbon source, which shows the feasibility of the approach.
In addition to the mutator strain strategy, a further approach employed in the present study was the simultaneous expression of the Weimberg pathway in the cytosol and mitochondria. This was based on the reasoning that the iron-sulfur cluster biogenesis on XylD may be improved in the organelle and that the AKG is an intermediate of the TCA cycle. In the strain AHY02, all enzymes of the pathway were tagged with mitochondrial targeting signals in addition to a full cytosolically localized pathway. The localization of the mitochondrial variants was confirmed by fluorescence microscopy. Together with AHY02, CEN.PK2-1C wild type strain was also included as a control for evolution. When a selection pressure on xylose was applied, both strains - AHY02 and CEN.PK2-1C - were able to grow in the course of evolution. Deletion of the xylulokinase (XKS1) gene was found to be detrimental for both evolved strains in xylose-containing media. This suggests that the evolution of the endogenous oxidoreductive and noxPPP genes is responsible for growth of the evolved cells. For the evolved strain AHY02, it could also be possible that the Weimberg pathway genes supported to growth in addition to the oxidoreductive route. To elucidate the underlying molecular mechanisms, genome sequencing and reverse engineering approaches would be necessary in future.
In addition to screening for growth on xylose as a sole carbon source, a less stringent screening system was created to examine even a minor flux of xylose towards AKG. For this, all genes necessary for conversion of isocitrate to AKG where deleted, yielding a glutamate auxotrophic strain. In this system, the cells can grow on other carbon sources, whereas xylose is only provided as a source of AKG for the synthesis of glutamate...
Identification of new natural products from nematode-associated bacteria using mass spectrometry
(2023)
This work aims to find unknown natural products produced by bacteria, that live in close association with nematodes and to elucidate their structure by using mass spectrometry.
The first chapter of this work is dedicated to the detection of hitherto unknown natural products by using a metabolomics approach and subsequent structure elucidation of said compounds. This chapter includes metabolomics analysis of Xenorhabdus szentirmaii wild type and knockout mutants, overproduction of the target compound, identification of derivatives from other strains and MS based structure elucidation.
The second and third chapters are about natural products that protect C. elegans from B. thuringiensis infections.
The second chapter deals with natural products that protect the nematode host without killing the pathogen. I deployed molecular biology methods to generate deletion and overproduction strains of a target compound, identified it via LC-MS/MS analysis and used LC-MS/MS and lipidomics to analyse the chemical properties of the active compound.
The third chapter aims at finding natural products, which are produced by Pseudomonas strains MYb11 and MYb12, respectively. These natural products display the ability to protect C. elegans by killing B. thuringiensis. I identified said compounds via fractionation and subsequent bioactivity testing. After identification, I generated production strains of the target compounds and elucidated the structure of the bioactive derivative.
The last chapter deals with the structure elucidation of peptides produced by an unusual GameXPeptide synthetase in Xenorhabdus miraniensis. I analysed producer strains of GameXPeptides using LC-MS and elucidated the structural differences between the known GameXPeptides, produced by P. luminescens TT01, and the unusual ones produced by X. miraniensis.
Bacterial biosynthetic assembly lines, such as non-ribosomal peptide synthetases (NRPS) and polyketide synthases, are often subject of synthetic biology – because they produce a variety of natural products invaluable for modern pharmacotherapy. Acquiring the ability to engineer these biosynthetic assembly lines allows the production of artificial non-ribosomal peptides (NRP), polyketides, and hybrids thereof with new or improved properties. However, traditional bioengineering approaches have suffered for decades from their very limited applicability and, unlike combinatorial chemistry, are stigmatized as inefficient because they cannot be linked to the high-throughput screening platforms of the pharmaceutical industry. Although combinatorial chemistry can generate new molecules cheaper, faster, and in greater numbers than traditional natural product discovery and bioengineering approaches, it does not meet current medical needs because it covers only a limited biologically relevant chemical space. Hence, methods for high-throughput generation of new natural product-like compound libraries could provide a new avenue towards the identification of new lead compounds. To this end, prior to this work, we introduced an artificial synthetic NRPS type, referred to as type S NRPS, to provide a first-of-its-kind bicombinatorial approach to parallelized high-throughput NRP library generation. However, a bottleneck of these first two generations of type S NRPS was a significant drop in production yields. To address this issue, we applied an iterative optimization process that enabled titer increases of up to 55-fold compared to the non-optimized equivalents, restoring them to wild-type levels and beyond.
