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Molecular Mechanisms of Bortezomib Action: Novel Evidence for the miRNA−mRNA Interaction Involvement
(2020)
Bortezomib is an anti-tumor agent, which inhibits 26S proteasome degrading ubiquitinated
proteins. While apoptotic transcription-associated activation in response to bortezomib has been
suggested, mechanisms related to its influence on post-transcriptional gene silencing mediated
regulation by non-coding RNAs remain not fully elucidated. In the present study, we examined
changes in global gene and miRNA expression and analyzed the identified miRNA–mRNA interactions
after bortezomib exposure in human neuroblastoma cells to define pathways affected by this agent in
this type of cells. Cell viability assays were performed to assess cytotoxicity of bortezomib. Global gene
and miRNA expression profiles of neuroblastoma cells after 24-h incubation with bortezomib were
determined using genome-wide RNA and miRNA microarray technology. Obtained results were
then confirmed by qRT-PCR and Western blot. Further bioinformatical analysis was performed
to identify affected biological processes and pathways. In total, 719 genes and 28 miRNAs were
downregulated, and 319 genes and 61 miRNAs were upregulated in neuroblastoma cells treated with
bortezomib. Possible interactions between dysregulated miRNA/mRNA, which could be linked to
bortezomib-induced neurotoxicity, affect neurogenesis, cellular calcium transport, and neuron death.
Bortezomib might exert toxic effects on neuroblastoma cells and regulate miRNA–mRNA interactions
influencing vital cellular functions. Further studies on the role of specific miRNA–mRNA interactions
are needed to elucidate mechanisms of bortezomib action.
Abstract
Background
Duchenne muscular dystrophy (DMD) is a progressive muscle‐wasting disease caused by mutations in the dystrophin gene, which leads to structural instability of the dystrophin–glycoprotein‐complex with subsequent muscle degeneration. In addition, muscle inflammation has been implicated in disease progression and therapeutically addressed with glucocorticosteroids. These have numerous adverse effects. Treatment with human immunoglobulin G (IgG) improved clinical and para‐clinical parameters in the early disease phase in the well‐established mdx mouse model. The aim of the present study was to confirm the efficacy of IgG in a long‐term pre‐clinical study in mdx mice.
Methods
IgG (2 g/kg body weight) or NaCl solution as control was administered monthly over 18 months by intraperitoneal injection in mdx mice beginning at 3 weeks of age. Several clinical outcome measures including endurance, muscle strength, and echocardiography were assessed. After 18 months, the animals were sacrificed, blood was collected for analysis, and muscle samples were obtained for ex vivo muscle contraction tests, quantitative PCR, and histology.
Results
IgG significantly improved the daily voluntary running performance (1.9 m more total daily running distance, P < 0.0001) and slowed the decrease in grip strength by 0.1 mN, (P = 0.018). IgG reduced fatigability of the diaphragm (improved ratio to maximum force by 0.09 ± 0.04, P = 0.044), but specific tetanic force remained unchanged in the ex vivo muscle contraction test. Cardiac function was significantly better after IgG, especially fractional area shortening (P = 0.012). These results were accompanied by a reduction in cardiac fibrosis and the infiltration of T cells (P = 0.0002) and macrophages (P = 0.0027). In addition, treatment with IgG resulted in a significant reduction of the infiltration of T cells (P ≤ 0.036) in the diaphragm, gastrocnemius, quadriceps, and a similar trend in tibialis anterior and macrophages (P ≤ 0.045) in gastrocnemius, quadriceps, tibialis anterior, and a similar trend in the diaphragm, as well as a decrease in myopathic changes as reflected by a reduced central nuclear index in the diaphragm, tibialis anterior, and quadriceps (P ≤ 0.002 in all).
Conclusions
The present study underscores the importance of an inflammatory contribution to the disease progression of DMD. The data demonstrate the long‐term efficacy of IgG in the mdx mouse. IgG is well tolerated by humans and could preferentially complement gene therapy in DMD. The data call for a clinical trial with IgG in DMD.
Dengue virus (DV) is a positive-strand RNA virus of the Flavivirus genus. It is one of the most prevalent mosquito-borne viruses, infecting globally 390 million individuals per year. The clinical spectrum of DV infection ranges from an asymptomatic course to severe complications such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), the latter because of severe plasma leakage. Given that the outcome of infection is likely determined by the kinetics of viral replication and the antiviral host cell immune response (HIR) it is of importance to understand the interaction between these two parameters. In this study, we use mathematical modeling to characterize and understand the complex interplay between intracellular DV replication and the host cells' defense mechanisms. We first measured viral RNA, viral protein, and virus particle production in Huh7 cells, which exhibit a notoriously weak intrinsic antiviral response. Based on these measurements, we developed a detailed intracellular DV replication model. We then measured replication in IFN competent A549 cells and used this data to couple the replication model with a model describing IFN activation and production of IFN stimulated genes (ISGs), as well as their interplay with DV replication. By comparing the cell line specific DV replication, we found that host factors involved in replication complex formation and virus particle production are crucial for replication efficiency. Regarding possible modes of action of the HIR, our model fits suggest that the HIR mainly affects DV RNA translation initiation, cytosolic DV RNA degradation, and naïve cell infection. We further analyzed the potential of direct acting antiviral drugs targeting different processes of the DV lifecycle in silico and found that targeting RNA synthesis and virus assembly and release are the most promising anti-DV drug targets.
Over the past 10 years, the crisis of sepsis has remained a great challenge. According to data from 2016, the sepsis-related mortality rate remains high. In addition, sepsis consumes extensive medical resources in intensive care units, and anti-inflammatory agents fail to improve sepsis-associated hyperinflammation and symptoms of immunosuppression. The specific immune mechanism of sepsis remains to be elucidated. Reactive oxygen species (ROS) are triggered by energy metabolism and respiratory dysfunction in sepsis, which not only cause oxidative damage to tissues and organelles, but also directly and indirectly promote NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. NLRP3 inflammasomes enlarge the inflammatory response and trigger apoptosis of immune cells to exacerbate sepsis progression. Inhibiting the negative effects of ROS and NLRP3 inflammasomes therefore provides the possibility of reversing the excessive inflammation during sepsis. In this review, we describe the interaction of ROS and NLRP3 inflammasomes during sepsis, provide prevention strategies, and identify fields that need further study.
Bats belong to the most gregarious and diverse mammals with highly complex social behaviors. Despite extensive research on their ecology and social behavior in some bat species, gained insights are restricted to only few of the more than 1300 species. In the recent past, bats have also become a central topic of a different branch of research: Since the 1990s bats came to the fore of virologists and immunologists due to the bats’ apparent importance as reservoir hosts and vectors of several (mostly tropical) diseases. While this research is focused mainly on emerging infectious diseases linked to bats, and their zoonotic potential, little has been invested regarding the link between disease transmission and bat social systems.
In my work, I aim at filling this gap by merging automated daily roosting observations, social network analysis, and a virological screening in Natterer’s bats (Myotis nattereri). In a collaborative approach, my co-workers and I analyzed the social structure of individually marked Natterer’s bats, their astrovirus detection rate and transmission pathways within their colony, as well as roosting interactions between different co-occurring con- and heterospecific bat colonies.
We discovered Natterer’s bats to display a very divergent social network structure that contradicts the findings of previous studies on large fission-fusion groups. Contrary to the modular social network structure found in e.g. primates or other bats species, the social network of Natterer’s bats consists of only one highly interconnected community. Moreover, although the close proximity between bat hosts in the colony should strongly promote direct transmission, we found indications that astrovirus infections follow at least partly an indirect transmission pathway via contaminated roost use. Lastly, our results prove that co-occurring con- and heterospecific bat colonies, e.g. as in this study Natterer’s bats, brown long-eared bats and Bechstein’s bats, can influence each other in their roost use by avoiding conspecific roosts and by being attracted towards those of heterospecifics. This holds implication for the transmission of parasites and pathogens within and between different colonies with opportunities for spillovers. To conclude, this multidisciplinary study led to valuable insights in the hitherto hidden mechanisms within and among bat colonies.
Abstract
Ecosystems with highly pulsed water supply must be better understood as climate change may increase frequency and severity of intense storms, droughts and floods. Here we collected data over 3 years (2016–2018) in the episodic wetland outflow channel (Aluize), Banhine National Park, in which the system state changed from dry to wet to dry. Field sampling included vegetation records, small‐scale vegetation zoning, the seed bank and water and soil quality. The same main plant species were found in both dry and wet conditions across the riverbed of the outflow channel. We found only very few diaspores of plants in the soil after prolonged drought. In the subsequent flooded state, we examined very dense vegetation on the water surface, which was dominated by the gramineous species Paspalidium obtusifolium. This species formed a compact floating mat that was rooted to the riverbed. The Cyperaceae Bolboschoenus glaucus showed high clonal growth in the form of root tubers, which likely serve as important food reservoir during drought. Soil and water analyses do not indicate a limitation by nutrients. We outline how resident people may change the plant community structure with an increasing practice of setting fire to the meadows in the dried‐up riverbed to facilitate plant regrowth as food for their livestock.
