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The collisionless tearing mode is investigated by means of the delta-f PIC code EUTERPE solving the gyrokinetic equation. In this thesis the first simulations of electromagnetic non-ideal MHD modes in a slab geometry with EUTERPE are presented. Linear simulations are carried out in the cases of vanishing and finite temperature gradients. Both cases are benchmarked using a shooting method showing that EUTERPE simulates the linearly unstable tearing mode to a very high accuracy. In the case of finite diamagnetic effects and values of the linear stability parameter Delta of order unity analytic predictions of the linear dispersion relation are compared with simulation results. The comparison validates the analytic results in this parameter range. Nonlinear single-mode simulations are performed in the small- to medium-Delta range measuring the dependency of the saturated island half width on the equilibrium current width. The results are compared with an analytic prediction obtained with a kinetic electromagnetic model. In this thesis the first simulation results in the regime of fast nonlinear reconnection~(medium- to high-Delta range) are presented using the standard gyrokinetic equation. In this regime a nonlinear critical threshold has been found dividing the saturated mode from the super-exponential phase for medium-Delta values. This critical threshold has been proven to occur in two slab equilibria frequently used for reconnection scenarios. Either changing the width of the equilibrium current or the wave number of the most unstable mode makes the threshold apparent. Extensive parameter studies including the variation of the domain extensions as well as the equilibrium current width are dedicated to a comprehensive overview of the critical threshold in a wide range of parameters. Additionally, a second critical threshold for high-Delta equilibria has been observed. A detailed comparison between a compressible gyrofluid code and EUTERPE is carried out. The two models are compared with each other in the linear regime by measuring growth rates over wave numbers of the most unstable mode for two setups of parameters. Analytical scaling predictions of the dispersion relation relevant to the low-Delta regime are discussed. Employing nonlinear simulations of both codes the saturated island half width and oscillation frequency of the magnetic islands are compared in the small-Delta range. Both models agree very well in the limit of marginal instability and differ slightly with decreasing wave vector. Recently, the full polarisation response in the quasi-neutrality equation was implemented in EUTERPE using the Padé approximation of the full gyrokinetic polarisation term. Linear simulation results including finite ratios of ion to electron temperature are benchmarked with the dispersion relation obtained from a hybrid model. Finite temperature effects influence the saturated island width slightly with increasing ion to electron temperature ratio which has been verified by both models.
Abstract
Climate change is increasing the frequency and intensity of drought events in many boreal forests. Trees are sessile organisms with a long generation time, which makes them vulnerable to fast climate change and hinders fast adaptations. Therefore, it is important to know how forests cope with drought stress and to explore the genetic basis of these reactions. We investigated three natural populations of white spruce (Picea glauca) in Alaska, located at one drought‐limited and two cold‐limited treelines with a paired plot design of one forest and one treeline plot. We obtained individual increment cores from 458 trees and climate data to assess dendrophenotypes, in particular the growth reaction to drought stress. To explore the genetic basis of these dendrophenotypes, we genotyped the individual trees at 3000 single nucleotide polymorphisms in candidate genes and performed genotype–phenotype association analysis using linear mixed models and Bayesian sparse linear mixed models. Growth reaction to drought stress differed in contrasting treeline populations. Therefore, the populations are likely to be unevenly affected by climate change. We identified 40 genes associated with dendrophenotypic traits that differed among the treeline populations. Most genes were identified in the drought‐limited site, indicating comparatively strong selection pressure of drought‐tolerant phenotypes. Contrasting patterns of drought‐associated genes among sampled sites and in comparison to Canadian populations in a previous study suggest that drought adaptation acts on a local scale. Our results highlight genes that are associated with wood traits which in turn are critical for the establishment and persistence of future forests under climate change.
Forests influence the climate of our Earth and provide habitat and food for many species and resources for human use. These valuable ecosystems are threatened by fast environmental changes caused by human-induced climte change. Negative growth responses and higher tree mortality rates were associated with increasing physiological stress induced by global warming. Especially boreal forests at high latitudes in the arctic region are threatened, a region predicted to undergo the highest increase in temperature during the next decades. Therefore, it is important to assess the adaptation potential in trees. For this purpose, I studied natural populations of white spruce (Picea glauca (Moench) Voss) in Alaska. In this thesis, I present three scientific papers in which my co-authors and I studied the phenotypic plasticity and genetic basis of tree growth, wood anatomy and drought tolerance as well as the genetic structure of white spruce populations in contrasting environments. We established three sites representing two cold-limited treelines and one drought-limited treeline with a paired plot design including one plot located at the treeline and one plot located in a closed-canopy forest, respectively. Additionally, the study design included one forest plot as reference. Within the entire project, in total 3,000 trees were measured, genotyped and dendrochronological data was obtained. I used several approaches to estimate the neutral and adaptive genetic diversity and phenotypic plasticity of white spruce as a model organism to explore the adaptation potential of trees to climate change.
In the first chapter, I combined neutral genetic markers with dendrochronological and climatic data to investigate population structure and individual growth of white spruce. Several individual-based dendrochronological approaches were applied to test the influence of genetic similarity and microenvironment on growth performance. The white spruce populations of the different sites showed high gene flow and high genetic diversity within and low genetic differentiation among populations, rather explained by geographic distance. The individual growth performances showed a high plasticity rather influenced by microenvironment than genetic similarity.
In the second chapter, I investigated the populations of the drought and cold-limited treeline sites to decipher the underlying genetic structure of drought tolerance using different genotype-phenotype association analyses. Based on tree-ring series and climatic data, growth declines caused by drought stress were identified and the individual reaction to the drought stress event was determined. A subset of 458 trees was genotyped, using SNPs in candidate genes and associated with the individual drought response. Most of the associations were revealed by an approach which took into account small-effect size SNPs and their interactions. Populations of the contrasting treelines responded differently to drought stress events. Populations further showed divergent genetic structures associated with drought responsive traits, most of them in the drought-limited site, indicating divergent selection pressure.
In the third chapter, my co-authors and I studied xylem anatomical traits at one of the cold-limited treeline sites to investigate whether genetic or spatial grouping affected the anatomy and growth of white spruce. Annual growth and xylem anatomy were compared between spatial groups and between genetic groups and individuals. Overall, wood traits were rather influenced by spatial than genetic grouping. Genetic effects were only found in earlywood hydraulic diameter and latewood density. Environmental conditions indirectly influenced traits related to water transport.
In conclusion, white spruce showed a high genetic diversity within and a low genetic differentiation among populations influenced by high gene flow rates. Genetic differences among populations are rather caused by geographical distance and therefore genetic drift. Differing selection pressure at the treeline ecotones presumably lead to divergent genetic structures underlying drought-tolerant phenotypes among the populations. Thus, adaptation to drought most likely acts on a local scale and involves small frequency shifts in several interacting genes. The identified genes with adaptive growth traits can be used to further exlore local adaptation in white spruce. Tree growth and wood anatomical traits are rather influenced by the environment than genetics and showed a high phentoypic plasticity. The high genetic diverstiy and phenotypic plasticity of white spruce may help the species to cope with rapid environmental changes. Still, additional work is needed to further explore adaptation processes to estimate how tree species reacted to rapid climate change. The presented thesis shed some light on the adaptation potential of trees by the example of white spruce using several approaches.
Objective: To compare the effectiveness and complications of intraligamentary anesthesia
(ILA) with conventional inferior alveolar nerve block (IANB) during injection and dental
treatment of mandibular posterior teeth.
Materials and Methods: In this randomized, prospective clinical trial, 72 patients (39 males, 33
females) patients scheduled for dental treatment of mandibular posterior teeth, were randomly
allocated to ILA group (n=35) received ILA injection or IANB group (n=37) received the
conventional IANB. Our primary outcome was to assess pain and stress (discomfort) during the
injection and dental treatment, using the Numeric Rating Scale (NRS) from 0 to 10 (0 = no
pain, 10= the worst pain imaginable). Whereas; recording 24 hours postoperative complications
were our Secondary outcomes.
Results: Patients in ILA group reported significantly less pain during injection when compared
with IANB group (p=0.03). While pain during dental treatment was similar in both groups
(p=0.2). Patients in both groups also reported similar low values of discomfort during treatment
(p= 0.7). Although no signs of nerve contact or any other postoperative complications were
observed, five patients in IANB group (none in ILA group) reported temporary irritations
Conclusion: This study showed equivalent effectiveness of both intraligamentary anesthesia
and conventional inferior alveolar nerve block, for pain control during routine dental treatment
of mandibular posterior teeth. Nevertheless, ILA showed significantly less pain during
injection. No major postoperative complications in both groups were observed.
Clinical Relevance: ILA could be considered as an effective alternative for routine dental
treatment.
Proteasomes comprise a family of proteasomal complexes essential for maintaining protein homeostasis. Accordingly, proteasomes represent promising therapeutic targets in multiple human diseases. Several proteasome inhibitors are approved for treating hematological cancers. However, their side effects impede their efficacy and broader therapeutic applications. Therefore, understanding the biology of the different proteasome complexes present in the cell is crucial for developing tailor-made inhibitors against specific proteasome complexes. Here, we will discuss the structure, biology, and function of the alternative Proteasome Activator 200 (PA200), also known as PSME4, and summarize the current evidence for its dysregulation in different human diseases. We hereby aim to stimulate research on this enigmatic proteasome regulator that has the potential to serve as a therapeutic target in cancer.
Polyethylene terephthalate (PET) is a mass-produced petroleum-based non-biodegradable plastic that contributes to the global plastic pollution. Recently, biocatalytic degradation has emerged as a viable recycling approach for PET waste, especially with thermophilic polyester hydrolases such as a cutinase (LCC) isolated from a leaf-branch compost metagenome and its variants. To improve the enzymatic PET hydrolysis performance, we fused a chitin-binding domain (ChBD) from Chitinolyticbacter meiyuanensis SYBC-H1 to the C-terminus of the previously reported LCCICCG variant, demonstrating higher adsorption to PET substrates and, as a result, improved degradation performance by up to 19.6% compared to with its precursor enzyme without the binding module. For compare hydrolysis with different binding module, the catalytic activity of LCCICCG-ChBD, LCCICCG-CBM, LCCICCG-PBM and LCCICCG-HFB4 were further investigated with PET substrates of various crystallinity and it showed measurable activity on high crystalline PET with 40% crystallinity. These results indicated that fusing a polymer-binding module to LCCICCG is a promising method stimulating the enzymatic hydrolysis of PET.