Non-ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural products, including many clinically used drugs. Engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) at high titre is an ongoing challenge. Here we describe a strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 290 mg l-1. Extending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into up to three 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.
Adhesion to host cells is the first and most crucial step in infections with pathogenic Gram negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). TAA-producing bacteria are the causative agent of many human diseases and TAA targeted anti-adhesive compounds might counteract such bacterial infections. The modularly structured Bartonella adhesin A (BadA) is one of the best characterised TAAs and serves as an attractive adhesin to study the domain-function relationship of TAAs during infection. BadA is a major virulence factor of B. henselae and is essential for the initial attachment to host cells via adhesion to extracellular matrix proteins. B. henselae is the causative agent of cat scratch disease and adheres to fibronectin using its long BadA fibres. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations.
Human, feline, and laboratory adapted B. henselae isolates display genomic and phenotypic differences. By analysing the genomes of eight B. henselae strains using long-read sequencing, a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes was identified. Moreover, numerous conserved flanking genes were characterised, however, their influence on the regulation of badA expression and modification remains to be explored. It seems that B. henselae G 5436 is the evolutionary ancestor of the other B. henselae strains analysed in this work. The diversity of the badA island among the B. henselae strains indicates that the downstream badA-like domain region might be used as a ‘toolbox’ for rearrangements in the badA gene. Overall, it is suggested that badA-domain duplications, insertions, and/or deletions are the result of active phase variation via site-specific recombination and contribute to rapid host adaptation in the scope of pathogenicity, immune evasion, and/or enhanced long-term colonisation.
The model strain B. henselae Marseille expresses a badA gene that includes 30 repetitive neck/stalk domains, each consisting of several predicted structural motifs. To further elucidate the motif sequences that mediate fibronectin binding, various modified badA constructs were generated. Their ability to bind fibronectin was assessed via whole-cell ELISA and fluorescence microscopy. In conclusion, it is suggested that BadA adheres to fibronectin in a cumulative fashion with quick saturation via unpaired β-strands appearing in structural motifs present in BadA neck/stalk domains 19, 27, and other homologous domains. Furthermore, antibodies targeting a 15-mer amino acid sequence in the DALL motif of BadA neck/stalk domain 27 were able to reduce fibronectin binding of the B. henselae mutant strain S27. Moreover, this DALL motif sequence is conserved in the genome of all analysed B. henselae strains. The identification of common binding motifs between BadA and fibronectin supports the development of new anti-adhesive compounds that might inhibit the initial adherence of B. henselae and other TAA-producing pathogens during infection.
mRNA localization to subcellular compartments has been reported across all kingdoms of life and it is generally believed to promote asymmetric protein synthesis and localization. In striking contrast to previous observations, we show that in S. cerevisiae the B-type cyclin CLB2 mRNA is localized and translated in the yeast bud, while the Clb2 protein, a key regulator of mitosis progression, is concentrated in the mother nucleus. Using single-molecule RNA imaging in fixed (smFISH) and living cells (MS2 system), we show that the CLB2 mRNA is transported to the yeast bud by the She2-She3 complex, via an mRNA ZIP-code situated in the coding sequence. In CLB2 mRNA localization mutants, Clb2 protein synthesis in the bud is decreased resulting in changes in cell cycle distribution and genetic instability. Altogether, we propose that CLB2 mRNA localization acts as a sensor for bud development to couple cell growth and cell cycle progression, revealing a novel function for mRNA localization.