Abstract
Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.
The definition of Green Chemistry was first formulated at the beginning of the 1990s – 30 years ago and states as follows: “design of chemical products and processes to reduce or eliminate the use and generation of hazardous substances” (Poliakoff et al. 2002). Biocatalysis is one of the examples of “green” chemistry as it is relying on natural or modified enzymes. Today, biocatalysis is a standard technology for the production of chemicals (Straathof et al. 2002).
In this PhD thesis, the implications of biocatalysis using different class of enzymes are discussed: two cytochrome P450 monoxygenases, two kinases and one lyase are shown as tools for the production of bioactive compounds.
The P450 enzymes have a central role in the oxidative metabolism of a wide variety of compounds including the synthesis of endogenous substrates such as steroids and fatty acids. Moreover, P450s catalyze the hydroxylation of non-activated carbon atoms in a regio- and stereospecific fashion avoiding use of protecting groups and several, time-consuming chemical steps.
Here, the recombinant expression and biocatalytic characterization of bacterial CYP107D1 for the regio- and stereoselective hydroxylation of two steroid compounds is reported. Since the natural electron transfer partners of these P450s are unknown, PdX and PdR from P. putida were employed to supply CYP107D1 with the necessary electrons for catalysis. This three-component system was used in bioconversions of two bile acids: LCA and DCA. P450 CYP107D1 exhibits high regio- and stereoselectivity for the tested steroids, giving 6β-hydroxylated products. The properties of the CYP107D1 make this multifaceted P450 monooxygenase an attractive enzyme for the production of novel drug metabolites. Moreover, the crystal structure of the enzyme is known, which provides the basis for developing a protein-engineering strategy aimed at catalytic properties of the CYP107D1
The second enzyme described in the thesis is the self-sufficient cytochrome P450 monooxygenase from Fusarium graminarium (FG067). From the overall structure, it resembles the well investigated CYP102 from Bacillus megaterium (CYP BM3) and the P450 from Fusarium oxysporum (CYPfoxy). In this study, two different strategies to recombinantly produce the fungal P450 monooxygenase P450-FG067, namely (a) producing in E. coli and (b) producing in P. pastoris were investigated. The P450 FG_067 from Fusarium graminarium was successfully overexpressed in P. pastoris. The enzyme was functionally active, converted fatty acid substrates of carbon chain length C10-16 with regiospecificity of the hydroxylating position ω -1, ω - 2 and ω-3, with the highest affinity for capric acid. The hydroxylation at different positions of the fatty acid chain is needed for different chemical industries. For example, ω-HFAs can be used as starting materials for the synthesis of polymers, with high resistance to heat or chemicals (Xiao et al. 2018). Therefore, the application of recombinant enzyme such as self-sufficient P450 FG_067 for a commercial production of HFAs is in high industrial demand.
In this thesis, two kinases were used for the producton of phosphorylated metabolites. Kinases catalyzing N-phosphorylation, which are of synthetic interest because of tedious chemical procedures in selective chemical N-phosphorylations. A highly active and stabile arginine kinase, obtained by cloning and expressing the argK gene from Limulus polyphemus in E. coli, was used in the one-step synthesis of Nω-phospho-L-arginine using the phosphoenolpyruvate/pyruvate kinase system for ATP regeneration. Applying arginine kinase in biocatalysis opens up new opportunities for the selective biocatalytic N-phosphorylation of interesting low-molecular-weight compounds and metabolites.
Another kinase investigated in this thesis was shikimate kinase. The highly active and stable shikimate kinase AroL was achieved by synthesizing the codon-optimized aroL gene and expressing it in high yield in E. coli. Next, shikimate kinase was used in an one-step synthesis of shikimate-3-phosphate using the phosphoenolpyruvate/pyruvate kinase system for ATP regeneration. Development of the described biocatalytic preparation of shikimate-3-phosphate is a superior route incomparison to a tedious multi-step and low yield classical synthesis of this compound. The biocatalytic phosphorylation is of great interest for a commercial production of metabolites and metabolite-like structures.
The last investigeted enzyme in this PhD thesis was argininosuccinate lyase from Saccharomyces cerevisiae. The argininosuccinate lyase was cloned and overexpressed in E. coli as a highly active and stable biocatalyst. A simple and straightforward biocatalytic asymmetric Michael addition reaction has been established for the synthesis of the key metabolite N-(([(4S)-4-amino-4-carboxybutyl]amino)imino methyl)-L-aspartic acid, commonly referred to as L-argininosuccinate. This one-step addition reaction was developed by running part of the urea cycle in reverse. The use of this argininosuccinate lyase and reaction monitoring by NMR enabled the development of a biocatalytic asymmetric Michael addition reaction as a novel green chemistry route with high molecular economy for the synthesis of this important metabolite at gram scale.
Recent advances in the field of scientific research have helped to understand the structure and functional activities of enzymes, which has in turn led to an increase in their stability, activity and substrate specificity. Nowadays, biocatalysis provide more sustainable, efficient, and less polluting methods for the production of fine chemicals and advanced pharmaceutical intermediates. The biocatalysts used in this thesis are introduced as a technology for the efficient synthesis of biologically active compounds, which is greener, reduces pollution and costs compared to chemical synthesis. In summary, the pharmaceutical industry should use the advantage of the progress of biochemistry to obtain biocatalysts in the production of fine chemicals on an industrial scale, improving the quality of end products and saving costs.
Abstract
Background
Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo‐epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram‐negative Escherichia coli.
Objectives
To assess the role of PF4, anti‐PF4/H antibodies and FcγRIIa in killing of Gram‐positive bacteria by platelets.
Methods
Binding of PF4 to protein‐A deficient Staphylococcus aureus (SA113Δspa) and non‐encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti‐PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P‐expression, PAC‐1 binding) and phosphatidylserine (PS)‐exposure were analyzed.
Results
Monoclonal and patient‐derived anti‐PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6‐fold increased fluorescence signal for human anti‐PF4/H antibodies to 24.0‐fold increase for KKO). Staphylococcus aureus (5.5 × 104cfu/mL) was efficiently killed by platelets (2.7 × 104cfu/mL) or their releasate (2.9 × 104cfu/mL). Killing was not further enhanced by PF4 or anti‐PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP‐6 stimulation and exposed high levels of PS.
Conclusions
Anti‐PF4/H antibodies seem to have only a minor role for direct killing of Gram‐positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability.
Plasma medical oncology: Immunological interpretation in head and neck squamous cell carcinoma
(2020)
Over the past several years, various important articles focusing on cancer therapy
approaches in head and neck squamous cell carcinomas (HNSCCs) using cold
atmospheric plasma (CAP) have been published (SEMMLER et al. 2020 [53],
METELMANN et al. 2018 [44], KEIDAR et al. 2011 [33]). This doctoral thesis presents
selected results from a prospective observational clinical study in CAP therapy of
palliative HNSCC patients, carried out at the Department of Oral and Maxillofacial
Surgery/Plastic Surgery of the Greifswald University Medicine. For oral and
maxillofacial surgeons, ulcerated surfaces of locally advanced head and neck squamous
cell carcinomas (UICC IV) offer a challenging treatment assignment with microbial
contamination and tumour progression. The clinical attempt appears to eradicate
microbial contamination and to initiate tumour regression. This doctoral thesis will
describe the processes of human tumour biology and tumour immunology in HNSCCs
and the extent of present knowledge concerning plasma medical oncology as an
anticancer modality. In the introduction of the doctoral thesis clinical results of plasma
therapy in locally advanced HNSCCs (UICC IV) are set out. This mainly includes the
investigation of a therapeutic concept, the treatment phases, the tumour size development
and the morphological changes of the infected tumour surface following cold atmospheric
plasma therapy. In the main part, a detailed immunological interpretation is proposed on
the basis of present preclinical and clinical immunological knowledge. Finally,
unexplored questions in plasma medical oncology are highlighted. This is highly
significant for future plasma research and clinical anticancer therapy.