The utilization of fluorescein-guided biopsies has recently been discussed to improve and expedite operative techniques in the detection of tumor-positive tissue, as well as to avoid making sampling errors. In this study, we aimed to report our experience with fluorescein-guided biopsies and elucidate distribution patterns in different histopathological diagnoses in order to develop strategies to increase the efficiency and accuracy of this technique. We report on 45 fluorescence-guided stereotactic biopsies in 44 patients (15 female, 29 male) at our institution from March 2016 to March 2021, including 25 frame-based stereotactic biopsies and 20 frameless image-guided biopsies using VarioGuide®. A total number of 347 biopsy samples with a median of 8 samples (range: 4–18) per patient were evaluated for intraoperative fluorescein uptake and correlated to definitive histopathology. The median age at surgery was 63 years (range: 18–87). Of the acquired specimens, 63% were fluorescein positive. Final histopathology included glioblastoma (n = 16), B-cell non-Hodgkin lymphoma (n = 10), astrocytoma, IDH-mutant WHO grade III (n = 6), astrocytoma, IDH-mutant WHO grade II (n = 1), oligodendroglioma, IDH-mutant and 1p/19q-codeleted WHO grade II (n = 2), reactive CNS tissue/inflammation (n = 4), post-transplantation lymphoproliferative disorder (PTLD; n = 2), ependymoma (n = 1), infection (toxoplasmosis; n = 1), multiple sclerosis (n = 1), and metastasis (n = 1). The sensitivity for high-grade gliomas was 85%, and the specificity was 70%. For contrast-enhancing lesions, the specificity of fluorescein was 84%. The number needed to sample for contrast-enhancing lesions was three, and the overall number needed to sample for final histopathological diagnosis was five. Interestingly, in the astrocytoma, IDH-mutant WHO grade III group, 22/46 (48%) demonstrated fluorescein uptake despite no evidence for gadolinium uptake, and 73% of these were tumor-positive. In our patient series, fluorescein-guided stereotactic biopsy increases the likelihood of definitive neuropathological diagnosis, and the number needed to sample can be reduced by 50% in contrast-enhancing lesions.
Ribozymes for Aminoacylation
(2012)
Aminoacyl-tRNA synthetases (aaRS) are at the heart of modern translation, catalyzing the accurate biosynthesis of aminoacyl-tRNAs. According to the RNA world hypothesis, the early translation system should have aminoacylation ribozymes for RNA aminoacylation. For this, an aaRS ribozyme system, consisting of the KK13 ribozyme and the C3a ribozyme was successfully designed, which can perform both amino acid activation and aminoacyl transfer reaction. Generation of such aminoacylation ribozyme system would fill up the gap between the RNA world and the modern biological world. In addition, two types of diversified aminoacylation ribozymes, symmetrical ribozymes and self-assembling ribozymes were successfully developed, which may have great meaning in the origin of life.
Sturgeons are among the most ancient linages of actinopterygians. At present, many sturgeon species are critically endangered. Surrogate production could be used as an affordable and a time-efficient method for endangered sturgeons. Our study established a method for identifying and isolating type A spermatogonia from different developmental stages of testes using flow cytometric cell sorting (FCM). Flow cytometric analysis of a whole testicular cell suspension showed several well-distinguished cell populations formed according to different values of light scatter parameters. FCM of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. Results showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter and high side scatter parameter values) contains the highest amount of type A spermatogonia. The sorted cell populations were characterized by expression profiles of 10 germ cell specific genes. The result confirmed that setting up for the P1 gate could precisely sort type A spermatogonia in all tested testicular developmental stages. The P2 gate, which was with lower forward scatter and side scatter values mostly, contained type B spermatogonia at a later maturing stage. Moreover, expressions of plzf, dnd, boule, and kitr were significantly higher in type A spermatogonia than in later developed germ cells. In addition, plzf was firstly found as a reliable marker to identify type A spermatogonia, which filled the gap of identification of spermatogonial stem cells in sterlet. It is expected to increase the efficiency of germ stem cell culture and transplantation with plzf identification. Our study thus first addressed a phenotypic characterization of a pure type A spermatogonia population in sterlet. FCM strategy can improve the production of sturgeons with surrogate broodstock and further the analysis of the cellular and molecular mechanisms of sturgeon germ cell development.
The glioblastoma multiforme (GBM) not only presents the most common tumor of the central nervous system in adults, it is also the most aggressive brain tumor. Although patients suffering from GBM standardly receive a combination of multiple treatments including surgery, radiotherapy and chemotherapy, its prognosis is still poor with a median survival time of only 12-15 months. Therefore, new and effective treatment methods are urgently needed.
A signaling molecule which is both involved in proliferation, migration and invasion of a broad range of healthy and malignant cells is the lipid mediator sphingosine-1-phosphate (S1P). Previous studies have confirmed that sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) is involved in the regulation of proliferation, invasion, metastasis, vascular maturation and angiogenesis of GBM cells, and is closely related to the occurrence and development of tumors. Thus, ACT-209905 (provided by Actelion Pharmaceuticals) as a selective S1PR1 modulator was applied to gain insights into the molecular processes activated by S1PR1 in GBM cells using two human (LN18, U87MG) and one murine (GL261) GBM cell line.
In our in vitro cell viability analyses, we found that ACT-209905 significantly reduced viability of LN18 cells in a concentration dependent manner. A combined administration of ACT-209905 with S1PR2 inhibitors (Compound 16, Compound 16ME – both provided by ONO Pharmaceuticals, and JTE-013 – commercially available) showed a stronger effect than the single administration demonstrating that both S1PR1 and S1PR2 are involved in growth of GBM cells and may interact with each other. Our results also demonstrated that ACT-209905 can induce apoptosis in GBM cells since caspase 3 activity was induced by the S1PR1 modulator which might therefore play an important role in inhibiting the proliferation of GBM cells. Further, we found a significant inhibitory effect of ACT-209905 on the migration and invasion of LN18 and U87MG GBM cells arguing for a participation of S1PR1 signaling in migration and invasion of GBM cells, too. Stimulation of S1P receptors results in the activation of several kinases such as AKT1 and ERK1/2, correspondingly our immunoblot analyses showed a strong activation of both kinases by S1P which was reduced by ACT-209905 in LN18 cells but not in GL261 cells suggesting that different pathways are activated by S1P in these GBM cell lines. Further studies have to be performed to clarify the role of AKT1 and ERK1/2 in the inhibitory effects of ACT-209905 on GBM proliferation, migration and invasion.
Currently, GBM stem cells are discussed as a reason for resistance against the radiochemotherapy and the recurrence of the tumor. Our immunoblot analyses showed that Nestin and CD133, two marker proteins for GBM stem cells, were higher expressed in GBM cells treated with ACT-209905 compared to control or S1P treated LN18 cells. Further investigations in the future might contribute to the elucidation of an involvement of the S1P receptors in the stem cell behavior of GBM cells. Paradoxically to the up-regulation of CD133 and Nestin by ACT-209905, treatment of LN18 stem-like neurospheres with ACT-209905 showed a significant cytotoxic effect of the compound which was even more pronounced in the stem-like neurosphere cells compared to the adherent parental LN18 cells.
Overall, the studies of this work improve our understanding of the complex mechanisms of S1P signaling in GBM cells and might drive the development of its pharmacological modulation as a new therapeutic principle in GBM. Furthermore, an extended knowledge about the molecular effects of ACT-209905 on GBM cells will broaden the understanding for possible future applications and clinical indications.
Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of (R)-selective transaminases were rationally designed, produced in E. coli, purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus, for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of (R)-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme.
With the aim to discover and create suitable biocatalysts for the synthesis of chiral amines in a faster and more efficient way, this thesis includes protein engineering studies (Article I), explores transaminase substrate specificities (Articles II and IV), and an ultrahigh-throughput growth system-based for the directed evolution of amine-forming enzymes (Article III).
The protein engineering studies described in Article I deal with the creation of a (R)-amine transaminase activity in the α-amino acid transaminase scaffold to expand our knowledge of the evolutionary relationship between amine transaminase and α-amino acid transaminase. Article II describes the broadening of the limited substrate scope of transaminases to enable the conversion of bulky substrates. In Article III, a growth selection system is described for an ultra-high throughput screening strategy to accelerate the identification of desired mutants, which can be widely applied to the directed evolution of amine-forming enzymes.
Global climate change is occurring all over the world, but in the Arctic the climate is changing more rapidly and drastically than in many other parts of our planet. Many species that are already at their climatic limit need to adapt to recent climate conditions or migrate in order to not go extinct. The possibilities of adaption include phenotypic plasticity and adaptation to various extents. This is also the case for white spruce P. glauca, which belongs to the conifers and thus in the largest group of gymnosperms still living today. Among the approx. 600 extant conifer species white spruce is one of the most widespread trees in North American boreal forests. Its range extends from 69° N in the Canadian Northwest Territories to the Great Lakes at about 44° N, where it occurs from sea level to an altitude of about 1520 m (Burns and Honkala, 1990). Site related, climate-dependent differences in white spruce reproduction can be seen as a strategy to survive under the harsh climatic conditions at Alaska's treelines: Besides sexual reproduction, the vegetative propagation occurs in the white spruce as an additional reproductive mechanism. This can be realized by "layering" when the lower branches of the tree crown touch the ground and develop roots to later grow as a separate individual with or without a connection to the mother tree. Known as other mechanisms of vegetative propagation are also the rooting of fallen trees which were not completely uprooted, and the "root suckering", in which new shoots sprout from the roots of the tree. However, the latter was not yet observed in the genus Picea. With the help of short, repetitive, non-coding sequences in the genome, which are therefore not subject to selection and are called microsatellites, these clones can be determined by genotyping.
For this purpose, using different polymorphic microsatellites, an individual multilocus genotype is created for each tree, by means of which it can be compared with all other trees of the same species.
In the first part of this work (article I), the occurrence of clones in three study areas at Alaskan treelines are examined and the reasons for their appearance in variable numbers are discussed. For this purpose, 2571 white spruces (P. glauca) were genotyped and their position was determined via differential GPS in the field. The percentage of clonal trees is higher in areas with harsh climatic conditions and correlates with the height of the lowest branches of the tree crown. This suggests that the vegetative propagation of white spruce is a backup strategy for times when climatic conditions hamper sexual reproduction. The correlation between clone numbers and tree crown height suggests "layering" as the main mechanism for cloning whereas selection for vegetative reproduction seems to be very unlikely shown by the results for genetic differentiation between the clonal and the singleton trees in this study.
In the second part of this work (articles II and III), the influence of environmental factors and phenotypic traits on the mycobiome of the needles (including all fungi living on (epiphytic) and in (endophytic) the needles) in our study areas in Alaska was investigated. The mycobiome of the white spruce needles was chosen as a proxy for the parasite infection rate by fungi and thus serves as a fitness parameter. For this purpose, all epiphytic and endophytic fungal species were analyzed by a metabarcoding analysis.
In article II, 48 trees of one study area at Alaska’s northern treeline (Brooks Range) were examined for differences in mycobiome due to genetic differentiation, phenotypic characteristics and / or habitat characteristics. The trees used for this study were sampled from two adjacent plots on a south-facing mountain slope with an elevation gradient from 875 to 950 meters above sea level. It could be shown that, in contrast to the trees genotype, the height above sea level, the mountain slope, as well as the height and age of the trees have a significant impact on the mycobiome. The genetic differentiation between the tree individuals, however, showed no significant effect.