Deviance detection describes an increase of neural response strength caused by a stimulus with a low probability of occurrence. This ubiquitous phenomenon has been reported for multiple species, from subthalamic areas to auditory cortex. While cortical deviance detection has been well characterised by a range of studies covering neural activity at population level (mismatch negativity, MMN) as well as at cellular level (stimulus-specific adaptation, SSA), subcortical deviance detection has been studied mainly on cellular level in the form of SSA. Here, we aim to bridge this gap by using noninvasively recorded auditory brainstem responses (ABRs) to investigate deviance detection at population level in the lower stations of the auditory system of a hearing specialist: the bat Carollia perspicillata. Our present approach uses behaviourally relevant vocalisation stimuli that are closer to the animals' natural soundscape than artificial stimuli used in previous studies that focussed on subcortical areas. We show that deviance detection in ABRs is significantly stronger for echolocation pulses than for social communication calls or artificial sounds, indicating that subthalamic deviance detection depends on the behavioural meaning of a stimulus. Additionally, complex physical sound features like frequency- and amplitude-modulation affected the strength of deviance detection in the ABR. In summary, our results suggest that at population level, the bat brain can detect different types of deviants already in the brainstem. This shows that subthalamic brain structures exhibit more advanced forms of deviance detection than previously known.
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.
Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.
The interaction of microplastics with freshwater biota and their interaction with other stressors is still not very well understood. Therefore, we investigated the ingestion, excretion and toxicity of microplastics in the freshwater gastropod Lymnaea stagnalis.
MP ingestion was analyzed as tissues levels in L. stagnalis after 6–96 h of exposure to 5–90 μm spherical polystyrene (PS) microplastics. To understand the excretion, tissue levels were determined after 24 h of exposure followed by a 12 h–7 d depuration period. To assess the toxicity, snails were exposed for 28 d to irregular PS microplastics (<63 μm, 6.4–100,000 particles mL−1), both alone and in combination with copper as additional stressor. To compare the toxicity of natural and synthetic particles, we also included diatomite particles. Microplastics ingestion and excretion significantly depended on the particle size and the exposure/depuration duration. An exposure to irregular PS had no effect on survival, reproduction, energy reserves and oxidative stress. However, we observed slight effects on immune cell phagocytosis. Exposure to microplastics did not exacerbate the reproductive toxicity of copper. In addition, there was no pronounced difference between the effects of microplastics and diatomite. The tolerance towards microplastics may originate from an adaptation of L. stagnalis to particle-rich environments or a general stress resilience. In conclusion, despite high uptake rates, PS fragments do not appear to be a relevant stressor for stress tolerant freshwater gastropods considering current environmental levels of microplastics.
Functional genomics studies in model organisms and human cell lines provided important insights into gene functions and their context-dependent role in genetic circuits. However, our functional understanding of many of these genes and how they combinatorically regulate key biological processes, remains limited. To enable the SpCas9-dependent mapping of gene-gene interactions in human cells, we established 3Cs multiplexing for the generation of combinatorial gRNA libraries in a distribution-unbiased manner and demonstrate its robust performance. The optimal number for combinatorial hit calling was 16 gRNA pairs and the skew of a library’s distribution was identified as a critical parameter dictating experimental scale and data quality. Our approach enabled us to investigate 247,032 gRNA-pairs targeting 12,736 gene-interactions in human autophagy. We identified novel genes essential for autophagy and provide experimental evidence that gene-associated categories of phenotypic strengths exist in autophagy. Furthermore, circuits of autophagy gene interactions reveal redundant nodes driven by paralog genes. Our combinatorial 3Cs approach is broadly suitable to investigate unexpected gene-interaction phenotypes in unperturbed and diseased cell contexts.