Cardiovascular diseases are the most common cause of death in industrial nations. The basis of these diseases is a dysfunction in the interaction between the cells the heart is composed of. The main types of cells making up the human heart are cardiomyocytes that build the myocardium and provide the contraction properties, endothelial cells that delimit the blood flowing through the inner chambers and coronary arteries from the myocardial tissue, and fibroblasts, which build the connective tissue. A common process in the development of cardiovascular diseases is the formation of fibrosis due to injury of the endothelium and subsequent infiltration of the cardiac tissue by immune cells, and inflammatory agents like cytokines. Cytokines exert different functions in cardiac cells. Tumor necrosis factor α (TNFα) is an inducer of apoptosis. Transforming growth factor ß (TGFß) is known for activation of proliferation. Other cytokines like C-X-C motif chemokine 11 (CXCL11), interleukin-6 (IL-6), or brain-derived neurotrophic factor (BDNF) have not yet been investigated or their impact on such cells is unknown. Eventually, however, fibrotic scar tissue arises from the transition from fibroblasts to myofibroblasts leading to a stiffening of the cardiac muscle and impaired pump function. In order to prevent the occurrence of these events the balance of proliferation, migration, and differentiation of cardiac cells needs to be controlled very delicately.
The mechanisms controlling these interactions are still not well understood, which is why this work aimed at the elucidation of molecular mechanisms within the three main cell types that might play a role in the regulation of cardiac function. A proteomic approach using mass spectrometry was used to identify alterations in protein levels that could provide hints about the involved pathways and find new players as candidates for more detailed investigation. Initially, the proteomic composition of HL-1 cardiomyocytes, L929 fibroblasts, and human umbilical vein endothelial cells (HUVECs) that were cultivated in standard growth conditions without stress was investigated. Half of the total protein intensity was made up by only 42 to 53 proteins, depending on the cell type. More than a third of all proteins were identified in all three cell types, which may be proteins performing common cell functions. Indeed, the proteins displaying the highest abundance seem to be predominantly involved in such common cellular functions as the regulation of glucose metabolism or the cytoskeleton. More specific functions like heart development and muscle contraction were found enriched in cardiomyocytes as were mitochondrial proteins. The proportion of proteins with extracellular localization and function was higher in fibroblasts and endothelial cells.
Secondly, the impact of cytokines on the proliferative behavior and the proteomic composition of cardiomyocytes and fibroblasts was analyzed. HL-1 cardiomyocytes and L929 fibroblasts were treated with different concentrations of cytokines with a cytotoxic, proliferative, or yet unknown effect on these cells. While HL-1 cells exhibited no macroscopic reaction to any of the cytokines used, cytotoxic/growth inhibitory (TNFα, CXCL11) and proliferative (TGFß, IL6, BDNF) effects were observed for L929 cells. The latter also showed CXCL11-induced upregulated EIF2 signaling, pointing to a higher need of protein synthesis.
The third aim was the examination of proteome adaptations in endothelial cells due to different kinds of stress, as these cells are the first line of defense against inflammatory agents or injury and therefore prone to wounding. The role of the growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in wounding and starvation was another object of this study as they are known for their angiogenic and cell survival supporting properties. Additionally, the impact of the cellular sex on the response to stress and growth factors was examined, because a person’s sex plays an important role in susceptibility, risk factors, and outcome of cardiovascular diseases. This has mainly been attributed to the different hormone levels, especially the higher levels of estrogen in premenopausal women, which exerts cardioprotective properties, but also genetic background was reported to play an important role. Only few studies that examined the molecular properties of HUVECs considered the cellular sex and if so, the genetic bias of unrelated samples was not taken into account. This is why Lorenz and colleagues at the Charité in Berlin collected HUVECs from newborn twins of opposite sex, cultivated them without stress in standard growth medium, exposed them to wounding and serum starvation, and investigated the impact of the growth factors and the sex on migrational behavior and metabolic issues. The current work focused on the alterations of not only the intra- but also the extracellular proteome, because paracrine signaling is crucial for intercellular communication in order to cope with stress. General differences between male and female cells were observed for proteins encoded on the X chromosome with higher levels in females (DDX3X, UBA1, EIF1AX, RPS4X, HDHD1), except for one protein with higher levels in male cells (G6PD). A Y-chromosomal protein was, for the first time, identified in endothelial cells (DDX3Y). Wounding, starvation, and growth factor treatment led to alterations and sex-specific different levels in an unexpectedly high number of proteins, with VEGF showing a stronger impact than bFGF. Many proteins with alterations observed without taking the sex into account, were actually only changed in male or female cells. Some proteins were regulated in opposite directions, or growth factors inhibited their secretion in a sex-specific way by unknown mechanisms. Tissue factor pathway inhibitor 2 (TFPI2) should be emphasized as a protein with sex-specific differences, especially in the extracellular space and with increased levels after starvation and VEGF treatment. These observations suggest a temporal lack in TFPI2 synthesis and secretion in male cells, which might explain the enhanced adaptation of females to wounding.
The results of this work lay the basis for future investigation by providing a database of intra- and extracellular proteome changes due to different environmental circumstances. It strongly suggests the investigation of male and female HUVECs, and other cells, separately to avoid the impact of the sex observed in this work. Essentially, the observations suggest a number of candidate proteins for more detailed investigations of endothelial and cardiovascular diseases.
The Immunomodulator 1-Methyltryptophan Drives Tryptophan Catabolism Toward the Kynurenic Acid Branch
(2020)
Background: Animal model studies revealed that the application of 1-methyltryptophan (1-MT), a tryptophan (TRP) analog, surprisingly increased plasma levels of the TRP metabolite, kynurenic acid (KYNA). Under inflammatory conditions, KYNA has been shown to mediate various immunomodulatory effects. Therefore, the present study aims to confirm and clarify the effects of 1-MT on TRP metabolism in mice as well as in humans.
Methods: Splenocytes from Balb/C or indoleamine 2,3-dioxygenase knockout (IDO1−/−) mice or whole human blood were stimulated with 1-MT for 6, 24, or 36 h. C57BL/6 mice received 1-MT in drinking water for 5 days. Cell-free supernatants and plasma were analyzed for TRP and its metabolites by tandem mass spectrometry (MS/MS).
Results: 1-MT treatment induced an increase in TRP and its metabolite, KYNA in Balb/C, IDO−/− mice, and in human blood. Concurrently, the intermediate metabolite kynurenine (KYN), as well as the KYN/TRP ratio, were reduced after 1-MT treatment. The effects of 1-MT on TRP metabolites were similar after the in vivo application of 1-MT to C57BL/6 mice.
Conclusions: The data indicate that 1-MT induced an increase of KYNA ex vivo and in vivo confirming previously described results. Furthermore, the results of IDO−/− mice indicate that this effect seems not to be mediated by IDO1. Due to the proven immunomodulatory properties of KYNA, a shift toward this branch of the kynurenine pathway (KP) may be one potential mode of action by 1-MT and should be considered for further applications.
Mathematical phylogenetics provides the theoretical framework for the reconstruction and analysis of phylogenetic trees and networks. The underlying theory is based on various mathematical disciplines, ranging from graph theory to probability theory.
In this thesis, we take a mostly combinatorial and graph-theoretical position and study different problems concerning phylogenetic trees and networks.
We start by considering phylogenetic diversity indices that rank species for conservation. Two such indices for rooted trees are the Fair Proportion index and the Equal Splits index, and we analyze how different they can be from each other and under which circumstances they coincide. Moreover, we define and investigate analogues of these indices for unrooted trees.
Subsequently, we study the Shapley value of unrooted trees, another popular phylogenetic diversity index. We show that it may fail as a prioritization criterion in biodiversity conservation and is outcompeted by an existing greedy approach. Afterwards, we leave the biodiversity setting and consider the Shapley value as a tree reconstruction tool. Here, we show that non-isomorphic trees may have permutation-equivalent Shapley transformation matrices and identical Shapley values, implying that the Shapley value cannot reliably be employed in tree reconstruction.
In addition to phylogenetic diversity indices, another class of indices frequently discussed in mathematical phylogenetics, is the class of balance indices. In this thesis, we study one of the oldest and most popular of them, namely the Colless index for rooted binary trees. We focus on its extremal values and analyze both its maximum and minimum values as well as the trees that achieve them.
Having analyzed various questions regarding phylogenetic trees, we finally turn to phylogenetic networks. We focus on a certain class of phylogenetic networks, namely tree-based networks, and consider this class both in a rooted and in an unrooted setting.
First, we prove the existence of a rooted non-binary universal tree-based network with n leaves for all positive integers n, that is, we show that there exists a rooted non-binary tree-based network with $n$ leaves that has every non-binary phylogenetic tree on the same leaf set as a base tree.
Finally, we study unrooted tree-based networks and introduce a class of networks that are necessarily tree-based, namely edge-based networks. We show that edge-based networks are closely related to a family of graphs in classical graph theory, so-called generalized series-parallel graphs, and explore this relationship in full detail.
In summary, we add new insights into existing concepts in mathematical phylogenetics, answer open questions in the literature, and introduce new concepts and approaches. In doing so, we make a small but relevant contribution to current research in mathematical phylogenetics.