Based on article II we examined the mycobiome composition of a total of 96 trees in 2 plots (16 trees each) at three sites in Alaska over a distance of 500 kilometers. Additionally, we sampled needles of two different ages for each tree (current year and three years old needles) summing up to 192 samples in total. The incentive of this study (article III) was to investigate the influence of origin and age of spruce needles on their mycobiome and if there is a genetic predisposition that is related to the fungal species community. In addition, the sampling design was improved by collecting needles from all four orientations (North, South, East and West) and sampling trees at a standardized distance to each other to avoid systematic errors. Comparable to article II the influence of the trees genetics on the species community of the epiphytic and endophytic fungi of the white spruce needles seems to be very unlikely. In contrast, a significant influence of the geographic origin and the needle age on the species structure of the needle inhabiting fungal species was found. The phenotypic tree traits height and dbh (diameter at breast height) had only minor influence and did in fact explain less than 2% of the mycobiome variance. Using Illumina sequencing, 10.2 million reads from the nucleotide sequence between the internal transcribed spacer (ITS) genes could be obtained, which yielded in 1575 ribotypes (called operational taxonomic unit, OTU) for the fungi. These were compared with a reference database to compare and assign them to known fungal species. For example, 942 OTUs with >95% similarity could be identified as known species, with 1975 samples identified on genus level and 2683 when determined to family level. The most pronounced difference between the two studies (article II and III) were due to the fungal species of the class of Pucciniomycetes, more specifically the genus Chrysomyxa which belongs to the rust fungi and is plant pathogenic. In the study of article II (sampling in 2012), Pucciniomycetes accounted for only a minor portion of the assigned DNA sequences. In the second study (article III, sampling in 2015) they accounted for more than half of all basidiomycetes found, which in turn contain 20.0% of all DNA sequences, the second largest phylum found beside Ascomycetes (51.4%).
Mass spectrometry-based Proteome analysis of porcine cells infected with African swine fever virus
(2023)
ASFV, a highly contagious, pathogenic and lethal pathogen of swine, poses a major threat to domestic and wild suids worldwide as neither vaccines nor treatments are available. Compared to other well-characterized similarly complex viruses like herpesviruses or adenoviruses, the understanding of ASFV biology is poor.
To improve the understanding of ASFV biology, following the establishment of a robust protocol for the isolation of primary monocyte-derived porcine macrophages (moMΦ) and their infection with ASFV for mass spectrometry (MS)-based proteome analysis was performed.
Under both conditions, naïve and infected, the isolated cells showed cell type-specific characteristics like phagocytosis and antigen presentation and protein expression patterns, including the expression of swine leucocyte antigens and CD markers. Furthermore, moMΦ could be reproducibly infected with ASFV isolates of different genotypes and pathogenicity.
The ASFV protein expression patterns in moMΦ correlate well with those observed in established cell lines at transcript and protein level. The expression of 27 ASFV proteins was confirmed at the protein level. Among them, 9 members of multi-gene families (MGF) and 12 novel open reading frames (nORFs) were recently predicted based on transcription start site mapping.
The direct comparison of closely related ASFV genotype II isolates revealed no virulence-associated protein expression patterns beyond those expected based on the genome sequences of the isolates.
Using different MS quantification strategies, it was shown that ASFV affects both static protein expression levels and protein synthesis. These changes in protein expression impact proteins and pathways known to be targeted by ASFV, including CD-markers, ER-stress and cell death pathways, and cellular antiviral responses. Beyond these observations that further validated the moMΦ infection model, novel effects of the ASFV infection on the cellular proteome were noticed.
These effects include the decreased expression levels of cathepsins, especially cathepsins D (CTSD), H (CTSH) and L (CTSL) as well as the transient activation of MAPK14/p38 prior to its strong downregulation. In addition to MAPK14/p38 further members of the MAPK14/p38 signaling pathway, like MAPKAPK2, were affected by ASFV infection.
As these modulations of the cellular proteome would in general result in decreased pro-inflammatory responses, it did stand out that the synthesis of interferon-response related genes including MX1 and ISG15 evaded the ASFV-induced global reduction of protein synthesis. In contrast, the synthesis of genes involved in RNA processing and splicing was significantly impaired. In total, the regulations of individual host proteins assessed in the context of the whole cellular proteome integrate well with each other and other cellular responses to ASFV infection and may help to improve the understanding of host-virus interactions.
Overall, this thesis provides novel insights into the expression of ASFV-encoded ORFs of different isolates and the host response to ASFV infection. It points out that the current knowledge of the ASFV coding capacity, temporal protein expression patterns, protein functionality, post-translational modifications and host interactions is still sketchy as many aspects of ASFV replication have yet to be understood. The established moMΦ-model to study ASFV infections in vitro provides a powerful tool for future applications to increase the understanding of ASFV biology.
Cerebral palsy (CP) remains one of the most common debilitating diseases in the world.
Factors such as neuromuscular incoordination, use of soft diet and delayed oral
clearance of food make maintenance of optimum oral hygiene a challenging task in
these children. The compromised oral hygiene in turn make these children highly
vulnerable to dental disease. Maintaining optimal oral/dental health in CP children is of
utmost importance as these children usually suffer from several associated general
health problems in addition to their primary condition. Poor dental health further
compromises their general health. Nevertheless, CP children often suffer from dental
diseases including dental caries more than healthy children. This underscores the need
for improvement in oral health of the CP children. The CP children are dependent on
those around them for their oral hygiene maintenance and dietary intake. CP children
spend most of their time with health care workers in special centers for them or with
their parents/care takers at home. Therefore, the attitude of special health care workers
and parents towards their dental health is of prime importance. The purpose of this
project included to determine the attitude of special health care workers towards their
dental health and oral health comprehension by parents of CP children. In addition, a
study on caries experience of CP children and associated risk factors was also completed.
The final objective was to identify various potential areas of improvement with ultimate
goal of improving dental health of the CP children.
The study involving special health care workers in a Disabled Children’s Center revealed
that oral health knowledge and practices among the workers could generally be labeled
as satisfactory, however some weak areas (such as trend towards symptom-oriented
utilization of oral health care and lack of knowledge about fluoridated water) were
identified. There is a need to enhance the workers’ knowledge in these areas.
The study involving parents of the CP children showed mixed results. Some areas of
strong oral health comprehension (such as importance of good dental health, harmful
effects of sweetened foods), others satisfactory (such as need for routine dental visits,
use of fluoride) and some weak areas (use of flavored fizzy drinks, bottled/canned juices
and sweetened/flavored milks, seeking early treatment) were identified among parents
of CP children. The results strongly indicated a need for enhanced efforts towards
improvement of oral health comprehension in the parents of the CP children.
The study about caries experience and risk factors in CP children has provided useful
information in an area where literature has been scarce. Almost all the studied children
had clinical dental caries, and most of the caries were untreated. Very few children in
the present study had optimal oral hygiene. This study confirmed the strong correlation
between high dental caries experience and poor oral hygiene. Routine dental check-up
visits and topical fluoride application clearly resulted in lower caries experience. Bottled
juices and crispy potato chips were the two predictors for high caries experience.
As stated before, the goal of the project was to identify various potential areas of
improvement in oral health attitude of special health care workers, oral health
comprehension of the parents of CP children and risk factors for dental caries in CP
children. It is contemplated that by addressing the identified weak areas, an
improvement in dental health and care of the CP children is expected. It is also expected
that the collected information will help in improvement of clinical preventive as well as
restorative services for the CP children. CP children and their parents lead a challenging
life. This project was an effort to assist these children and their parents/care takers with
ultimate goal of improving their oral health and in turn their overall quality of life.
Abstract
The KV7 potassium channel openers flupirtine and retigabine have been valuable options in the therapy of pain and epilepsy. However, as a result of adverse reactions, both drugs are currently no longer in therapeutic use. The flupirtine‐induced liver injury and the retigabine linked tissue discolouration do not appear related at first glance; nevertheless, both events can be attributed to the triaminoaryl scaffold, which is affected by oxidation leading to elusive reactive quinone diimine or azaquinone diimine metabolites. Since the mechanism of action, i. e. KV7 channel opening, seems not to be involved in toxicity, this study aimed to further develop safer replacements for flupirtine and retigabine. In a ligand‐based design strategy, replacing amino substituents of the triaminoaryl core with alkyl substituents led to carba analogues with improved oxidation resistance and negligible risk of quinoid metabolite formation. In addition to these improved safety features, some of the novel analogues exhibited significantly improved KV7.2/3 channel opening activity, indicated by an up to 13‐fold increase in potency and an efficacy of up to 176 % compared to flupirtine, thus being attractive candidates for further development.
KV7 channel openers have proven their therapeutic value in the treatment of pain as well as epilepsy and, moreover, they hold the potential to expand into additional indications with unmet medical needs. However, the clinically validated but meanwhile discontinued KV7 channel openers flupirtine and retigabine bear an oxidation‐sensitive triaminoraryl scaffold, which is suspected of causing adverse drug reactions via the formation of quinoid oxidation products. Here, we report the design and synthesis of nicotinamide analogs and related compounds that remediate the liability in the chemical structure of flupirtine and retigabine. Optimization of a nicotinamide lead structure yielded analogs with excellent KV7.2/3 opening activity, as evidenced by EC50 values approaching the single‐digit nanomolar range. On the other hand, weighted KV7.2/3 opening activity data including inactive compounds allowed for the establishment of structure–activity relationships and a plausible binding mode hypothesis verified by docking and molecular dynamics simulations.
The potassium channel opening drugs flupirtine and retigabine have been withdrawn from the market due to occasional drug-induced liver injury (DILI) and tissue discoloration, respectively. While the mechanism underlying DILI after prolonged flupirtine use is not entirely understood, evidence indicates that both drugs are metabolized in an initial step to reactive ortho- and/or para-azaquinone diimines or ortho- and/or para-quinone diimines, respectively. Aiming to develop safer alternatives for the treatment of pain and epilepsy, we have attempted to separate activity from toxicity by employing a drug design strategy of avoiding the detrimental oxidation of the central aromatic ring by shifting oxidation toward the formation of benign metabolites. In the present investigation, an alternative retrometabolic design strategy was followed. The nitrogen atom, which could be involved in the formation of both ortho- or para-quinone diimines of the lead structures, was shifted away from the central ring, yielding a substitution pattern with nitrogen substituents in the meta position only. Evaluation of KV7.2/3 opening activity of the 11 new specially designed derivatives revealed surprisingly steep structure–activity relationship data with inactive compounds and an activity cliff that led to the identification of an apparent “magic methyl” effect in the case of N-(4-fluorobenzyl)-6-[(4-fluorobenzyl)amino]-2-methoxy-4-methylnicotinamide. This flupirtine analogue showed potent KV7.2/3 opening activity, being six times as active as flupirtine itself, and by design is devoid of the potential for azaquinone diimine formation.