Die kontinuierliche Messung des CO2-Gaswechsels homogener Sedimente einzelliger Grünalgen hat ergeben, daß das Kohlendioxyd während der ersten Belichtungsphase zunächst an einen primären, in den verdunkelten Zellen bereits vorhandenen CO2-Acceptor (AI) angelagert wird. AI ist nur im Licht zur Aufnahme und lockeren Bindung von Kohlendioxyd befähigt und gibt die während kurzer Lichtperioden (4-30 sec) aufgenommene CO2-Menge in der anschließenden Dunkelperiode sehr schnell wieder ab. Im Verlauf längerer Belichtungszeiten (> 30 sec) übergibt AI das locker gebundene Kohlendioxyd an den inzwischen in zunehmender Konzentration gebildeten CO2-Acceptor des Calvin - Zyklus (Ribulosediphosphat = AII). Die mit der Aufnahme und lockeren Bindung von Kohlendioxyd an den aktivierten Acceptor AI und der CO2-Weitergabe an AII zusammenhängenden Übergangserscheinungen werden eingehend diskutiert.
The change in allele frequencies within a population over time represents a fundamental process of evolution. By monitoring allele frequencies, we can analyze the effects of natural selection and genetic drift on populations. To efficiently track time-resolved genetic change, large experimental or wild populations can be sequenced as pools of individuals sampled over time using high-throughput genome sequencing (called the Evolve & Resequence approach, E&R). Here, we present a set of experiments using hundreds of natural genotypes of the model plant Arabidopsis thaliana to showcase the power of this approach to study rapid evolution at large scale. First, we validate that sequencing DNA directly extracted from pools of flowers from multiple plants -- organs that are relatively consistent in size and easy to sample -- produces comparable results to other, more expensive state-of-the-art approaches such as sampling and sequencing of individual leaves. Sequencing pools of flowers from 25-50 individuals at ∼40X coverage recovers genome-wide frequencies in diverse populations with accuracy r > 0.95. Secondly, to enable analyses of evolutionary adaptation using E&R approaches of plants in highly replicated environments, we provide open source tools that streamline sequencing data curation and calculate various population genetic statistics two orders of magnitude faster than current software. To directly demonstrate the usefulness of our method, we conducted a two-year outdoor evolution experiment with A. thaliana to show signals of rapid evolution in multiple genomic regions. We demonstrate how these laboratory and computational Pool-seq-based methods can be scaled to study hundreds of populations across many climates.
The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.
The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.
UV inactivated KAPPA can be reactivated like other temperate phages by plating on uvirradiated host cells (indicator). The capacity of the indicator Serratia HY for multiplication of unirradiated KAPPA was about 0.1% survivors (colony formers). The induction of clear plaque (c·) mutants by irradiating extracellular KAPPA and plating on untreated indicator can be increased further about 2 to 4 times by using UV irradiated indicator. The increase of the number of c mutants under the latter conditions, with increasing UV dose given to the phage, was never a firstorder reaction. The highest frequency of c mutants obtained was about 4.5 per cent. Plating of unirradiated KAPPA on irradiated indicator (lowest survival fraction was 0.01%) never increased the spontaneous mutation rate to c. Two c mutants studied in detail belong to two different cistrons as shown in a complementation test (map distance about 5.3%). Only one of both was revertible to the phenotype c+ spontaneously and with a higher rate by UV. However, as shown in crossing experiments with the wild type, the backmutants do not have the original genotype but originated from mutations in at least two different intragenic suppressor loci; the map distances between them and the original c mutation were 0.64% and 0.13 per cent. Host range (h) and virulent (v) mutants could not be induced by irradiation of the free phage and plating on untreated indicator. This indicates that the UV induced high mutability of the c loci in KAPPA represents an exceptional case of behavior (UV-hot spot). Some unstable h mutants could be isolated by plating irradiated phage on irradiated indicator.