Drained peatlands are significant sources of the greenhouse gas (GHG) carbon dioxide.Rewetting is a proven strategy used to protect carbon stocks; however, it can lead to increasedemissions of the potent GHG methane. The response to rewetting of soil microbiomes as drivers ofthese processes is poorly understood, as are the biotic and abiotic factors that control communitycomposition. We analyzed the pro- and eukaryotic microbiomes of three contrasting pairs ofminerotrophic fens subject to decade-long drainage and subsequent long-term rewetting. Abiotic soilproperties including moisture, dissolved organic matter, methane fluxes, and ecosystem respirationrates were also determined. The composition of the microbiomes was fen-type-specific, but allrewetted sites showed higher abundances of anaerobic taxa compared to drained sites. Based onmulti-variate statistics and network analyses, we identified soil moisture as a major driver ofcommunity composition. Furthermore, salinity drove the separation between coastal and freshwaterfen communities. Methanogens were more than 10-fold more abundant in rewetted than in drainedsites, while their abundance was lowest in the coastal fen, likely due to competition with sulfatereducers. The microbiome compositions were reflected in methane fluxes from the sites. Our resultsshed light on the factors that structure fen microbiomes via environmental filtering.
In 2010, the identification of 17 novel (R)-ATAs represented a breakthrough for the biocatalytic asymmetric synthesis of chiral amines, because only one (R)-ATA was described before. These novel ATAs were identified in a bioinformatic approach by studying the substrate acceptance of BCATs and DATAs to deduce the unknown substrate coordination of (R)-ATAs. Article I describes an alternative approach for the identification of (R)-ATA activity by reengineering the substrate- recognition site of α-AATs. While the engineering of the eBCAT led to the formation of an initial (R)-amine acceptance only, the (R)-ATA activity was successfully introduced in the DATA scaffold. These results demonstrate the transformation of an α-AAT in a moderately active (R)-ATA for the first time and highlight the evolutionary relationship between α-AATs and ATAs. Despite the availability of different ATAs nowadays, their substrate spectrum is limited due to the natural composition of their active sites. Several protein-engineering studies showed the widening of the substrate spectrum and the acceptance of bulky substrates by screening large mutant libraries to identify beneficial variants. In Article II, we developed an in silico engineering approach for amine transaminases to improve the conversion of bulky substrates and to reduce the number of variants to be tested in the laboratory. The resulting double-mutants of the (S)-ATA from C. violaceum displayed a >200-fold improved activity towards the bulky benchmark substrate. These variants expand the available biocatalytic toolbox for the synthesis of bulky amines, and the developed framework paves the way for rational protein-engineering protocols.
By studying unconventional transaminase substrates, we explored the potential of the available in- house transaminase toolbox in Articles III, IV, V, and VI. In Article III, we showed the transamination of a β-keto ester, leading to the synthesis of β-phenylalanine. The described cascade in Article IV enables the synthesis of amino carbohydrates. In addition, Article V describes an enzymatic cascade for the synthesis of amino fatty acids, which was extended in Article VI to obtain fatty amines.
The findings of this thesis clearly contribute to the understanding of the substrate scope and specificity of amine transaminases and expand the application of this versatile biocatalyst beyond classical ketone substrates.
Streptococcus pneumoniaeinfections lead to high morbidity and mortality rates worldwide.Pneumococcal polysaccharide conjugate vaccines significantly reduce the burden of disease but havea limited range of protection, which encourages the development of a broadly protective protein-basedalternative. We and others have shown that immunization with pneumococcal lipoproteins that lackthe lipid anchor protects against colonization. Since immunity againstS. pneumoniaeis mediatedthrough Toll-like receptor 2 signaling induced by lipidated proteins, we investigated the effects ofa lipid modification on the induced immune responses in either intranasally or subcutaneouslyvaccinated mice. Here, we demonstrate that lipidation of recombinant lipoproteins DacB and PnrAstrongly improves their immunogenicity. Mice immunized with lipidated proteins showed enhancedantibody concentrations and different induction kinetics. The induced humoral immune responsewas modulated by lipidation, indicated by increased IgG2/IgG1 subclass ratios related to Th1-typeimmunity. In a mouse model of colonization, immunization with lipidated antigens led to a moderatebut consistent reduction of pneumococcal colonization as compared to the non-lipidated proteins,indicating that protein lipidation can improve the protective capacity of the coupled antigen. Thus,protein lipidation represents a promising approach for the development of a serotype-independentpneumococcal vaccine.
Inflammatory Joint Disease Is a Risk Factor for Streptococcal Sepsis and Septic Arthritis in Mice
(2020)
Septic arthritis is a medical emergency associated with high morbidity and mortality, yet hardly any novel advances exist for its clinical management. Despite septic arthritis being a global health burden, experimental data uncovering its etiopathogenesis remain scarce. In particular, any interplay between septic arthritis and preceding joint diseases are unknown as is the contribution of the synovial membrane to the onset of inflammation. Using C57BL/6 mice as a model to study sepsis, we discovered that Group A Streptococcus (GAS) – an important pathogen causing septic arthritis - was able to invade the articular microenvironment. Bacterial invasion resulted in the infiltration of immune cells and detrimental inflammation. In vitro infected fibroblast-like synoviocytes induced the expression of chemokines (Ccl2, Cxcl2), inflammatory cytokines (Tnf, Il6), and integrin ligands (ICAM-1, VCAM-1). Apart from orchestrating immune cell attraction and retention, synoviocytes also upregulated mediators impacting on bone remodeling (Rankl) and cartilage integrity (Mmp13). Using collagen-induced arthritis in DBA/1 × B10.Q F1 mice, we could show that an inflammatory joint disease exacerbated subsequent septic arthritis which was associated with an excessive release of cytokines and eicosanoids. Importantly, the severity of joint inflammation controlled the extent of bone erosions during septic arthritis. In order to ameliorate septic arthritis, our results suggest that targeting synoviocytes might be a promising approach when treating patients with inflammatory joint disease for sepsis.
Background: Physical inactivity is one of the main risk factors for cardiovascular disease,
which remains a major cause of death in Germany and around the globe. Thus, investigating
prevalences, population trends, high-risk groups, and intervention effects of physical activity
(PA) and sedentary time (ST) is highly relevant to public health. To receive reliable data, a
key issue in research is to apply an appropriate study design including the carefully
considered use of assessments. Otherwise, bias to PA and ST data may be introduced. The
present thesis investigates three often overlooked issues related to the impact of measurement
on PA and ST research data. The first aim was to examine whether mere measurement alters
PA and ST over the course of twelve months (study 1). The second aim was to identify
potential socio-demographic and cardiometabolic moderators of the mere-measurement effect
(study 2). The third aim was to present design, protocol, and preliminary results of an interim
analysis of a randomized controlled trial (RCT) aiming to test whether a video demonstration
of PA intensity levels reduces the lack of agreement between self-reported and objectively
measured PA (study 3).
Methods: Studies 1 and 2 were based on data of a trial to test the feasibility of a brief tailored
letter intervention to increase PA and to reduce ST during leisure time. Among a sample of
subjects with no history of myocardial infarction, stroke, or vascular interventions, a number
of 175 individuals aged 40 to 65 years participated in the study. At baseline, participants
received standardized measurement of blood pressure and waist circumference, blood sample
taking, and seven-day accelerometry. At baseline and after one, six, and twelve months,
participants completed the International Physical Activity Questionnaire (IPAQ). A random
subsample received a brief tailored letter intervention at months one, three, and four. A
number of 153 participants were included in study 1 using all available data across 12 months.
Changes in PA and ST were analyzed using latent growth modeling. For study 2, baseline and
one-month follow-up data of 175 participants were used. Dependence of one-month changes
in PA and ST on socio-demographic and cardiometabolic variables was analyzed using linear
regression models. In study 3, individuals aged between 40 and 75 years were recruited at a
shopping mall in Greifswald, Germany. Participants received seven-day accelerometry and
were invited to the cardiovascular examination center of the University Medicine Greifswald.
After random allocation to experimental and control group, they completed the selfadministered
IPAQ – Short Form via tablet-computer. The experimental group additionally
received a video demonstration of PA intensity levels before answering the questionnaire. A number of 131 participants were analyzed to receive preliminary results of an interim analysis
in order to verify the presumptions made for the a priori power calculation and to decide on
early stopping of the study. The difference between the study groups in the agreement
between self-reported and accelerometer-based PA was analyzed using a two-sample t-test.
Results: In study 1, results revealed no change in leisure-time PA, an increase in transportrelated
PA (p = .023), and a tendency towards a reduction of ST (p = .060) between baseline
and one-month assessment. Further, ST decreased between six and twelve months (p = .037).