Optomechanical (om) systems are characterized by their nonlinear light-matter interaction. This is responsible for unique dynamic properties and allows the detection of a variety of classical and quantum mechanical phenomena on a microscopic as well as on a macroscopic scale. In this work we have studied the dynamic behavior of two laser-driven om systems, the single om cell ("cavity optomechanics / membrane-in-the-middle setup") and a two-dimensional hexagonal array of these cells ("om graphene"). The first case was motivated by the possibility to detect the transition from quantum mechanics to classical mechanics directly on the basis of the dynamic behavior. For this we focus on multistability effects of the optical and mechanical degrees of freedom, that are modeled by harmonic oscillators. Our description is based on the quantum optical master equation, which takes into account the environmental interaction assuming a vanishing temperature. As a consequence of decoherence, the dynamics occur near the semiclassical limit, i.e. it is characterized by quantum fluctuations. The quantum-to-classical transition is realized formally by rescaling the equations of motion. In the classical limit, quantum fluctuations disappear and the mean field equations were evaluated by analytical and numerical methods. We found that classical multistability is characterized by stationary signatures on the route to chaos, as well as by the coexistence of single-periodic orbits for the mechanical degree of freedom. The latter point was extensively evaluated by means of a self-consistent approach. For the dynamics in the quantum regime quantum fluctuations cannot be neglected. For this purpose, the master equation was solved by means of a numerical implementation of the Quantum State Diffusion (QSD) method. Based on Wigner and autocorrelation functions, we were able to show that quantum multistability is a dynamic effect: chaotic dynamics is suppressed and there is a time-dependent distribution of the phase space volume on classical simple-periodic orbits. The results can be interpreted within a semiclassical picture, which makes use of the single QSD quantum trajectory. Accordingly, the quantum-classical transition is explained as a time-scale effect, which is determined by tunneling probabilities in an effective mean-field potential. The subject of the second part of the work is the transport of low-energy Dirac quasiparticles in om graphene, propagating as light and sound waves. For this purpose, we investigated the scattering of a plane light wave by laser-induced photon-phonon coupling planar and circular barriers. The starting point is the om Dirac equation, which results from the continuum approximation of the Hamiltonian description of the two-dimensional array near the semiclassical limit. This work was motivated by the rich and interesting relativistic transport and tunneling phenomena found for electrons in graphene, which now appear in a new way. The reason is the presence of the new spin degree of freedom, which distinguishes the optical and mechanical excitations. In this spin space, the om interaction can be understood as a potential, which in our analysis consists of a time-independent and a time-dependent sinusoidal part. For the first case of a static barrier, the transport is elastic and is characterized by stationary scattering signatures. After solving the scattering problem via continuity conditions we were able to identify different scattering regimes depending on scattering parameters. In addition to relativistic phenomena such as Klein tunneling, simple parameter variation allows to use the barrier as a resonant light-sound interconverter and angle-dependent emitter. For the oscillating barrier, the transport is inelastic and is characterized by dynamic scattering signatures. To solve the time-periodic scattering problem, we have applied the Floquet theory for an effective two-level system. As a result of the barrier oscillation, photons and phonons can get and give away energy portions in the form of integer multiples of the oscillation frequency. The interference of short (classical) and long-wave (quantum) components leads to mixing of the scattering regimes. This allows to use the barrier as a time-periodic light-sound interconverter with interesting radiation characteristics. In addition, we have argued that the oscillating barrier provides the necessary energetic conditions for detecting zitterbewegung.
The transcription factor EB (TFEB) promotes protein degradation by the autophagy and lysosomal pathway (ALP) and overexpression of TFEB was suggested for the treatment of ALP-related diseases that often affect the heart. However, TFEB-mediated ALP induction may perturb cardiac stress response. We used adeno-associated viral vectors type 9 (AAV9) to overexpress TFEB (AAV9-Tfeb) or Luciferase-control (AAV9-Luc) in cardiomyocytes of 12-week-old male mice. Mice were subjected to transverse aortic constriction (TAC, 27G; AAV9-Luc: n = 9; AAV9-Tfeb: n = 14) or sham (AAV9-Luc: n = 9; AAV9-Tfeb: n = 9) surgery for 28 days. Heart morphology, echocardiography, gene expression, and protein levels were monitored. AAV9-Tfeb had no effect on cardiac structure and function in sham animals. TAC resulted in compensated left ventricular hypertrophy in AAV9-Luc mice. AAV9-Tfeb TAC mice showed a reduced LV ejection fraction and increased left ventricular diameters. Morphological, histological, and real-time PCR analyses showed increased heart weights, exaggerated fibrosis, and higher expression of stress markers and remodeling genes in AAV9-Tfeb TAC compared to AAV9-Luc TAC. RNA-sequencing, real-time PCR and Western Blot revealed a stronger ALP activation in the hearts of AAV9-Tfeb TAC mice. Cardiomyocyte-specific TFEB-overexpression promoted ALP gene expression during TAC, which was associated with heart failure. Treatment of ALP-related diseases by overexpression of TFEB warrants careful consideration.
The focus of the first two articles was the engineering and application of enzymes for the conversion of the bio-based resources glycerol and its oxidation product glyceraldehyde for the production of the value added product glyceric acid. Article III focuses on the cloning, exploration and engineering of a polyol dehydrogenase, which later on was used as cofactor recycling system in order to produce ε-caprolactone from cyclohexanol as presented in arti-cle IV. The following paragraphs will give a short outline of each article. ARTICLE I: ASYMMETRIC SYNTHESIS OF D-GLYCERIC ACID BY AN ALDITOL OXIDASE AND DIRECTED EVOLUTION FOR ENHANCED OXIDATIVE ACTIVITY TOWARDS GLYCEROL. GERSTENBRUCH, S., WULF, H., MUßMANN, N., O’CONNELL, T., MAURER, K.-H. & BORNSCHEUER, U. T. (2012). Appl. Microbiol. Biotechnol. 96, 1243-1252. The alditol oxidase of Streptomyces coelicolor A3(2) (AldO) was used to catalyze the oxida-tion of glycerol to glyceraldehyde and glyceric acid. The enantioselectivity for the FAD-de-pendent glycerol oxidation was elucidated and different strategies were used to enhance the substrate specificity towards glycerol. Directed evolution by error-prone PCR led to an AldO double mutant with 1.5-fold improved activity for glycerol. Further improvement of activity was achieved by combination of mutations, leading to a quadruple mutant with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. In small-scale biotransformation concentrations up to 2.0 g•l-1 D-glyceric acid could be reached using whole cells. Investi¬gation of the effects of the introduced mutations led to a further identification of es¬sential amino acids with respect to enzyme functionality and structural stability. ARTICLE II: KINETIC RESOLUTION OF GLYCERALDEHYDE USING AN ALDEHYDE DEHYDROGENASE FROM DEINOCOCCUS GEOTHERMALIS DSM 11300 COMBINED WITH ELECTROCHEMICAL COFACTOR RECYCLING. WULF, H., PERZBORN, M., SIEVERS, G., SCHOLZ, F. & BORNSCHEUER, U. T. (2012). J. Mol. Catal. B Enzym. 74, 144-150. Two aldehyde dehydrogenases (ALDH) from Escherichia coli BL21 and Deinococcus geother-malis were cloned, characterized and evaluated according to their applicability for a bio-catalysis setup with electrolytic cofactor recycling. Both ALDHs turned out to have a sim¬ilar substrate scope and favor short to medium chain aldehydes and both oxidize glyceralde¬hyde to D-glyceric acid. The ALDH variant of D. geothermalis shows higher specific activity towards glyceraldehyde and has an elevated optimum temperature compared to the BL21 enzyme. Due to the higher specific activity of the ALDH of D. geothermalis, this enzyme was used to conduct a kinetic resolution of glyceraldehyde with electrolytic NAD+ recycling at a glassy carbon foam electrode with ABTS as redox mediator yielding in 1.8 g•l-1 glyceric acid. ARTICLE III: PROTEIN ENGINEERING OF A THERMOSTABLE POLYOL DEHYDROGENASE. WULF, H.*, MALLIN, H.*, BORNSCHEUER U.T. (2012). Enzyme Microb. Technol. 51, 217-224 (*equally contributed). The new enzyme polyol dehydrogenase PDH-11300 from D. geothermalis was extensively characterized regarding its temperature optimum and thermostability. A peptide stretch responsible for substrate recognition from the PDH-11300 was substituted by this particular stretch of a homolog enzyme, the galactitol dehydrogenase from Rhodobacter sphaeroides (PDH-158), resulting in a chimeric enzyme (PDH-loop). The substrate scopes were deter-mined and basically the chimeric enzyme represented the average of both wild-type en-zymes. A rather unexpected finding was the notably increased T5060, by 7°C to 55.3°C, and an increased specific activity against cyclohexanol. Finally, the cofactor specificity was suc¬cess-fully altered from NADH to NADPH by an Asp55Asn mutation, which is located at the NAD+ binding cleft, without influencing the catalytic properties of the dehydrogenase. ARTICLE IV: A SELF-SUFFICIENT BAEYER-VILLIGER BIOCATALYSIS SYSTEM FOR THE SYNTHESIS OF Ɛ-CAPROLACTONE FROM CYCLOHEXANOL. MALLIN, H. *, WULF, H. *, BORNSCHEUER U.T. (2013). Enzyme Microb. Technol., online, DOI: 10.1016/j.enzmictec.2013.01.007 (*equally contributed). The application of the engineered PDH-loopN mutant [1] (Article III) for the production of ε-caprolactone from cyclohexanol was investigated in a co-immobilization approach with the cyclohexanone monooxygenase from Acinetobacter calcoaceticus. Biotransformation with solubilized enzymes led to an isolated yield of 55% pure ε-caprolactone with no residual cy-clohexanol to be detected. During the immobilization experiments a higher enzyme ratio in favor of the CHMO led to higher reaction velocities. Similarly, the addition of soluble fresh CHMO during reuse of co-immobilization batches significantly increased the activity identi-fying the CHMO as the bottleneck in this reaction setup.
Fast screening of enzyme variants is crucial for tailoring biocatalysts for the asymmetric synthesis of non-natural chiral chemicals, such as amines. However, most existing screening methods either are limited by the throughput or require specialized equipment. Herein, we report a simple, high-throughput, low-equipment dependent, and generally applicable growth selection system for engineering amine-forming or converting enzymes and apply it to improve biocatalysts belonging to three different enzyme classes. This results in (i) an amine transaminase variant with 110-fold increased specific activity for the asymmetric synthesis of the chiral amine intermediate of Linagliptin; (ii) a 270-fold improved monoamine oxidase to prepare the chiral amine intermediate of Cinacalcet by deracemization; and (iii) an ammonia lyase variant with a 26-fold increased activity in the asymmetric synthesis of a non-natural amino acid. Our growth selection system is adaptable to different enzyme classes, varying levels of enzyme activities, and thus a flexible tool for various stages of an engineering campaign.