Vampire bats are the only mammals that feed exclusively on blood. To uncover genomic changes associated with this dietary adaptation, we generated a haplotype-resolved genome of the common vampire bat and screened 27 bat species for genes that were specifically lost in the vampire bat lineage. We found previously unknown gene losses that relate to reduced insulin secretion (FFAR1 and SLC30A8), limited glycogen stores (PPP1R3E), and a unique gastric physiology (CTSE). Other gene losses likely reflect the biased nutrient composition (ERN2 and CTRL) and distinct pathogen diversity of blood (RNASE7) and predict the complete lack of cone-based vision in these strictly nocturnal bats (PDE6H and PDE6C). Notably, REP15 loss likely helped vampire bats adapt to high dietary iron levels by enhancing iron excretion, and the loss of CYP39A1 could have contributed to their exceptional cognitive abilities. These findings enhance our understanding of vampire bat biology and the genomic underpinnings of adaptations to blood feeding.
In natural environments, background noise can degrade the integrity of acoustic signals, posing a problem for animals that rely on their vocalizations for communication and navigation. A simple behavioral strategy to combat acoustic interference would be to restrict call emissions to periods of low-amplitude or no noise. Using audio playback and computational tools for the automated detection of over 2.5 million vocalizations from groups of freely vocalizing bats, we show that bats (Carollia perspicillata) can dynamically adapt the timing of their calls to avoid acoustic jamming in both predictably and unpredictably patterned noise. This study demonstrates that bats spontaneously seek out temporal windows of opportunity for vocalizing in acoustically crowded environments, providing a mechanism for efficient echolocation and communication in cluttered acoustic landscapes.
One Sentence Summary: Bats avoid acoustic interference by rapidly adjusting the timing of vocalizations to the temporal pattern of varying noise.
Die Einwirkung von UV-Strahlen (254 mµ) auf Bakterien und auf DNS führt zur Bildung einer Reihe von Photoprodukten. Thymin bildet ein Thymin-Dimeres und mindestens zwei weitere Photoprodukte. Aus Cytosin entstehen Uracil und ebenfalls mindestens zwei weitere Photoprodukte. Das Thymin-Dimere läßt sich durch Bestrahlung mit UV-Licht in wäßriger Lösung zu 87% wieder in Thymin zurückverwandeln. Bei den übrigen Photoprodukten gelingt diese Reaktion nicht.
Die durch UV-Strahlen verursachten biologischen Schäden in der Bakterienzelle dürften weitgehend auf die Bildung von dimerem Thymin zurückzuführen sein. Demgegenüber sind die übrigen Photoprodukte, die erst bei höherer UV-Dosis auftreten, nur von untergeordneter biologischer Bedeutung.
Die von der Strahlendosis abhängige Bildung der Thymin-Photoprodukte in Zellen von E. coli wurde quantitativ untersucht.
Eine Denaturierung der nativen DNS durch Erhitzen oder durch Abspalten der Purine zur Apurinsäure hat zur Folge, daß die Bildung des Thymin-Dimeren und eines der übrigen Thymin-Photoprodukte besonders stark begünstigt wird.