Time trends of the intervention group did not differ significantly from those of the
assessment-only group. Results of study 2 revealed that men increased transport-related PA
more than women (p = .031) and men with higher triglycerides increased transport-related PA
less than men with lower triglycerides (p = .043). Men with higher systolic blood pressure
reduced ST more than those with lower systolic blood pressure (p = .028). However, this
linear association ceased to exist at a level of approximately 145 mmHg. A similar
relationship was found for glycated hemoglobin and ST in men. In study 3, preliminary
results of the interim analysis revealed a lower formal mean difference in the video group (M
= 21.8 min/day, SD = 108.9) compared to the control group (M = 41.0 min/day, SD = 117.4,
t(129) = 0.97, p = .166). The p-value lay between the significance (p < .010) and futility (p >
.269) boundaries of the test simulations.
Conclusions: Results of the present thesis have three implications for considering the impact
of PA and ST assessments in cardiovascular research. First, mere-measurement effects within
a feasibility trial were found in transport-related PA and ST suggesting to interfere with
potential intervention effects. Thus, measurement effects should be considered when planning
studies and interventions and when interpreting outcomes. Second, male sex and more
favorable triglycerides levels in men were associated with a higher increase of transportrelated
PA whereas worse health in men was associated with a higher reduction of ST. Thus,
using the mere-measurement effect for prevention purposes may require researchers and
practitioners to tailor PA and ST intervention components to individuals’ health condition.
Third, the design and protocol of the RCT seems appropriate to test the effect of a novel video
on the gap between self-reported and accelerometer-based PA. Preliminary results point to the
efficacy of the video.
Exploring Virulence Factors and Alternative Therapies against Staphylococcus aureus Pneumonia
(2020)
Abstract
Higher biodiversity can stabilize the productivity and functioning of grassland communities when subjected to extreme climatic events. The positive biodiversity–stability relationship emerges via increased resistance and/or recovery to these events. However, invader presence might disrupt this diversity–stability relationship by altering biotic interactions. Investigating such disruptions is important given that invasion by non‐native species and extreme climatic events are expected to increase in the future due to anthropogenic pressure. Here we present one of the first multisite invader × biodiversity × drought manipulation experiment to examine combined effects of biodiversity and invasion on drought resistance and recovery at three semi‐natural grassland sites across Europe. The stability of biomass production to an extreme drought manipulation (100% rainfall reduction; BE: 88 days, BG: 85 days, DE: 76 days) was quantified in field mesocosms with a richness gradient of 1, 3, and 6 species and three invasion treatments (no invader, Lupinus polyphyllus, Senecio inaequidens). Our results suggest that biodiversity stabilized community productivity by increasing the ability of native species to recover from extreme drought events. However, invader presence turned the positive and stabilizing effects of diversity on native species recovery into a neutral relationship. This effect was independent of the two invader's own capacity to recover from an extreme drought event. In summary, we found that invader presence may disrupt how native community interactions lead to stability of ecosystems in response to extreme climatic events. Consequently, the interaction of three global change drivers, climate extremes, diversity decline, and invasive species, may exacerbate their effects on ecosystem functioning.
Interleukin-33 (IL-33) is a mucosal alarmin belonging to the IL-1 cytokine family and is now recognized to have a key role in innate and adaptive immunity, contributing to tissue homeostasis and response to environmental stresses. In addition, IL-33 has also been shown to work as a positive regulator that initiates and maintains a Th2 immune response. In the context of pregnancy, it has been recently demonstrated that upon certain stress conditions, such as an infection induced inflammation, IL-33 is released from the uterine mucosa and triggers decidual B cells to produce anti-inflammatory molecules, which in turn restore immune homeostasis and prevents the development of preterm birth. In this study we therefore performed a detailed characterization of IL-33 receptor (Il1rl1 or ST2) expression in B cells during normal pregnancy, as well as in a mouse model of preterm birth. We observed that splenic B cells significantly up-regulate the expression of Il1rl1 during pregnancy and identified the B1 B cell population as the main ST2-expressing B cell subset. A further kinetic analysis showed that percentages of ST2-expressing B1 B cells are significantly augmented on days 12 and 14 of pregnancy, both in the spleen and peritoneal cavity of pregnant mice, and then drop toward the end of pregnancy to the levels observed in non-pregnant animals. Furthermore, using a mouse model of LPS-induced preterm birth, we demonstrated that not only are the percentages of ST2-expressing B1 B cells significantly enlarged in the spleen during the acute phase of preterm birth, but decidual B cells also significantly up-regulate ST2 expression as compared to term-pregnant mice. Overall, our results suggest a functional role of ST2 expression in B cells during pregnancy and reinforce the importance of the IL-33/ST2 axis in B cells as a critical mechanism to control inflammation-induced preterm birth.
This thesis deals with the process considerations and optimizations of a whole-cell enzyme cascade reaction for the synthesis of ɛ-caprolactone. The enzyme cascade synthesis of ɛ-caprolactone has been conceptualized and verified using a dehydrogenase and a monooxygenase. The advantage of this enzyme combination is the closed-loop co-factor regeneration. Dehydrogenase and monooxygenase expressed in discrete whole cells were applied in defined ratio to conceptualize the cascade reaction. This necessitates the use of separate co-factor regeneration system due to impermeability of the E. coli cell wall to the co-factor. Article I deal with the design and optimization of dehydrogenase and monooxygenase co-expression in a same E. coli cell. In Article II, the cascade reaction was upscaled and a fed-batch process was realized. Following which, the important reaction metrices were analyzed and optimized. Article III extends the two-enzyme cascade with a lipase. The use of lipase helps to overcome the product inhibition of monooxygenase by ɛ-caprolactone.
Changes in the environment will alter the growth rate of trees and forests. Different disciplines assess such growth rates differently, for example, with tree-ring width data, forest inventories or with carbon-flux data from eddy covariance towers. Such data is used to quantify forests biomass increment, forest’s carbon sequestration or to reconstruct environmental variables before instrumental records. However, raw measurement data is typically not considered to be representative for the average growth rate of trees or forests. Depending on the research question, the effects of certain environmental variables or effects of tree and forest structure have to be removed first. It can be challenging to define and quantify a growth trend that can answer a specific research question because trees and forests grow and respond to environmental change in multiple ways simultaneously, for example, with altered radial increment, height growth, and stand density. Further challenges pose time-lagged feedback loops, for example, between height and radial increment or between stand density and radial increment. Generally, different environments will lead to different tree and forest structures, but because of tree’s longevity this adaptation to the new environment will take decades or even centuries. Consequently, there can be an offset between the present forest structure and what we term the potential natural forest (PNF): Similar to the potential natural vegetation (PNV), the PNF represents that forest that would develop under the current environmental conditions in the absence of human intervention. Because growth rates are affected by the tree and forest structure, growth-trend estimates will differ between the present and the potential forest. Consequently, if the legacy effects of the past are not of interest, the PNF is the theoretical baseline to correct and estimate growth trends.
Introduction: Following behavioral recommendations is key to successful containment of the COVID-19 pandemic. Therefore, it is important to identify causes and patterns of non-compliance in the population to further optimize risk and health communication.
Methods: A total of 157 participants [80% female; mean age = 27.82 years (SD = 11.01)] were surveyed regarding their intention to comply with behavioral recommendations issued by the German government. Latent class analysis examined patterns of compliance, and subsequent multinomial logistic regression models tested sociodemographic (age, gender, country of origin, level of education, region, and number of persons per household) and psychosocial (knowledge about preventive behaviors, risk perception, stigmatizing attitudes) predictors.
Results: Three latent classes were identified: high compliance (25%) with all recommendations; public compliance (51%), with high compliance regarding public but not personal behaviors; and low compliance (24%) with most recommendations. Compared to high compliance, low compliance was associated with male gender [relative risk ratio (RRR) = 0.08 (0.01; 0.85)], younger age [RRR = 0.72 (0.57; 0.93)], and lower public stigma [RRR = 0.21 (0.05; 0.88)]. Low compliers were also younger than public compliers [RRR = 0.76 (0.59; 0.98)].
Discussion: With 25% of the sample reporting full compliance, and 51% differing in terms of public and personal compliance, these findings challenge the sustainability of strict regulatory measures. Moreover, young males were most likely to express low compliance, stressing the need for selective health promotion efforts. Finally, the positive association between public stigma and compliance points to potential othering effects of stigma during a pandemic, but further longitudinal research is required to examine its impact on health and social processes throughout the pandemic.
Abstract
Background and purpose:Diagnosing a patient with headache as a migraineur is critical for state-of-the-art migrainemanagement. Screening tools are imperative means to improve the diagnostic yield in the primary care settings andspecialized clinics. This study aims to translate and assess the diagnostic accuracy of a German version of theID Migraine™as a widely used and efficient screening instrument.