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.
Body-size variability results from a variety of extrinsic and intrinsic factors (environmental and biological influences) underpinned by phylogeny. In ostracodes it is assumed that body size is predominantly controlled by ecological conditions, but investigations have mostly focused on local or regional study areas. In this study, we investigate the geographical size variability (length, height, and width) of Holocene and Recent valves of the salinity-tolerant ostracode species Cyprideis torosa within a large geographical area (31°–51° latitude, and 12°–96° longitude). It is shown that distant local size clusters of Cyprideis torosa are framed within two large-scale geographical patterns. One pattern describes the separation of two different size classes (i.e., morphotypes) at around ∼42° N. The co-occurrence of both size morphotypes in the same habitats excludes an environmental control on the distribution of the morphotypes but rather could point to the existence of two differentiated lineages. Generally, correlations between valve size and environmental parameters (salinity, geographical positions) strongly depend on the taxonomic resolution. While latitude explains the overall size variability of C. torosa sensu lato (i.e., undifferentiated for morphotypes), salinity-size correlations are restricted to the morphotype scale. Another large-scale pattern represents a continuous increase in valve size of C. torosa with latitude according to the macroecological pattern referred as Bergmann trend. Existing explanations for Bergmann trends insufficiently clarify the size cline of C. torosa which might be because these models are restricted to intraspecific levels. The observed size-latitude relationship of C. torosa may, therefore, result from interspecific divergence (i.e., size ordered spatially may result from interspecific divergence sorting) while environmental influence is of minor importance. Our results imply that geographical body-size patterns of ostracodes are not straightforward and are probably not caused by universal mechanisms. Consideration of phylogenetic relationships of ostracodes is therefore necessary before attempting to identify the role of environmental controls on body size variability.
Calcitic valves of non-marine ostracodes are important geochemical archives. Investigations of the relationship between the ranges of oxygen and carbon isotope values of modern ostracode populations and their host water provide important information on local or regional conditions and influences. Here we present the first δ18Oostracode and δ13C of the freshwater ostracode species Cytheridella ilosvayi along with the isotopic composition of the waters in which the ostracodes calcified, δDwater, δ18Owater, δ13CDIC values—covering a large geographical range (Florida to Brazil). With this data we extended a newly developed approach based on the estimation of δ18O values of monthly equilibrium calcites as references for the interpretation of δ18Oostracode values. The expected apparent oxygen isotope fractionation between CaCO3 and H2O is correlated with temperature with smaller values occurring at higher temperatures as valid at isotope equilibrium (δ18Ocalcite_eq). Uncertainties about the expected equilibrium calcites derive from incomplete knowledge of high-frequency variations of the water bodies caused by interplay of mixing, evaporation, and temperature. Coincidence between δ18Oostracode and δ18Ocalcite_eq is restricted to few months indicating a seasonal calcification of Cytheridella. There is a characteristic pattern in its difference between mean δ18Oostracode and δ18Ocalcite_eq which implies that Cytheridella provides a synchronous life cycle in its geographical range with two calcification periods in spring (May, June) and autumn (October). This ubiquitous life cycle of Cytheridella in the entire study area is considered to be phylogenetically inherited. It might have originally been adapted to environmental conditions but has been conserved during the migration and radiation of the group over the Neotropical realm.
The stratospheric aerosol layer plays an important role in the radiative balance of Earth primarily through scattering of solar radiation. The magnitude of this effect depends critically on the size distribution of the aerosol. The aerosol layer is in large part fed by volcanic eruptions strong enough to inject gaseous sulfur species into the stratosphere. The evolution of the stratospheric aerosol size after volcanic eruptions is currently one of the biggest uncertainties in stratospheric aerosol science. We retrieved aerosol particle size information from satellite solar occultation measurements from the Stratospheric Aerosol and Gas Experiment III mounted on the International Space Station (SAGE III/ISS) using a robust spectral method. We show that, surprisingly, some volcanic eruptions can lead to a decrease in average aerosol size, like the 2018 Ambae and the 2021 La Soufrière eruptions. In 2019 an intriguing contrast is observed, where the Raikoke eruption (48∘ N, 153∘ E) in 2019 led to the more expected stratospheric aerosol size increase, while the Ulawun eruptions (5∘ S, 151∘ E), which followed shortly after, again resulted in a reduction in the values of the median radius and absolute distribution width in the lowermost stratosphere. In addition, the Raikoke and Ulawun eruptions were simulated with the aerosol climate model MAECHAM5-HAM. In these model runs, the evolution of the extinction coefficient as well as of the effective radius could be reproduced well for the first 3 months of volcanic activity. However, the long lifetime of the very small aerosol sizes of many months observed in the satellite retrieval data could not be reproduced.
Myxomycetes or Myxogastria (supergroup Amoebozoa) are one of several Protistean groups dispersing via airborne spores. The model organism for the group, so far exclusively studied in a laboratory environment, is Physarum polycephalum. Here, molecular evolution, distribution and the ecology of spores dispersal was investigated for the non-model species Physarum albescens. This nivicolous myxomycete fruits with snow melt in most mountain ranges of the northern hemisphere and disperses via spherical, dark-colored and melanin-rich spores. Fruit body development and subsequent spore dispersal occurs within a short time window of a few days. At this time, the fruiting plasmodium is fully exposed to the harsh environment if the protecting snow melts away. The spores, with a diameter of 10–13 µm of the typical size for myxomycetes, can potentially reach all suitable habitats worldwide, which led to the assumption that not only Ph. albescens but most myxomycete species should be ubiquitously distributed over the world.
In the first part of this study (article 1), the question was, if spore dispersal can realize a gene flow sufficient to meet the above-mentioned assumption. A total of 324 accessions of Ph. albescens, collected all over the northern hemisphere, was sequenced for 1-3 genetic markers (SSU, EF1A, COI), and 98 specimens were further analyzed using the genotyping by sequencing technique. As a result, at least 18 reproductively isolated units, which can be seen as cryptic biological species, emerged as phylogroups in a three-gene phylogeny, but as well in a SNP-based phylogeny and were confirmed by a recombination analysis between the three markers. However, this evolutive radiation is not simply caused by geographic fragmentation due to low dispersal capability: within a certain region, multiple phylogroups coexisted next to each other, although some appeared to be regional endemics. Most likely, mutations in mating-type genes, as shown to exist for the cultivable Ph. polycephalum, are the main drivers of speciation. This challenges the hypothesis of ubiquitous distribution of Ph. albescens and corroborates the results of the few available studies for other myxomycete species. In addition, groups of clonal specimens, mostly but not always restricted to a certain slope or valley indicated that sexual and asexual reproduction coexists in the natural populations of Ph. albescens.
In the second part (articles 2), the fundamental niche for Ph. albescens was described using all available records for the species. The resulting set of 537 unique occurrence points was subjected to a correlative spatial approach using the software MaxEnt. In dependence on the predictor variables three species distribution models emerged which differed only in details. The first consisted of only 19 bioclimatic variables and an elevation map from the WorldClim dataset. The second was corrected for pseudo-absences resulting from missing survey activities, and the third was expanded with an additional categorical environment variable on snow cover. High mean AUC (area under the curve) values above 0.97 could be reached with all three models. Variables for snow cover, precipitation of the coldest quarter (of the year), and elevation correlated highly to predict the distribution of Ph. albescens. Only in mid-northern latitudes, elevation alone was a good predictor, but it would cause false-positive predictions in arid mountain ranges and failed to explain occurrence in lowland sites at higher latitudes. Mountains in humid climates showed the highest incidences, confirming recent studies that long-lasting snow covers combined with mild summers are crucial for the ecological guild of nivicolous myxomycetes, with Ph. albescens as a typical species.
Spore size is crucial for dispersal ability and should thus be a character under strong selection. In addition, spores carrying two nuclei with opposite mating types should have a colonization advantage. This was the hypothesis for the last part of this study (articles 3 and 4), which investigated this trait in a quantitative manner. This required a method to analyze thousands of spores automatically (article 3) and with high precision for size and the number of nuclei enclosed. Human errors should be excluded, to reveal even subtle differences in the resulting spore size distributions. Two challenges had to be met for this approach. First, a preparation technique was developed to reduce false segmentations due to overlaying spores by aligning spores on one common plane with a high-frequency vibration device. Second, the segmentation process was automated to allow separating spores that are densely packed in the respective images. A machine learning algorithm was set up and trained to reliable identify and measure dark-colored spores. The technique produced consistent results with high accuracy, and the large number of spores allowed to compile spore size distributions, to check for the constancy of this character, which is impossible with manual measurements limited to low numbers.
The resulting spore size distributions, obtained from over 80 specimens (article 4), were mostly narrow, which is in accordance with our hypothesis. Spore size was as well fairly constant within fructifications from one colony. However, mean spore size within different accessions of Ph. albescens showed large variation (ca. 10%, a range often indicated to key out different morphospecies of myxomycetes), and this was explained only by a minor part with differences between biospecies. Not much smaller (8%) was the variation within a group of clonal specimens collected within 25 m distance. This points to a strong influence of environmental factors even at a micro spatial scale, perhaps caused by microclimatic differences and high phenotypic plasticity for spore size. The influence of large-scale covariates like altitude or latitude was negligible. However, spore size correlated with the variance in this trait, indicating that oversized spores may be caused by detrimental environmental conditions. Two aberrations in spore development were found: First, a few specimens showed a multimodal distribution for spore size with two or even three discernible spore populations. The estimated volumes of those populations correspond to a multiple of the first and most abundant conspicuous spore size population. Second, not all spores were uninucleate as to be expected for meiotic products. This was revealed by fluorescence signals from staining the same spores with DAPI, with a second machine learning algorithm trained to identify the nuclei in a spore. A few specimens showed a significant proportion of binucleated spores in the size range of normal-sized ones, and these specimens were not the ones with multimodal spore size distributions. This indicates that the negative impacts (inbreeding) of multinucleate spores should outweigh a possible colonization advantage and is in accordance with the high genetic diversity found in the worldwide population of Ph. albescens, indicating predominantly sexual reproduction in wild populations of myxomycetes.
