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Arsenic trioxide is a toxic metalloid and carcinogen that is also used as an anticancer drug, and for this reason it is important to identify the routes of arsenite uptake by cells. In this study the ability of hexose transporters to facilitate arsenic trioxide uptake in Saccharomyces cerevisiae was examined. In the absence of glucose, strains with disruption of the arsenite efflux gene ACR3 accumulated high levels of (73)As(OH)(3). The addition of glucose inhibited uptake by approximately 80%. Disruption of FPS1, the aquaglyceroporin gene, reduced glucose-independent uptake by only about 25%, and the residual uptake was nearly completely inhibited by hexoses, including glucose, galactose, mannose, and fructose but not pentoses or disaccharides. A strain lacking FPS1, ACR3, and all genes for hexose permeases except for HXT3, HXT6, HXT7, and GAL2 exhibited hexose-inhibitable (73)As(OH)(3) uptake, whereas a strain lacking all 18 hexose transport-related genes (HXT1 to HXT17 and GAL2), FPS1 and ACR3, exhibited <10% of wild type (73)As(OH)(3) transport. When HXT1, HXT3, HXT4, HXT5, HXT7, or HXT9 was individually expressed in that strain, hexose-inhibitable (73)As(OH)(3) uptake was restored. In addition, the transport of [(14)C]glucose was inhibited by As(OH)(3). These results clearly demonstrate that hexose permeases catalyze the majority of the transport of the trivalent metalloid arsenic trioxide.
Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1
(2011)
The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-Å 3D map of the 1.7-MDa amphipol-solubilized supercomplex I1III2IV1 from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.
Background: In addition to controlled post-translational modifications proteins can be modified with highly reactive compounds. Usually this leads to a compromised functionality of the protein. Methylglyoxal is one of the most common agents that attack arginine residues. Methylglyoxal is also regarded as a pro-oxidant that affects cellular redox homeostasis by contributing to the formation of reactive oxygen species. Antioxidant enzymes like catalase are required to protect the cell from oxidative damage. These enzymes are also targets for methylglyoxal-mediated modification which could severely affect their catalytic activity in breaking down reactive oxygen species to less reactive or inert compounds.
Results: Here, bovine liver catalase was incubated with high levels of methylglyoxal to induce its glycation. This treatment did not lead to a pronounced reduction of enzymatic activity. Subsequently methylglyoxal-mediated arginine modifications (hydroimidazolone and dihydroxyimidazolidine) were quantitatively analysed by sensitive nano high performance liquid chromatography/electron spray ionisation/tandem mass spectrometry. Whereas several arginine residues displayed low to moderate levels of glycation (e.g., Arg93, Arg365, Arg444) Arg354 in the active centre of catalase was never found to be modified.
Conclusions: Bovine liver catalase is able to tolerate very high levels of the modifying α-oxoaldehyde methylglyoxal so that its essential enzymatic function is not impaired.
Electronic supplementary material: The online version of this article (doi:10.1186/s13104-015-1793-5) contains supplementary material, which is available to authorized users.
Abstract: Simple cells in primary visual cortex were famously found to respond to low-level image components such as edges. Sparse coding and independent component analysis (ICA) emerged as the standard computational models for simple cell coding because they linked their receptive fields to the statistics of visual stimuli. However, a salient feature of image statistics, occlusions of image components, is not considered by these models. Here we ask if occlusions have an effect on the predicted shapes of simple cell receptive fields. We use a comparative approach to answer this question and investigate two models for simple cells: a standard linear model and an occlusive model. For both models we simultaneously estimate optimal receptive fields, sparsity and stimulus noise. The two models are identical except for their component superposition assumption. We find the image encoding and receptive fields predicted by the models to differ significantly. While both models predict many Gabor-like fields, the occlusive model predicts a much sparser encoding and high percentages of ‘globular’ receptive fields. This relatively new center-surround type of simple cell response is observed since reverse correlation is used in experimental studies. While high percentages of ‘globular’ fields can be obtained using specific choices of sparsity and overcompleteness in linear sparse coding, no or only low proportions are reported in the vast majority of studies on linear models (including all ICA models). Likewise, for the here investigated linear model and optimal sparsity, only low proportions of ‘globular’ fields are observed. In comparison, the occlusive model robustly infers high proportions and can match the experimentally observed high proportions of ‘globular’ fields well. Our computational study, therefore, suggests that ‘globular’ fields may be evidence for an optimal encoding of visual occlusions in primary visual cortex.