Methods:
The Functional Assessment of Chronic Illness Therapy translation methodology was used to translate theoriginal three-itemID Migraine™, including a fourth question for aura, from the English language into the German language.Diagnostic accuracy of the GermanID Migraine™and predictors of false screening results were assessed among patientspresenting to a headache outpatient clinic of a tertiary care center in Germany over a 6-month period.
Results:
The translation procedure yielded a harmonized GermanID Migraine™and its diagnostic accuracy was assessedin 105 patients (80 female, 46.5+17.2 years of age), including 79 patients (75.2%) with migraine. The three-item GermanID Migraine™provides a sensitivity of 99%, specificity of 68%, and positive and negative predictive values of 90% and 95%,respectively, using a cutoff of2. Positive and negative predictive values in a general headache population are estimated tobe 74% and 98%, respectively. The aura question identified 18 out of 20 migraineurs with aura.
Conclusions:
The GermanID Migraine™is an accurate screening tool for migraine even in a challenging population of aspecialized outpatient clinic. Its diagnostic accuracy indicates a potential utility for screening in primary health care.
Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.
Background: Alexithymia is a personality trait characterized by difficulties in identifying and describing emotions and associated with various psychiatric disorders. Neuroimaging studies found evidence for morphological and functional brain alterations in alexithymic subjects. However, the neurobiological mechanisms underlying alexithymia remain incompletely understood. Methods: We study the association of alexithymia with cortical correlation networks in a large community-dwelling sample of the Study of Health in Pomerania. Our analysis includes data of n = 2,199 individuals (49.4% females, age = 52.1 ± 13.6 years) which were divided into a low and high alexithymic group by a median split of the Toronto Alexithymia Scale. Cortical correlation networks were constructed based on the mean thicknesses of 68 regions, and differences in centralities were investigated. Results: We found a significantly increased centrality of the right paracentral lobule in the high alexithymia network after correction for multiple testing. Several other regions with motoric and sensory functions showed altered centrality on a nominally significant level. Conclusions: Finding increased centrality of the paracentral lobule, a brain area with sensory as well as motoric features and involvement in bowel and bladder voiding, may contribute to explain the association of alexithymia with functional somatic disorders and chronic pain syndromes.
High resolution palaeo-ecological analysis of an Arctic ice-wedge polygon mire (Kytalyk, NE Siberia)
(2020)
Ice-wedge polygon mires are typical features of the Artic and therefore especially affected by climate change. They show, caused by soil-ice action, an amazing regular polygonal structure in meter dimension of higher and lower elevated dry and wet parts, and to this microtopography adapted vegetation. Polygon mires play, analogous to other mires, an important role in carbon sequestration, water balance, wildlife habitat and archive value with local to global significance. By storing enormous amounts of the global soil carbon polygon mires are crucial for our climate. Despite this relevance by covering large areas, polygon mires are comparatively poorly scientifically investigated and understood. It is still difficult to make forecasts on how polygon mires will develop under a changing climate in the Arctic, especially because internal factors and self-organisation complicate the understanding of their functioning. Therefore the investigation of modern and past polygon mires is necessary. This dissertation presents high resolution palaeo-ecological studies of a Northeast Siberian model polygon: ice-wedge polygon Lhc11 located in the Indigirka Lowlands at the scientific station Kytalyk. During field work in July 2011 the study site, covering an area of 26 × 21 m was divided into 546 plots, in which vegetation composition and microtopographical elevation characteristics were assessed and surface samples were collected. For palaeoecological analysis a 105.5 cm long peat section was excavated from the same site. Cluster analysis revealed five plant communities, which are clearly separated with respect to ground surface height, frost surface height and coverages of open water and vegetation, confirming the pattern already identified in other studies of Arctic ice-wedge polygons. The correct recognition of these patterns is crucial in palaeoecological studies in order to reconstruct landscape elements and their dynamics. This recognition requires insight in the short-distance relationships between surface elevation/wetness, vegetation and pollen deposition. The applied pollen-vegetation reference study shows that in general modern pollen deposition in polygon Lhc11 corresponds well with actual vegetation, allowing accurate reconstruction of local site conditions from fossil palynomorph sequences, including the reconstruction of the dynamics of closely spaced microtopographical elements. We conducted an evaluation of common palaeo proxies to compare their wetness reconstruction potential. The analysed proxies macrofossils, pollen, testate amoebae, geochemistry and sediment properties show similar wetness trends. Macrofossils provided the most detailed wetness reconstruction, spanning several wetness classes from very dry to wet, because they could be identified to genus or species level. However, as the proxies sometimes show contradictory results, a multi-proxy approach is preferable over a single proxy interpretation as it allows the reconstruction of environmental development in a broader palaeoecological context. For a better understanding of polygon dynamics and former greenhouse gas fluxes, more detailed and better quantified palaeo-microtopographical information is required. Therefore we developed a new transfer approach for modelling past Ground Surface Heights (GSH) in polygon mires from plant fossils. Based on the composition of modern vegetation we constructed two sets of potential fossil types (plant macrofossils and pollen), an extensive and a more restricted one. We applied Canonical Correspondence Analysis to model the relationships between potential fossil types and measured GSH. Both models show a strong relationship between modelled and measured GSH values and a high accuracy in prediction. Finally, we used the models to predict GSH values for Holocene peat samples. We found a fair correspondence with expert-based multi-proxy reconstruction of wetness conditions, even though only a minor part of the encountered fossils were represented in the GSH models, illustrating the robustness of the approach. The method can thus be used to reconstruct palaeoenvironmental conditions in a more objective way and can serve as a template for further palaeoecological studies. The 4000 years lasting history of the Lhc11 polygon site started with the establishment of a low-centre polygon in a drained thermokarst lake basin. Polygon Lhc11 formed part of a low-centre polygon for about 2000 years, experiencing enormous environmental influences discernible by incidence of silt, charred detritus, change of fossils composition and strongly declined peat accumulation rates and finally developed into a mature and degradation stage, into a low-high-centre polygon, currently characterized by high elevation differences. In the context of less studied but large-scale polygon mire occurrence, the high-resolution analysed ice-wedge polygon Lhc11 delivers insights into state and dynamics of a representative Siberian polygon site, in terms of modern and past vegetation and elevation characteristics. Furthermore the present study provides facilities for palaeoecological polygon studies including a new quantitative elevation modelling approach and provides valuable datasets for future research, e.g. greenhouse gas emissions and therefore contributes to a better understanding of these climate relevant ecosystems.
Background: This randomized controlled trial investigated if uni- and bihemispheric transcranial direct current stimulation (tDCS) of the motor cortex can enhance the effects of visuo-motor grip force tracking task training and transfer to clinical assessments of upper extremity motor function.
Methods: In a randomized, double-blind, sham-controlled trial, 40 chronic stroke patients underwent 5 days of visuo-motor grip force tracking task training of the paretic hand with either unilateral or bilateral (N = 15/group) or placebo tDCS (N = 10). Immediate and long-term (3 months) effects on training outcome and motor recovery (Upper Extremity Fugl-Meyer, UE-FM, Wolf Motor Function Test, and WMFT) were investigated.
Results: Trained task performance significantly improved independently of tDCS in a curvilinear fashion. In the anodal stimulation group UE-FM scores were higher than in the sham group at day 5 (adjusted mean difference: 2.6, 95%CI: 0.6–4.5, p = 0.010) and at 3 months follow up (adjusted mean difference: 2.8, 95%CI: 0.8–4.7, p = 0.006). Neither training alone, nor the combination of training and tDCS improved WMFT performance.
Conclusions: Visuo-motor grip force tracking task training can facilitate recovery of upper extremity function. Only minimal add-on effects of anodal but not dual tDCS were observed.
Clinical Trial Registration: https://clinicaltrials.gov/ct2/results?recrs=&cond=&term=NCT01969097&cntry=&state=&city=&dist=, identifier: NCT01969097, retrospectively registered on 25/10/2013.
Abstract
Peatlands are lands with a peat layer at the surface, containing a large proportion of organic carbon. Such lands cover ≈1 000 000 km2 in Europe, which is almost 10% of the total surface area. In many countries, peatlands have been artificially drained over centuries, leading to not only enormous emissions of CO2 but also soil subsidence, mobilization of nutrients, higher flood risks, and loss of biodiversity. These problems can largely be solved by stopping drainage and rewetting the land. Wet peatlands do not release CO2, can potentially sequester carbon, help to improve water quality, provide habitat for rare and threatened biodiversity, and can still be used for production of biomass (“paludiculture”). Wisely adjusted land use on peatlands can substantially contribute to low‐emission goals and further benefits for farmers, the economy, society, and the environment.
Abstract
Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.
Overall, the present thesis provides tools for virus characterization. Importantly, the application of the developed tools contributed to the fundamental knowledge of selected, veterinary relevant viruses in terms of their underlying biology and virus-host interaction.