Background: Gastrointestinal hormones (GIHs) are crucial for the regulation of a variety of physiological functions and have been linked to hunger, satiety, and appetite control. Thus, they might constitute meaningful biomarkers in longitudinal and interventional studies on eating behavior and body weight control. However, little is known about the physiological levels of GIHs, their intra-individual stability over time, and their interaction with other metabolic and lifestyle-related parameters. Therefore, the aim of this pilot study is to investigate the intra-individual stability of GIHs in normal-weight adults over time. Methods: Plasma concentrations of ghrelin, leptin, GLP-1 (glucagon-like-peptide), and PP (pancreatic polypeptide) were assessed by enzyme-linked immunosorbent assay (ELISA) in 17 normal-weight, healthy adults in a longitudinal design at baseline and at follow-up six months later. The reliability of the measurements was estimated using intra-class correlation (ICC). In a second step, we considered the stability of GIH levels after controlling for changes in blood glucose and hemoglobin A1 (HbA1c) as well as self-reported physical activity and dietary habits. Results: We found excellent reliability for ghrelin, good reliability for GLP1 and PP, and moderate reliability for leptin. After considering glucose, HbA1c, physical activity, and dietary habits as co-variates, the reliability of ghrelin, GLP1, and PP did not change significantly; the reliability of leptin changed to poor reliability. Conclusions: The GIHs ghrelin, GLP1, and PP demonstrated good to excellent test–retest reliability in healthy individuals, a finding that was not modified after adjusting for glucose control, physical activity, or dietary habits. Leptin showed only moderate to poor reliability, which might be linked to weight fluctuations, albeit small, between baseline and follow-up assessment in our study sample. Together, these findings support that ghrelin, GLP1, and PP might be further examined as biomarkers in studies on weight control, with GLP1 and PP serving as anorexic markers and ghrelin as an orexigenic marker. Additional reliability studies in obese individuals are necessary to verify or refute our findings for this cohort.
Deteriorations in slow wave sleep (SWS) have been linked to brain aging and Alzheimer’s disease (AD), possibly due to its key role in clearance of amyloid-beta and tau (Aß/tau), two pathogenic hallmarks of AD. Spermidine administration has been shown to improve sleep quality in animal models. So far, the association between spermidine levels in humans and parameters of SWS physiology are unknown but may be valuable for therapeutic strategies. Data from 216 participants (age range 50–81 years) of the population-based Study of Health in Pomerania TREND were included in our analysis. We investigated associations between spermidine plasma levels, key parameters of sleep macroarchitecture and microarchitecture that were previously associated with AD pathology, and brain health measured via a marker of structural brain atrophy (AD score). Higher spermidine levels were significantly associated with lower coupling between slow oscillations and spindle activity. No association was evident for SWS, slow oscillatory, and spindle activity throughout non-rapid eye movement sleep. Furthermore, elevated spermidine blood levels were significantly associated with a higher AD score, while sleep markers revealed no association with AD score. The association between higher spermidine levels and brain health was not mediated by coupling between slow oscillations and spindle activity. We report that higher spermidine blood levels are associated not only with deteriorated brain health but also with less advantageous markers of sleep quality in older adults. Future studies need to evaluate whether sleep, spermidine, and Aß/tau deposition are interrelated and whether sleep may play a mediating role.
Introduction
Supplementation with spermidine may support healthy aging, but elevated spermidine tissue levels were shown to be an indicator of Alzheimer's disease (AD).
Methods
Data from 659 participants (age range: 21–81 years) of the population-based Study of Health in Pomerania TREND were included. We investigated the association between spermidine plasma levels and markers of brain aging (hippocampal volume, AD score, global cortical thickness [CT], and white matter hyperintensities [WMH]).
Results
Higher spermidine levels were significantly associated with lower hippocampal volume (ß = −0.076; 95% confidence interval [CI]: −0.13 to −0.02; q = 0.026), higher AD score (ß = 0.118; 95% CI: 0.05 to 0.19; q = 0.006), lower global CT (ß = −0.104; 95% CI: −0.17 to −0.04; q = 0.014), but not WMH volume. Sensitivity analysis revealed no substantial changes after excluding participants with cancer, depression, or hemolysis.
Discussion
Elevated spermidine plasma levels are associated with advanced brain aging and might serve as potential early biomarker for AD and vascular brain pathology.
Despite a plethora of therapeutic approaches, the injection of local anaesthetics itself remains one of the most painful and dreadful procedures among children. Stimulation of acupoint LI4 is associated with analgesic effects in dentistry. Goal of the study To investigate whether stimulation of LI4, added to standard therapy (ST), reduces pain and distress during injection of local anaesthetic (LA) in comparison with ST alone. Materials and Methods Children, scheduled for dental treatment in local anaesthesia on 2 separate days were enrolled in this trial, approved by local ethics commission. On one day each child received bilateral acupuncture of LI4 point, using indwelling fixed “New Pyonex” needles (0.2 x 1.5 mm; Seirin, Japan). The parents of the children were asked to stimulate the needles by massage. Standardized injection of LA was performed 5 min following acupuncture. The needles were withdrawn at the end of dental treatment. On the other day of treatment children received LA injection without acupuncture. The order of treatment days (acupuncture first or vice versa) was randomised. Primary endpoint was the pain intensity during LA injection reported by children on Visual Rating Scale from 0=no pain to 10=maximal pain imaginable (VRS-11). Secondary endpoints were parent- and dentist-assessed pain intensity (measured on Numeric Rating Scale 1-10), patients’ heart rate before and during dental treatment and satisfaction with received therapy (measured on Numerical Rating Scale 1-5.) Side effects of LI4 stimulation were also recorded. Results and Discussion The data of 49 children (22 females; age 10 ± 4 yrs; mean ± SD), who completed both visits, were analysed. Children reported less pain with than without acupuncture: 2.2 ± 2.5 vs. 3.9 ± 2.7; mean ± SD, p<0.001. Heart rate decreased after LI4 stimulation compared to ST alone throughout the dental treatment (p<0.05). LI4 stimulation was safe and raised better satisfaction with the treatment among children and parents, than ST alone (p<0.05). Other secondary endpoints were comparable between both sessions. Conclusion Stimulation of acupuncture point LI4 reduces pain and autonomous stress during injection of local anaesthetics in paediatric dentistry.
Abstract
Background
Early mobilization improves physical independency of critically ill patients at hospital discharge in a general intensive care unit (ICU)‐cohort. We aimed to investigate clinical and molecular benefits or detriments of early mobilization and muscle activating measures in a high‐risk ICU‐acquired weakness cohort.
Methods
Fifty patients with a SOFA score ≥9 within 72 h after ICU admission were randomized to muscle activating measures such as neuromuscular electrical stimulation or whole‐body vibration in addition to early protocol‐based physiotherapy (intervention) or early protocol‐based physiotherapy alone (control). Muscle strength and function were assessed by Medical Research Council (MRC) score, handgrip strength and Functional Independence Measure at first awakening, ICU discharge, and 12 month follow‐up. Patients underwent open surgical muscle biopsy on day 15. We investigated the impact of muscle activating measures in addition to early protocol‐based physiotherapy on muscle strength and function as well as on muscle wasting, morphology, and homeostasis in patients with sepsis and ICU‐acquired weakness. We compared the data with patients treated with common physiotherapeutic practice (CPP) earlier.
Results
ICU‐acquired weakness occurs within the entire cohort, and muscle activating measures did not improve muscle strength or function at first awakening (MRC median [IQR]: CPP 3.3 [3.0–4.3]; control 3.0 [2.7–3.4]; intervention 3.0 [2.1–3.8]; P > 0.05 for all), ICU discharge (MRC median [IQR]: CPP 3.8 [3.4–4.4]; control 3.9 [3.3–4.0]; intervention 3.6 [2.8–4.0]; P > 0.05 for all), and 12 month follow‐up (MRC median [IQR]: control 5.0 [4.3–5.0]; intervention 4.8 [4.3–5.0]; P = 0.342 for all). No signs of necrosis or inflammatory infiltration were present in the histological analysis. Myocyte cross‐sectional area in the intervention group was significantly larger in comparison with the control group (type I +10%; type IIa +13%; type IIb +3%; P < 0.001 for all) and CPP (type I +36%; type IIa +49%; type IIb +65%; P < 0.001 for all). This increase was accompanied by an up‐regulated gene expression for myosin heavy chains (fold change median [IQR]: MYH1 2.3 [1.1–2.7]; MYH2 0.7 [0.2–1.8]; MYH4 5.1 [2.2–15.3]) and an unaffected gene expression for TRIM63, TRIM62, and FBXO32.
Conclusions
In our patients with sepsis syndrome at high risk for ICU‐acquired weakness muscle activating measures in addition to early protocol‐based physiotherapy did not improve muscle strength or function at first awakening, ICU discharge, or 12 month follow‐up. Yet it prevented muscle atrophy.
Vitamin B6 deficiency during pregnancy translates into a severe vitamin B6 deficiency (plasma levels decreased by 97%) in new-born rats. Further, hallmarks are increased (+89%) concentrations of homocysteine, gross changes in gene methylation and expression, and metabolic alterations including lipid metabolism. This study focuses on determining the effects of vitamin B6-deficiency on cardiolipin composition and oxidative phosphorylation in liver. For this purpose, hepatic cardiolipin composition was analyzed by means of LC/MS/MS, and mitochondrial oxygen consumption was determined by using a Clark-type electrode in a rat model of vitamin B6 deficiency. Liver mitochondria from new-born rats with pre-term vitamin B6 deficiency responded with substantial alterations in cardiolipin composition that include the following changes in the amounts of cardiolipin incorporated fatty acids: increase in C16, decrease in C18, decrease in saturated fatty acid, as well as increase in amount of oxidized cardiolipin species. These changes were accompanied by significantly decreased capacity of oxidative phosphorylation. In conclusion, vitamin B6 deficiency in new born rats induces massive alterations of cardiolipin composition and function of liver mitochondria. These findings support the importance of sufficient periconceptional supply of vitamin B6 to prevent vitamin B6 deficiency.
Impact statement
Vitamin B6 (VitB6) is an active co-enzyme for more than 150 enzymes and is required for a great diversity of biosynthesis and metabolic reactions. There is an increased need for VitB6 during pregnancy and sufficient supply of VitB6 is crucial for the prevention of cleft palate and neural tube defects. We show that liver mitochondria from new-born rats with pre-term VitB6 deficiency respond with substantial alterations in cardiolipin (CL) composition and in the amount of oxidized CL species. These changes are associated with a decrease in the efficiency of oxidative phosphorylation. The results of this study support the significance of sufficient supply of VitB6 during pregnancy (and periconceptional) for diminishing the number of early abortions and minimizing malformation. The established link between VitB6 deficiency, CL composition, and mitochondrial respiration/energy production provides mechanistic insight as to how the VitB6 deficiency translates into the known pathophysiological and clinically relevant conditions.