Auf der Oberfläche von Erythrozyten, Thrombozyten und Neutrophilen befinden sich mehrere hundert verschiedene polymorphe, ungekoppelt vererbte Blutgruppenantigene. Dementsprechend birgt jede Bluttransfusion das Risiko einer Immunisierung gegen fremde Blutgruppenmerkmale. Auch während der Schwangerschaft können aufgrund väterlich vererbter Antigene Alloantikörper induziert werden. Deshalb muss das Blut vor jeder Transfusion oder während einer Schwangerschaft auf das Vorhandensein irregulärer erythrozytärer Antikörper untersucht werden. Dabei greifen die aktuellen diagnostischen Verfahren auf primäre, stabilisierte Testerythrozyten von Blutspendern zurück, deren relevante Blutgruppenantigene bekannt sind. Antikörperspezifitäten können anhand von Agglutinationsreaktionen der Testzellen mit dem zu untersuchenden Patientenplasma auf ein oder mehrere Antigene zurückgeführt werden. Ist jedoch ein Antikörper gegen ein häufiges, ein hochfrequentes oder ein nicht-polymorphes, ubiquitäres Antigen gerichtet, kann in Ermangelung Antigen-negativer Testzellen keine adäquate Diagnostik gewährleistet, die Verträglichkeit der Transfusion also nicht definitiv sichergestellt werden. Auch der medizinische Einsatz therapeutischer Antikörper, welche Antigene adressieren, die auch auf Erythrozyten exprimiert werden, führt zunehmend zu Problemen. Tests auf granulozytäre Antikörper sind mangelhaft bezüglich ihrer Robustheit, besitzen eine unzureichende Auflösung und sind zudem meist zeitaufwändig und daher teuer. Antikörper gegen humane Plättchenantigene spielen insbesondere in der Schwangerschaft eine Rolle; sie vermögen bei Neugeborenen thrombozytopenische Blutungen bis hin zu massiven Hirnblutungen zu verursachen, die zu schweren Entwicklungsstörungen führen können. Bisher erfolgt jedoch mangels geeigneter Reagenzien keine standardisierte pränatale Untersuchung auf thrombozytäre Antikörper. In dieser Arbeit wurde ein neuartiges Verfahren für die Identifikation und Differenzierung irregulärer Blutgruppenantikörper etabliert, welches auf gentechnisch hergestellten, xenogenen Testzellen basiert, die einzelne definierte humane Blutgruppenantigene auf ihrer Oberfläche präsentieren. Die nicht humanen Zellen co exprimieren Fluorochrome, anhand derer Antikörper-markierte Testzellen durchflusszytometrisch voneinander unterscheidbar sind. Weiterhin können die generierten Testzellen zur Depletion von Antikörpern aus polyagglutinierenden Plasmen unter Erhalt der anderen Antikörperspezifitäten verwendet werden. Diese Technologie könnte die konventionelle Diagnostik erheblich erleichtern und bietet zudem die Möglichkeit, therapeutische Antikörper (wie z. B. anti-CD38, anti CD47, etc.), die häufig zu Interferenzen mit der Routinediagnostik führen, spezifisch prädiagnostisch aus Patientenproben zu entfernen.
Hyperparasitic fungi on black mildews (Meliolales, Ascomycota) : hidden diversity in the tropics
(2022)
Hyperparasitism on plant-parasitic fungi is a widespread but rarely studied phenomenon. Here, for the first time, we compile in a checklist information provided by peer-reviewed literature for fungi growing on colonies of black mildews (Meliolales, Ascomycota), a species-rich group of tropical and subtropical plant-parasitic microfungi. The checklist contains information on 189 species of contact-biotrophic microfungi in 82 genera. They belong to seven morphological groups: dematiaceous hyphomycetes, moniliaceous hyphomycetes, pycnidioid, perithecioid, catathecioid, and apothecioid fungi. By the fact that species accumulation curves do not reach saturation for any tropical country, it is evident that the knowledge of the diversity of hyperparasitic fungi on Meliolales is incomplete. A network analysis of records of hyperparasitic fungi, their host fungi and host plants shows that genera of hyperparasitic fungi are generalists concerning genera of Meliolales. However, most species of hyperparasitic fungi are restricted to meliolalean hosts. In addition to hyperparasitic fungi, diverse further microorganisms use meliolalean colonies as ecological niche. Systematic positions of most species are unknown because DNA sequence data are lacking for species of fungi hyperparasitic on Meliolales. We discuss the specific challenges of obtaining DNA sequence data from hyperparasitic fungi. In order to better understand the diversity, evolution and biology of hyperparasitic fungi, it is necessary to increase sampling efforts and to undertake further morphological, molecular, and ecological studies.