By using in vitro models and full-genome sequencing, important new findings were gained that contributed to the deeper understanding of the selected viruses. Results show that in vitro models can be successfully modified to enable study of specific host factors that are important for viral entry. By genetically modifying a bovine cell line using CRISPR/CAS9 technology , a stable cell culture model was established that is now available to the research community, to study the virus-host interaction of pestiviruses. The model was further used to elucidate the adaptability of bovine viral diarrhea the virus and impact on infectivity and growth. By using deep sequencing, genetic changes that occurred during the adaption process of bovine viral diarrhea virus were identified and linked to the phenotype, allowing the characterization of genetic regions important for virus binding to the host cell.
Whole-genome analysis using deep-sequencing was further used to characterize circulating rabbit haemorrhagic disease virus (RHDV) strains from Germany. The study provides more than 50 full genomes of RHDV strains sampled between 2013 and 2020. Since the virus family is drastically under sampled, in particular in central Europe, these sequences represent a very valuable addition to the field. The investigation led further to the discovery of a novel recombinant virus strain in hares, that is likely still circulating today. This finding is of special interest, since it is the first detection of a recombination event between the genogroups RHDV and European brown hare syndrome virus (EBHSV) of Lagoviruses. It highlights the importance of full genome virus surveillance and the potential risk of virus variants that might evade diagnostic detection.
Serological assay were used to study the persistency of antibodies developed during a natural infection with Schmallenberg virus. It could be shown that these antibodies are long lasting and therefore, re-emergence of this virus in Europe is likely favoured by introduction of naïve animals into a herd and not by decreasing antibody-titers over time.
Overall, the discoveries described in this thesis underline the importance of adequate tools for virus characterization and they give valuable answers to fundamental questions regarding the biology of the different viruses.
inhibiting fear-related thoughts and defensive behaviors when they are no longer appropriate to the situation is a prerequisite for flexible and adaptive responding to changing environments. Such inhibition of defensive systems is mediated by ventromedial prefrontal cortex (vmpfc), limbic basolateral amygdala (BLA), and brain stem locus-coeruleus noradrenergic system (Lc-nAs). non-invasive, transcutaneous vagus nerve stimulation (tVnS) has shown to activate this circuit. Using a multiple-day single-cue fear conditioning and extinction paradigm, we investigated long-term effects of tVnS on inhibition of low-level amygdala modulated fear potentiated startle and cognitive risk assessments. We found that administration of tVnS during extinction training facilitated inhibition of fear potentiated startle responses and cognitive risk assessments, resulting in facilitated formation, consolidation and long-term recall of extinction memory, and prevention of the return of fear. these findings might indicate new ways to increase the efficacy of exposure-based treatments of anxiety disorders.
Purpose:
To examine the prevalence of the so-called bovine aortic arch variation (common origin of the brachiocephalic trunk and the left common carotid artery) in embolic stroke patients, compared with a control group.
Methods:
Aortic arch branching patterns were retrospectively evaluated in 474 individuals with (n = 152) and without (n = 322) acute embolic stroke of the anterior circulation. Contrast-enhanced CT scans of the chest and neck (arterial contrast phase, 1–2- mm slice thickness) were used to evaluate aortic arch anatomy. The stroke cohort included 152 patients who were treated for embolic strokes of the anterior circulation between 2008 and 2018. A total of 322 randomly selected patients who had received thoracic CT angiographies within the same time frame were included as a control group.
Results:
With a prevalence of 25.7%, the bovine aortic arch variant was significantly more common among patients suffering from embolic strokes, compared with 17.1% of control patients (p = 0.039, OR = 1.67, 95%CI = 1.05–1.97). Stroke patients were more likely to show the bovine arch subtype B (left common carotid artery originating from the brachiocephalic trunk instead of the aortic arch) (10.5% vs. 5.0%, p = 0.039, OR = 2.25, 95%CI = 1.09–4.63), while subtype A (V-shaped common aortic origin of the brachiocephalic trunk and the left carotid) was similarly common in both groups. There was no significant difference regarding the frequency of other commonly observed variant branching patterns of the aortic arch.
Conclusion:
The bovine aortic arch, particularly the bovine arch subtype B, was significantly more common among embolic stroke patients. This might be due to altered hemodynamic properties within the bovine arch.
The aim of our research is a stereoselective synthesis development of 4-aminocyclohexanol by the application of a keto reductase (KRED) and an amine transaminase (ATA). 4-Aminocyclohexanol is a valuable precursor for active pharmaceutical ingredients, for example, lomibuvir (a HCV protease inhibitor), ambroxol (a secretolytic agent) and other bioactive molecules. Today, the trans-4-aminocyclohexanol is accessed via Ni-catalyzed synthetic procedure giving moderate yields. In our project we perform cis- and trans-4-aminocyclohexanol synthesis from 1,4-cyclohexanedione (a bio-based precursor) by an one-pot approach combining sequentially a KRED and an ATA as catalysts. For this, we envisaged two multistep enzymatic procedures. The route A would involve 4-hydroxycyclohexanone formation from 1,4-cyclohexanedione via a KRED-catalyzed monoreduction and a further transamination mediated by an ATA towards 4-aminocyclohexanol. The route B would consist of switching the steps of the previous sequential approach, that is, a monoamination of the diketone to yield 4-aminocyclohexanone, and the subsequent reduction of the remaining carbonyl group. Only route A turned out to be feasible, and we performed 4-aminocyclohexanol synthesis at the preparative scale in the sequential and tandem modes. Depending on the ATA, both isomers can be obtained.
Background
In combination with systematic routine screening, brief alcohol interventions have the potential to promote population health. Little is known on the optimal screening interval. Therefore, this study pursued 2 research questions: (i) How stable are screening results for at‐risk drinking over 12 months? (ii) Can the transition from low‐risk to at‐risk drinking be predicted by gender, age, school education, employment, or past week alcohol use?
Methods
A sample of 831 adults (55% female; mean age = 30.8 years) from the general population was assessed 4 times over 12 months. The Alcohol Use Disorders Identification Test—Consumption was used to screen for at‐risk drinking each time. Participants were categorized either as low‐risk or at‐risk drinkers at baseline, 3, 6, and 12 months later. Stable and instable risk status trajectories were analyzed descriptively and graphically. Transitioning from low‐risk drinking at baseline to at‐risk drinking at any follow‐up was predicted using a logistic regression model.
Results
Consistent screening results over time were observed in 509 participants (61%). Of all baseline low‐risk drinkers, 113 (21%) received a positive screening result in 1 or more follow‐up assessments. Females (vs. males; OR = 1.66; 95% confidence intervals [95% CI] = 1.04; 2.64), 18‐ to 29‐year‐olds (vs. 30‐ to 45‐year‐olds; OR = 2.30; 95% CI = 1.26; 4.20), and those reporting 2 or more drinking days (vs. less than 2; OR = 3.11; 95% CI = 1.93; 5.01) and heavy episodic drinking (vs. none; OR = 2.35; 95% CI = 1.06; 5.20) in the week prior to the baseline assessment had increased odds for a transition to at‐risk drinking.
Conclusions
Our findings suggest that the widely used time frame of 1 year may be ambiguous regarding the screening for at‐risk alcohol use although generalizability may be limited due to higher‐educated people being overrepresented in our sample.
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Acquired inherited and/or somaticmutations drive its development. In order to prevent the formation of these mutations, precise and immediaterepair of any DNA damage is indispensable. Non-homologous end-joining (NHEJ) is the key mechanism of DNAdouble-strand break repair. Here, we report that miR-502 targets two components in pancreatic cell lines, Ku70and XLF of the C-NHEJ. Interestingly, we also observed an attenuated cell cycle response to gamma ionizingradiation (γ-IR) via diminished phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 in these cell lines.Altogether, pancreatic cells showed increased susceptibility toγ-IR via direct inhibition of DNA double-strandbreak repair and attenuation of the cell cycle response.
The non-renewable energy sources coal, oil and natural gas that contribute the major share of the world's energy, will be running out in the next 40-80 years. With the growing energy demands especially in developing countries, which is likely to surpass that of the developed countries in next 50 years, an alternate energy source is the need to the hour. The nuclear fusion energy is foreseen as one of the potential candidates to solve the current global energy crisis. One of the major challenges faced by the fusion community is the problem of power exhaust. With the larger fusion devices to be built in the future, the heat load on the plasma facing components are expected to grow higher. The present work explores two numerical studies performed on the Wendelstein 7-X, the world's largest stellarator type fusion device, to cope with this problem.