The introduction of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) enabled the separation and visualization of a substantial fraction of an organism’s entire proteome, and when mass spectrometry entered protein science, these proteins became even amenable to identification on a grand scale. Nevertheless, important classes of proteins elude a separation on classical 2 D gels, as the ones showing extremes in isoelectric point or molecular weight, and foremost very hydrophobic proteins naturally embedded in lipid membranes. This thesis aimed at the establishment and adaptation of alternatives to 2-D PAGE. New techniques allowing for an identification and quantification of critical protein classes were designed and adopted to physiological questions in the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. In a comprehensive study on cytoplasmic proteins of S. aureus COL the number of proteins identified by a 2-D gel based approach could be extended by 650 proteins employing gel free technologies. Application of these complementary methods resulted in the establishment of a comprehensive reference map of the cytosolic proteome in growing and non-growing S. aureus cells which can serve as basis for further physiological investigations. Gel free separation of complex protein digests was likewise used in a quantitative study on heat stress in B. subtilis. By implementation of the iTRAQ® technology four different physiological states could be relatively quantified in one experiment. A parallel generation of 2-D gel based data enabled the depiction of strengths and weaknesses of protein quantitation by both, spot intensities on 2-D gels and iTRAQ® signal intensities in MS/MS spectra. Furthermore, new insights into heat sensitivity of pivotal enzymes involved in amino acid biosynthesis could be delivered. The institution of gel free approaches and advancements in 2-D PAGE provide the tools to penetrate into yet unamenable scopes of proteomes. A review on proteome coverage in B. subtilis gives an overview on the strategies which have been explored for most comprehensive protein identification in various sub-proteomes. Although more than one third of B. subtilis’ open reading frames could be demonstrated on protein level, one has to be aware of the fact that it still is a long way to achieve complete coverage of its proteome. Integral membrane proteins make up about one quarter of the entirety of proteins in a cell. Despite their large portion they are clearly understudied due to the intricacy of identification. Their low abundance and non-accessibility of membrane-spanning domains represent major experimental difficulties. The establishment of a protocol efficiently depleting cytosolic proteins by membrane shaving and targeting trans-membrane peptides by novel digestion strategies essentially facilitated identification of highly hydrophobic integral membrane proteins. This protocol was not only successfully applied to the membrane proteome of growing S. aureus cells, but was shown to be applicable in B. subtilis as well. Both studies displayed the novel membrane shaving approach to be highly complementary to a previously established separation of membrane proteins via 1 D PAGE. A combination of the two techniques resulted in identification of about half of the theoretical membrane proteome in both bacteria, and hence layed the foundation for advanced and quantitative analyses. In this regard, 14N/15N metabolically labeled membrane samples of growing and non-growing cells of S. aureus COL were relatively quantified revealing a significant difference in amount for more than one third of the proteins. A corresponding experimental setup was used to compare the membrane proteomes of S. aureus SA113 and its mutant deficient in the lysylphosphatidylglycerol synthetase MprF. Interesting quantitative differences were obtained for proteins most likely involved in the regulation of cellular surface net charge as well as for virulence-associated proteins.
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.
Pentathiepins are polysulfur-containing compounds that exert antiproliferative and cytotoxic activity in cancer cells, induce oxidative stress and apoptosis, and inhibit glutathione peroxidase (GPx1). This renders them promising candidates for anticancer drug development. However, the biological effects and how they intertwine have not yet been systematically assessed in diverse cancer cell lines. In this study, six novel pentathiepins were synthesized to suit particular requirements such as fluorescent properties or improved water solubility. Structural elucidation by X-ray crystallography was successful for three derivatives. All six underwent extensive biological evaluation in 14 human cancer cell lines. These studies included investigating the inhibition of GPx1 and cell proliferation, cytotoxicity, and the induction of ROS and DNA strand breaks. Furthermore, selected hallmarks of apoptosis and the impact on cell cycle progression were studied. All six pentathiepins exerted high cytotoxic and antiproliferative activity, while five also strongly inhibited GPx1. There is a clear connection between the potential to provoke oxidative stress and damage to DNA in the form of single- and double-strand breaks. Additionally, these studies support apoptosis but not ferroptosis as the mechanism of cell death in some of the cell lines. As the various pentathiepins give rise to different biological responses, modulation of the biological effects depends on the distinct chemical structures fused to the sulfur ring. This may allow for an optimization of the anticancer activity of pentathiepins in the future.
Pentathiepins are cyclic polysulfides that exert antiproliferative and cytotoxic activity in cancer cells, induce oxidative stress and apoptosis, and potently inhibit GPx1. These properties render this class of compounds promising candidates for the development of anticancer drugs. However, the biological effects and how they intertwine to promote high cytotoxicity have not been systematically assessed throughout a panel of cancer cell lines from distinct tissues of origin. In this thesis, six novel pentathiepins were analyzed and constitute the second generation of compounds with additional properties such as fluorescence or improved water solubility to facilitate cellular testing. All compounds underwent extensive biological evaluation in 14 human cancer cell lines. These studies included investigations of the inhibitory potential with regards to GPx1 and cell proliferation, examined the cytotoxicity in human cancer cell lines, as well as the induction of oxidative stress and DNA strand breaks. Furthermore, selected hallmarks of apoptosis, ferroptosis, and autophagy were studied. Experimental approaches regarding these cellular mechanisms included observing morphological changes, detecting phosphatidyl serine exposure and caspase activity, and quantifying cleaved PARP1 and levels of LC3B II. In addition, the analysis of the cell cycle aimed to identify aberrations or arrests in cell division.
Five of the six tested pentathiepins proved to be potent inhibitors of the GPx1, while all six exerted high cytotoxic and antiproliferative activity, although to different extents. There was a clear connection observed between the potential to provoke oxidative stress and damage to DNA in the form of single- and double-strand breaks both extra- and intracellularly. Furthermore, various experiments supported apoptosis but not ferroptosis as the mechanism of cell death in four different cell lines. In particular, the externalization of PS, the detection of activated caspases, and the cleavage of PARP1 corroborated this conclusion. Additionally, indications for autophagy were found, but more investigations are required to verify the current data. The findings of this dissertation are mainly in line with the postulated mechanism of action proposed for pentathiepins and a previous publication from our group that described their biological activity. However, the influence of modulators such as oxygen and GSH on the biological effects was ambiguous and dependent on the compound. The expression profile of the cell lines concerning GPx1 and CAT did not influence the cellular response toward the treatment, whereas the cell doubling time correlated with the cytotoxicity.
As the various pentathiepins give rise to different biological responses, modulation of the biological effects depends on the distinct chemical structures fused to the sulfur ring. This may allow for future optimization of the anticancer activity of pentathiepins. An analysis of the structure-activity relationships revealed that the piperazine scaffold was associated with superior biological activity compared to the pyrrolo-pyrazine backbone. Furthermore, substituents with electron-withdrawing properties or those providing a free electron pair, such as fluorine or morpholine, were advantageous. These findings should help design and synthesize the next generation of pentathiepins, thereby expanding the library of compounds, allowing for the further deduction of structure-activity relationships and an improved understanding of their mechanism of action.
The genus Capripoxvirus of the family Poxviridae consists of the species lumpy skin disease virus, sheeppox virus and goatpox virus that affect cattle, sheep and goats, respectively. Whereas lumpy skin disease virus (LSDV) is transmitted mainly mechanically via blood-feeding insects and possibly hard ticks, the major transmission routes of sheeppox virus (SPPV) and goatpox virus (GTPV) are via direct contact and aerosols. Affected animals develop fever and display clinical signs such as ocular and nasal discharge, lymphadenopathy and characteristic lesions of the skin. Severe clinical course, especially in combination with respiratory signs, can result in the death of the affected animals. In endemic regions, mortality of capripox virus-induced diseases is low (1-10%). However, mortalities of up to 75% have been reported for LSDV and up to 100% for SPPV and GTPV in exotic breeds and high-producing dairy or beef animals. The loss of quality of the leather, reduced weight gain and milk yield as well as complete loss of affected animals have severe impact on national and global economies. Therefore, capripox virus-induced diseases have significant impact on both the affected individual animal as well as on the existence of small-scale farmers and large agricultural enterprises. However, until now, only live attenuated vaccines are commercially available. These attenuated vaccines are not authorized in the European Union and their administration would comprise the disease-free status of the respective country. Thus, reliable diagnostic tools for the detection and characterization of capripox viruses as well as safe and efficient control measures are of high importance.
The objectives of the present thesis were the development, validation and comparison of diagnostic tools, the establishment of challenge infection models and the performance of pathogenesis studies for all three capripox virus species, and the development and testing of different inactivated prototype vaccine candidates against LSDV.
First, new real-time quantitative polymerase chain reaction (qPCR) assays for robust detection and differentiation of LSDV field strains, LSDV vaccine strains, SPPV and GTPV were developed and extensively validated. In the following, two single assays were combined to duplex assays, one for the differentiation between LSDV field strains and LSDV vaccine strains, and the second for discrimination of SPPV and GTPV. Finally, a diagnostic workflow based on these new duplex assays in combination with already published methods was established. This workflow enables time-saving, robust and reliable detection, species-specific identification and genetic and phylogenetic characterization of all three capripox virus species. In addition, already existing serological examination methods (serum neutralization assay and commercial enzyme-linked immunosorbent assay) were compared regarding their sensitivity and specificity. Furthermore, pathogenesis studies with different capripox virus isolates were performed in the respective target species, and the suitability of selected virus isolates as challenge viruses for future vaccine studies was analyzed. Pathogenesis studies with isolates GTPV-“V/103” and LSDV-“Macedonia2016” revealed that both are proper candidates for challenge models. Finally, three different SPPV isolates (SPPV-“V/104”, SPPV-“India/2013/Surankote” and SPPV-“Egypt/2018”) were tested in sheep regarding their virulence to find a suitable challenge model for SPPV, and SPPV-“India/2013/Surankote” was chosen for future vaccine studies.
Once appropriate challenge models were established, different inactivated prototype vaccines against LSDV were developed, and vaccine safety as well as vaccine efficacy were tested in cattle. Eventually, a Polygen-adjuvanted inactivated LSDV-vaccine candidate was selected that is able to fully prevent cattle from any LSDV-related clinical signs after severe challenge infection. Furthermore, molecular and serological data indicate that this inactivated prototype vaccine is even able to induce a kind of “sterile immunity” against LSDV in those cattle. It has to be mentioned that a commercially available vaccine similar to this prototype vaccine would be a great advance for the control of LSDV.
In the future, additional studies addressing diagnostics and optimized control of capripox viruses should be performed. Firstly, probe-based real-time qPCR assays for the differentiation of SPPV and GTPV vaccine strains from their respective virulent field strains should be developed and included into the diagnostic workflow. Secondly, further tests of the inactivated prototype vaccine, e.g. determination of the minimum protective dose and the possibility of cross-protection in sheep and goats against SPPV and GTPV, respectively, should be performed.