The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here, we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in Arabidopsis thaliana, ribosomal cistrons are present in Fagus sylvatica only in four major regions, in line with FISH studies. On most assembled chromosomes, telomeric repeats were found at both ends, while centromeric repeats were found to be scattered throughout the genome apart from their main occurrence per chromosome. The genome-wide distribution of SNPs was evaluated using a second individual from Jamy Nature Reserve (Poland). SNPs, repeat elements and duplicated genes were unevenly distributed in the genomes, with one major anomaly on chromosome 4. The genome presented here adds to the available highly resolved plant genomes and we hope it will serve as a valuable basis for future research on genome architecture and for understanding the past and future of European Beech populations in a changing climate.
A method which serves to isolate the gonads from the sea cucumber (Holothuria polii) is outlined. Criteria that will secure a well determined status of maturity of the sperm are given. From this preparation a deoxyribonucleic acid is made, purified and analysed. It is concluded that the analytical data are in compliance with the theory of Crick and Watson. The ratio of Moles for this DNA while its nitrogen to phosphorus ratio on weight basis is 1,67.
Specialized surveillance mechanisms are essential to maintain the genetic integrity of germ cells, which are not only the source of all somatic cells but also of the germ cells of the next generation. DNA damage and chromosomal aberrations are, therefore, not only detrimental for the individual but affect the entire species. In oocytes, the surveillance of the structural integrity of the DNA is maintained by the p53 family member TAp63α. The TAp63α protein is highly expressed in a closed and inactive state and gets activated to the open conformation upon the detection of DNA damage, in particular DNA double-strand breaks. To understand the cellular response to DNA damage that leads to the TAp63α triggered oocyte death we have investigated the RNA transcriptome of oocytes following irradiation at different time points. The analysis shows enhanced expression of pro-apoptotic and typical p53 target genes such as CDKn1a or Mdm2, concomitant with the activation of TAp63α. While DNA repair genes are not upregulated, inflammation-related genes become transcribed when apoptosis is initiated by activation of STAT transcription factors. Furthermore, comparison with the transcriptional profile of the ΔNp63α isoform from other studies shows only a minimal overlap, suggesting distinct regulatory programs of different p63 isoforms.
Die akute myeloische Leukämie (AML) ist eine aggressive Erkrankung des Knochenmarks, welche die Hämatopoese beeinträchtigt und zu Knochenmarksversagen führt. Trotz des Fortschritts in der AML-Therapie bleibt die Prognose für die meisten Patienten schlecht, sodass neue Therapieansätze für die Behandlung dringend benötigt werden. Autophagie, ein kataboler Abbauprozess von zellulären Komponenten, ist nachweislich an der Entstehung von AML beteiligt. Als zentraler Regulator von Zellüberleben, Homöostase und Stoffwechsel, dient die Autophagie als Nährstoffquelle durch die Wiederverwertung von Makromolekülen während begrenzter Energieversorgung. AML-Zellen benötigen ein konstantes Nährstoff- und Energieniveau, um ihre Vermehrung aufrechtzuerhalten. Dies wird durch eine Umstellung von Stoffwechselwegen, insbesondere des mitochondrialen Stoffwechsels einschließlich der oxidativen Phosphorylierung (OXPHOS) und des Tricarbonsäurezyklus (TCA), erreicht.
Mehrere Studien haben die Hemmung der Autophagie für die Behandlung von Krebs als vielversprechenden Ansatz vorgestellt. Doch eine Monotherapie mit Autophagie-Inhibitoren erzielte nur eine geringfügige Wirksamkeit. Eine mögliche Erklärung hierfür ist die Entstehung von Kompensationsmechanismen, die zum Ausgleich der Autophagie-Hemmung in Krebszellen entstehen. Bis heute sind diese Kompensationsmechanismen kaum untersucht. Ziel dieser Arbeit ist es, ein geeignetes Autophagie-Gen zu identifizieren, mit dem sich die Rolle der Autophagie-Hemmung für das Überleben von AML-Zellen untersuchen lässt. Zusätzlich sollen die kompensatorischen Mechanismen, die durch die Autophagie-Hemmung in AML-Zellen entstehen können, untersucht werden, um neue metabolische Angriffspunkte zu identifizieren, die für Kombinationstherapien genutzt werden können.