The first project on `'Numerical Studies on the impact of Connection Length in Wendelstein 7-X'' identifies magnetic configuration with long connection lengths, which could bring down the peak heat fluxes onto the divertor to manageable levels, by greater role of cross-field transport which may assist to get a wider heat deposition profile. The second project on `'Development of Heating Scenario to Reduce the Impact of Bootstrap Currents in Wendelstein 7-X'' advocates a novel self-consistent approach to reach high plasma density at full heating power without overloading the divertor during the transient phase of the evolution of the toroidal plasma current, by controlling two parameters; density and power. The aim of both the projects is to contribute to tackling the challenge of the tremendous power exhaust from fusion plasma which, if solved, will be a large step closer to a fusion power plant.
Objective
Congenital hyperinsulinism (CHI) is a rare disease characterized by persistent hypoglycemia as a result of inappropriate insulin secretion, which can lead to irreversible neurological defects in infants. Poor efficacy and strong adverse effects of the current medications impede successful treatment. The aim of the study was to investigate new approaches to silence β-cells and thus attenuate insulin secretion.
Research Design and Methods
In the scope of our research, we tested substances more selective and more potent than the gold standard diazoxide that also interact with neuroendocrine ATP-sensitive K+ (KATP) channels. Additionally, KATP channel-independent targets as Ca2+-activated K+ channels of intermediate conductance (KCa3.1) and L-type Ca2+ channels were investigated. Experiments were performed using human islet cell clusters isolated from tissue of CHI patients (histologically classified as pathological) and islet cell clusters obtained from C57BL/6N (WT) or SUR1 knockout (SUR1-/-) mice. The cytosolic Ca2+ concentration ([Ca2+]c) was used as a parameter for the pathway regulated by electrical activity and was determined by fura-2 fluorescence. The mitochondrial membrane potential (ΔΨ) was determined by rhodamine 123 fluorescence and single channel currents were measured by the patch-clamp technique.
Results
The selective KATP channel opener NN414 (5 µM) diminished [Ca2+]c in isolated human CHI islet cell clusters and WT mouse islet cell clusters stimulated with 10 mM glucose. In islet cell clusters lacking functional KATP channels (SUR1-/-) the drug was without effect. VU0071063 (30 µM), another KATP channel opener considered to be selective, lowered [Ca2+]c in human CHI islet cell clusters. The compound was also effective in islet cell clusters from SUR1-/- mice, showing that [Ca2+]c is influenced by additional effects besides KATP channels. Contrasting to NN414, the drug depolarized ΔΨ in murine islet cell clusters pointing to severe interference with mitochondrial metabolism. An opener of KCa3.1 channels, DCEBIO (100 µM), significantly decreased [Ca2+]c in SUR1-/- and human CHI islet cell clusters. To target L-type Ca2+ channels we tested two already approved drugs, dextromethorphan (DXM) and simvastatin. DXM (100 µM) efficiently diminished [Ca2+]c in stimulated human CHI islet cell clusters as well as in stimulated SUR1-/- islet cell clusters. Similar effects on [Ca2+]c were observed in experiments with simvastatin (7.2 µM).
Conclusions
NN414 seems to provide a good alternative to the currently used KATP channel opener diazoxide. Targeting KCa3.1 channels by channel openers or L-type Ca2+ channels by DXM or simvastatin might be valuable approaches for treatment of CHI caused by mutations of KATP channels not sensitive to KATP channel openers.
The global prevalence of kidney diseases has been steadily rising over the last decades. Today, around 10% of the world population suffers from relevant chronic kidney disease. Podocytes are highly specialized and terminally differentiated cells residing in the filtering units of the kidneys, the so-called glomeruli. With their interdigitating foot-processes, these cells are a crucial part of the renal filtration barrier. As podocytes are post-mitotic, injury or loss of these cells results in an impairment of the filtration barrier with subsequent loss of global kidney function. Therefore, the question whether a relevant amount of podocytes can be regenerated and if this regeneration can be influenced is crucial for future therapeutic developments. As in vivo microscopic imaging of podocytes in higher animals like mice or rats is rather challenging, larval zebrafish have been applied as an animal model for podocyte development and kidney filtration. 48 hours post fertilization, zebrafish larvae develop a single filtering glomerulus with a similar morphology and molecular construction to that in mammals. For evaluation of podocyte morphology and filtration, we used transgenic zebrafish strains in which podocytes were labeled with fluorescence proteins. Additionally, podocytes expressed the bacterial enzyme nitroreductase fused to the fluorescence protein mCherry. In this model, application of the antibiotic metronidazole leads to podocyte-specific cell death. Through cross-breeding we established strains that additionally express an eGFP-labeled protein in the blood plasma. Using in vivo two-photon microscopy, we could image podocyte-loss induced impairments of the glomerular filtration barrier. Additionally, we tracked characteristic morphological changes of podocyte morphology including podocyte foot process effacement, development of sub-podocyte pseudocysts and finally detachment of whole cells from the glomerular basement membrane. These changes have been before described histologically in different animal models as well as in patient biopsies. Using the in vivo microscopy approach, we could clearly describe the temporal sequence of these alterations. Finally, we also tracked individual, non-detached podocytes over up to 24 hours, and found that these cells were non-migratory. These results show that early podocyte-regeneration through immigration of intra- or extraglomerular cells is unlikely within the first 24 hours of acute glomerular injury.
Reactive species play an essential role in orchestrating wound healing responses. They act as secondary messengers and drive redox-signaling pathways that are involved in the hemostatic, inflammatory, proliferative and remodeling phases of wound healing. Cold plasma produces a profusion of short- and long-lived redox species that promotes wound healing, however, until today, the knowledge of CAP mediated wound healing remained scarce. In this thesis, CAP mediated wound healing mechanism and their effect on extracellular matrix and adhesion molecules have been investigated. To this end, a keratinocyte cell line (HaCaT), skin fibroblast cell line (GM Fbs) and an in vitro coculture model including both HaCaT and GM Fbs at a 2:1 ratio, were employed to investigate the cross talk between these two skin cell types.
We examined the impact of CAP on extracellular matrix proteins and cell adhesion molecules in GM Fbs and observed a significant impact of cold plasma treatment on the expression level of collagen moieties, cell adhesion molecule like integrin, cadherin, versican, MMPs as well as extracellular matrix proteins.
Moreover, scratch assays with monocultures of HaCaT, GM Fbs and coculture of these two cell types were performed. We detected that, CAP accelerated the migratory capability of HaCaT cells cocultured with fibroblasts. In fact, compared to HaCaT monoculture, a significant acceleration on cell migration was observed in coculture upon CAP treatment. NAC, a potent antioxidant could abrogate this CAP-stimulated cell migration in coculture, further pointing towards the importance of well-orchestrated reactive species in wound healing. To better understand this CAP-mediated effect on cell migration, we examined the signaling pathways involved in tissue homeostasis and regeneration. We checked the HIPPO signaling pathway and observed an upregulation of several signaling molecules at transcriptional level in GM Fbs upon CAP treatment.
YAP is the central nuclear executer of HIPPO signaling pathway. YAP was upregulated in both HaCaT cells and GM Fbs. The major downstream effectors of the HIPPO signaling pathway (CTGF and Cyr61) were also upregulated in dermal fibroblasts at both transcriptional and protein level. However, administration of antioxidant NAC inhibited CAP-mediated wound healing and abrogated the gene expression of the HIPPO downstream effectors. These results confirm that the upregulation of YAP-CTGF-CYR61 axis is due to CAP-generated redox species. In HaCaT cells, both CTGF and Cyr61 was minimally transcribed. Even though CTGF was rarely detected in HaCaT cells on the protein level,Cyr61 remained undetected. This again shows the importance of the cross talk between fibroblasts and keratinocytes.
The coculture with the inclusion of fibroblasts showed an accelerated migration rate, compared to HaCaT monoculture which specifies a cross talk between these two cell types. Thus, monoculture of HaCaT cells were incubated with CAP-treated and untreated fibroblast conditioned medium. Interestingly, we observed that HaCaT cells exhibited an improved cell migration rate when incubated with CAP-treated fibroblast-conditioned media compared to that observed after incubation with untreated media. Upon investigation, an induction of CTGF and Cyr61 secretion was observed upon CAP treatment in the fibroblast-conditioned media. Furthermore, exposure to recombinant CTGF and Cyr61 could also significantly improve HaCaT cell migration which confirms that CAP mediated accelerated cell migration is due to activation of YAP-CTGF-Cyr61 axis.
In conclusion, this study revealed a completely new mechanical insight of CAP mediated wound healing. Along with several other ECM molecules, CAP activates a regenerative signaling pathway i.e., HIPPO signaling pathway in dermal fibroblasts at the onset of wound healing. Dermal fibroblasts drive a paracrine interaction by secreting CTGF and Cyr61 in close vicinity of wound, resulting in accelerated keratinocyte migration and wound healing in coculture.