Simple Summary
This German multicenter study investigated the importance of different supportive measures offered to patients with prostate cancer who undergo surgery (radical prostatectomy). A number of these supportive measures are required during the certification of a urologic hospital as prostate cancer center. However, a broad scientific basis evaluating these measures from the patient’s perspective is still lacking. In this study, patients were asked to rate the relevance of several supportive measures and to estimate the effective availability of these different supportive measures at their urologic clinic about 15 months after surgery. Our study highlights that only six of fifteen different supportive measures were rated as very relevant by patients. None of these six supportive measures were offered more intensively at the certified clinics compared to the non-certified clinics according to the patients. Our study helps to identify those supportive measures with the highest subjective impact on patients in this setting.
Abstract
Certification as a prostate cancer center requires the offer of several supportive measures to patients undergoing radical prostatectomy (RP). However, it remains unclear how patients estimate the relevance of these measures and whether the availability of these measures differs between certified prostate cancer centers (CERTs) and non-certified centers (NCERTs). In 20 German urologic centers, a survey comprising questions on the relevance of 15 supportive measures was sent to 1000 patients at a median of 15 months after RP. Additionally, patients were asked to rate the availability of these measures using a four-item Likert scale. The aim of this study was to compare these ratings between CERTs and NCERTs. The response rate was 75.0%. In total, 480 patients underwent surgery in CERTs, and 270 in NCERTs. Patients rated 6/15 supportive measures as very relevant: preoperative medical counselling concerning treatment options, a preoperative briefing answering last questions, preoperative pelvic floor exercises (PFEs), postoperative PFEs, postoperative social support, and postoperative rehabilitation addressing physical fitness recovery. These ratings showed no significant difference between CERTs and NCERTs (p = 0.133–0.676). In addition, 4/9 of the remaining criteria were rated as more detailed by patients in CERTs. IMPROVE represents the first study worldwide to evaluate a patient-reported assessment of the supportive measures accompanying RP. Pertinent offers vary marginally between CERTs and NCERTs.
Simple Summary
This multicenter study investigated the extent of patient’s decision regret (PatR) in patients with prostate cancer comparing different surgical modalities. Robot-assisted radical prostatectomy has replaced open radical prostatectomy as the surgical standard of care in many countries worldwide. However, a broad scientific basis evaluating the difference in patient-relevant outcomes between both approaches is still lacking. In this context, PatR is increasingly moving into the scientific focus. Our study shows a critical PatR in slightly more than one third of patients about 15 months after surgery. Patients who underwent robot-assisted surgery, and also patients without postoperative urinary stress incontinence, report significantly lower PatR. Likewise, this difference was also demonstrated for patients who decided together with their treating physician on the specific surgical procedure (consensual decision making). Our study helps to further establish PatR as an important endpoint in the setting of radical prostatectomy and identifies criteria which may be addressed to reduce PatR.
Abstract
Patient’s regret (PatR) concerning the choice of therapy represents a crucial endpoint for treatment evaluation after radical prostatectomy (RP) for prostate cancer (PCA). This study aims to compare PatR following robot-assisted (RARP) and open surgical approach (ORP). A survey comprising perioperative-functional criteria was sent to 1000 patients in 20 German centers at a median of 15 months after RP. Surgery-related items were collected from participating centers. To calculate PatR differences between approaches, a multivariate regressive base model (MVBM) was established incorporating surgical approach and demographic, center-specific, and tumor-specific criteria not primarily affected by surgical approach. An extended model (MVEM) was further adjusted by variables potentially affected by surgical approach. PatR was based on five validated questions ranging 0–100 (cutoff >15 defined as critical PatR). The response rate was 75.0%. After exclusion of patients with laparoscopic RP or stage M1b/c, the study cohort comprised 277/365 ORP/RARP patients. ORP/RARP patients had a median PatR of 15/10 (p < 0.001) and 46.2%/28.1% had a PatR >15, respectively (p < 0.001). Based on the MVBM, RARP patients showed PatR >15 relative 46.8% less frequently (p < 0.001). Consensual decision making regarding surgical approach independently reduced PatR. With the MVEM, the independent impact of both surgical approach and of consensual decision making was confirmed. This study involving centers of different care levels showed significantly lower PatR following RARP.
The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentrationdependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)- labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilatorstimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.
This thesis describes the implementation and first on-line application of a multi-reflection time-of-flight (MR-ToF) mass analyzer for high-resolution mass separation at the ISOLTRAP mass spectrometer at ISOLDE/CERN. On the one hand, the major objective was to improve ISOLTRAPs mass-measurement capabilities with respect to the ratio of delivered contaminating ions to ions of interest. On the other hand, the time necessary to purify wanted from unwanted species should be reduced as much as possible to enable access to even more exotic nuclei. The device has been set up, optimized and tested at the University of Greifswald before its move to ISOLTRAP. The achieved performance comprises mass resolving powers of up to 200000 reached at observation times of 30ms and a contamination suppression of about four orders of magnitude by use of a Bradbury-Nielsen gate. With the characteristics, it outperforms clearly the so far state-of-the-art purification method of a gas-filled Penning trap. To improve the utilization of the MR-ToF mass analyzer, the in-trap lift method has been developed. It simplifies the application and optimization of the device, which is a crucial time factor in an on-line experiment. The device was the first of its kind successfully applied to radioactive ion beams for a mass analysis, for a mass separation (in combination with the Bradbury-Nielsen gate) as a preparatory step for a subsequent Penning-trap mass measurement and as a high-precision mass spectrometer of its own. The later was recently used for the first mass measurement of the neutron-rich calcium isotopes 53Ca and 54Ca. The so-far achieved mass-resolving power of 200000 belongs to the highest reported for time-of-flight mass analyzers at all. The first successful application of the MR-ToF system as the only mass separator at ISOLTRAP resulted in the mass measurement of 82Zn. The new mass value has been compared to mass extrapolations of the most recent Hartree-Fock-Bogoliubov (HFB) mass models, HFB-19 to HFB-21, of the BRUSLIB collaboration. The mass of the nuclide is of high interest for the compositions and depth profile of the outer crust of neutron stars. In the classical model of the outer crust of a cold, non-accrediting and non-rotating neutron star, the sequence of nuclides found within this parts is determined mainly by the binding energy of exotic nuclides. The crustal compositions determined with the three HFB mass models differed with respect to the appearance of a layer of 82Zn, originating from different mass extrapolations of this mass. With the new experimental data, the extrapolations could be evaluated. It was found that the HFB-21 mass value differs less from the experimental data than the ones from HFB-19 and 20. Therefore, in the classical model, 82Zn does not appear anymore in the outer crust. Due to its high resolution and very fast measurement time, the MR-ToF mass analyzer will be an important instruments for future activities at ISOLTRAP, at the ISOLDE facility in general, and at other radioactive ion-beam facilities.
Urbanization is a major contributor to the loss of biodiversity. Its rapid progress is mostly at the expense of natural ecosystems and the species inhabiting them. While some species can adjust quickly and thrive in cities, many others cannot. To support biodiversity conservation and guide management decisions in urban areas, it is important to find robust methods to estimate the urban affinity of species (i.e. their tendency to live in urban areas) and understand how it is associated with their traits. Since previous studies mainly relied on discrete classifications of species' urban affinity, often involving inconsistent assessments or variable parameters, their results were difficult to compare. To address this issue, we developed and evaluated a set of continuous indices that quantify species' urban affinity based on publicly available occurrence data. We investigated the extent to which a species' position along the urban affinity gradient depends on the chosen index and how this choice affects inferences about the relationship between urban affinity and a set of morphological, sensory and functional traits. While these indices are applicable to a wide range of taxonomic groups, we examined their performance using a global set of 356 bat species. As bats vary in sensitivity to anthropogenic disturbances, they provide an interesting case study. We found that different types of indices resulted in different rankings of species on the urban affinity spectrum, but this had little effect on the association of traits with urban affinity. Our results suggest that bat species predisposed to urban life are characterized by low echolocation call frequencies, relatively long call durations, small body size and flexibility in the selection of the roost type. We conclude that simple indices are appropriate and practical, and propose to apply them to more taxa to improve our understanding of how urbanization favours or filters species with particular traits.
(1) Background: The aim of this study was to systematically compare TEM sections of mineralized human enamel and dentine prepared by focused ion beam (in situ lift-out) technique and ultramicrotomy through a combination of microscopic examination methods (scanning electron microscopy and transmission electron microscopy). In contrast with published studies, we compared the TEM preparation methods using the same specimen blocks as those for the ultramicrotomy and FIB technique. (2) Methods: A further evaluation of TEM sample preparation was obtained by confocal laser scanning microscopy and atomic force microscopy. In addition, ultramicrotome- and focused ion beam-induced artefacts are illustrated. (3) Results: The FIB technique exposed a major difference between non-decalcified enamel and dentine concerning the ultrastructural morphology compared to ultramicrotome-prepared sections. We found that ultramicrotomy was useful for cutting mineralized dentine, with the possibility of mechanical artefacts, but offers limited options for the preparation of mineralized enamel. FIB preparation produced high-quality TEM sections, showing the anisotropic ultrastructural morphology in detail, with minor structural artefacts. Our results show that the solution of artificial saliva and glutardialdehyde (2.5% by volume) is a very suitable fixative for human mineralized tissue. (4) Conclusions: The protocol that we developed has strong potential for the preparation of mineralized biomaterials for TEM imaging and analysis.
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.
Inflammasome activation and formation of ASC specks in patients with juvenile idiopathic arthritis
(2023)
Objective
The formation of large intracellular protein aggregates of the inflammasome adaptor ASC is a hallmark of inflammasome activation and characteristic of autoinflammation. Inflammasome activated cells release the highly proinflammatory cytokine IL-1β in addition to ASC specks into the extracellular space. Autoinflammatory activity has been demonstrated in systemic JIA, however minimal data exist on the role of inflammasomes in other JIA subtypes. We therefore investigated, if pyroptotic cells are present in the circulation of oligo- and poly-articular JIA.
Methods
Peripheral blood of JIA patients (n = 46) was investigated for ASC speck formation, a key step in inflammasome activation, by flow cytometry and immunofluorescence. Free ASC and proinflammatory cytokine levels were determined by ELISA and multiplex assay.
Results
Oligo-articular JIA patients showed a significantly increased proportion of ASC speck+ monocytes compared to poly-articular JIA patients. In serum free ASC alone is not sufficient to assess inflammasome activity and does not correlate with ASC speck+ monocytes. Compared to control several cytokines were significantly elevated in samples of JIA patients. JIA serum containing antinuclear antibodies, incubated with ASC specks boosts a secondary inflammation by IL-1β production in macrophages.
Conclusion
For the first time, we detect ex vivo inflammasome activation by ASC speck formation in oligo- and poly-articular JIA patients. Most notably, inflammasome activation was significantly higher in oligo- compared to poly-articular JIA patients. This data suggests that inflammasome derived autoinflammation may have a greater influence in the previously thought autoimmune oligo-articular JIA patients.