Zu Beginn der Arbeit wurde ein gezielter CRISPR/Cas9 Screen in zwei humanen AML-Zelllinien durchgeführt, um Autophagie-Gene zu identifizieren, deren Verlust eine Proliferationsstörung in AML-Zellen verursacht, welche überwunden werden kann. Validierungsexperimente zeigten, dass der Verlust von ATG3 das Zellwachstum signifikant verminderte. Außerdem zeigte die Messung des Autophagie-Fluxes, dass der Verlust von ATG3 die Autophagie stark beeinträchtigte. Dies wurde durch eine Western-Blot-Analyse, die eine beeinträchtigte LC3-Lipidierung zeigte, und durch eine Immunfluoreszenzanalyse der Autophagosomen-Bildung mittels konfokaler Mikroskopie, die eine geringere Anzahl von Autophagosomen in ATG3-defizienten Zellen ergab, bestätigt. Deshalb wurde der Knockdown von ATG3 in AML Zellen verwendet, um die Mechanismen, die zum Ausgleichen der Autophagie-Hemmung entstehen, zu untersuchen. Zuerst wurde die Zellproliferation in fünf verschiedenen AML Zelllinien über sieben Tage betrachtet. In allen Zellenlinien führte der Verlust von ATG3 mittels small hairpin RNA zu verminderter Zellproliferation. Diese Ergebnisse zeigen die wichtige Rolle von ATG3 in der Autophagie und dass Autophagie-Hemmung durch ATG3-Verlust das Wachstum von AML-Zellen beeinträchtigt.
Da der Verlust von ATG3 die Proliferation von AML-Zellen beeinträchtigte, wurde eine Zellzyklusanalyse durchgeführt. Eine reduzierte S-Phase bestätigte die verminderte Proliferation in ATG3-depletierten AML-Zellen, doch der Zellzyklus war grundsätzlich nicht gestoppt. Darüber hinaus ergab die Analyse der Apoptose, dass diese unter dem Verlust von ATG3 erhöht war, aber etwa 50% der Zellen blieben vital. Diese Beobachtungen deuten darauf hin, dass AML-Zellen trotz des Verlusts der ATG3-abhängigen Autophagie weiter proliferieren können.
Um die Mechanismen zur Kompensation der Autophagie-Hemmung zu untersuchen, wurden die Auswirkungen des ATG3-Verlusts auf die mitochondriale Homöostase untersucht. Die Mitophagie sowie das mitochondriale Membranpotenzial und die Masse unterschieden sich zwischen Kontroll- und ATG3-depletierten AML-Zellen nicht, was darauf hindeutet, dass die mitochondriale Homöostase durch den Verlust von ATG3 nicht beeinträchtigt ist. Als nächstes wurde die mitochondriale Funktion durch Messung des ATP-Spiegels und der OXPHOS untersucht. Die ATP-Level und die OXPHOS waren nach dem Verlust von ATG3 in AML-Zellen erhöht, was auf eine gesteigerte mitochondriale Aktivität bei Autophagie-Defizienz hinweist.
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.
Untersuchungen über die Redox-Eigenschaften der Haut nach Bestrahlung mit ultraviolettem Licht
(1959)
Eingehendere Untersuchungen über die mit Hilfe der Nadi - Reaktion nachweisbaren erhöhten Oxydationswirkungen an uv-bestrahlten Hautstellen führten zur Feststellung, daß eine Erhöhung der Peroxydase-Wirkung in Verbindung mit Vermehrung der peroxydischen Eigenschaften als Ursache anzusprechen ist. Aktivierung der Tyrosinase im bestrahlten Gebiet wurde sowohl an der albinotischen Mäusehaut als auch bei Froschschwimmhäuten nachgewiesen. Wellenlängen-Abhängigkeit und Bedeutung als Primäreffekt der UV-Strahlen werden diskutiert.