Author Summary: The statistics of our visual world is dominated by occlusions. Almost every image processed by our brain consists of mutually occluding objects, animals and plants. Our visual cortex is optimized through evolution and throughout our lifespan for such stimuli. Yet, the standard computational models of primary visual processing do not consider occlusions. In this study, we ask what effects visual occlusions may have on predicted response properties of simple cells which are the first cortical processing units for images. Our results suggest that recently observed differences between experiments and predictions of the standard simple cell models can be attributed to occlusions. The most significant consequence of occlusions is the prediction of many cells sensitive to center-surround stimuli. Experimentally, large quantities of such cells are observed since new techniques (reverse correlation) are used. Without occlusions, they are only obtained for specific settings and none of the seminal studies (sparse coding, ICA) predicted such fields. In contrast, the new type of response naturally emerges as soon as occlusions are considered. In comparison with recent in vivo experiments we find that occlusive models are consistent with the high percentages of center-surround simple cells observed in macaque monkeys, ferrets and mice.
Many studies about endocrine pollution in the aquatic environment reveal changes in the reproduction system of biota. We analysed endocrine activities in two rivers in Southern Germany using three approaches: (1) chemical analyses, (2) in vitro bioassays, and (3) in vivo investigations in fish and snails. Chemical analyses were based on gas chromatography coupled with mass spectrometry. For in vitro analyses of endocrine potentials in water, sediment, and waste water samples, we used the E-screen assay (human breast cancer cells MCF-7) and reporter gene assays (human cell line HeLa-9903 and MDA-kb2). In addition, we performed reproduction tests with the freshwater mudsnail Potamopyrgus antipodarum to analyse water and sediment samples. We exposed juvenile brown trout (Salmo trutta f. fario) to water downstream of a wastewater outfall (Schussen River) or to water from a reference site (Argen River) to investigate the vitellogenin production. Furthermore, two feral fish species, chub (Leuciscus cephalus) and spirlin (Alburnoides bipunctatus), were caught in both rivers to determine their gonadal maturity and the gonadosomatic index. Chemical analyses provided only little information about endocrine active substances, whereas the in vitro assays revealed endocrine potentials in most of the samples. In addition to endocrine potentials, we also observed toxic potentials (E-screen/reproduction test) in waste water samples, which could interfere with and camouflage endocrine effects. The results of our in vivo tests were mostly in line with the results of the in vitro assays and revealed a consistent reproduction-disrupting (reproduction tests) and an occasional endocrine action (vitellogenin levels) in both investigated rivers, with more pronounced effects for the Schussen river (e.g. a lower gonadosomatic index). We were able to show that biological in vitro assays for endocrine potentials in natural stream water reasonably reflect reproduction and endocrine disruption observed in snails and field-exposed fish, respectively.
Cockchafers of the genus Melolontha (Coleoptera: Scarabaeidae) can be severe pests in forestry, agriculture and horticulture. Gradation of the two most important species, the forest cockchafer M. hippocastani FABR. and the European cockchafer M. melolontha L., occurs currently in several parts of central Europe. Orientation behaviour of the adult beetles has been the focus of recent studies (REINECKE et al. 2002 a, b, 2005). However, especially the larvae are dreaded because their belowground damage is not visible directly after feeding. There are a lot of speculations about belowground living insects and their way of living, but until now there were not that many experimental investigations. A rather unknown topic is the orientation behaviour of soil living organisms, which is also subject of some publications (HORBER 1954, HAUSS & SCHÜTTE 1976, HASLER 1986, HIBBARD et al. 1994, JEWETT & BJOSTAD 1996, BERNKLAU & BJOSTAD 1998A, BERNKLAU & BJOSTAD 1998B, BERNKLAU et al. 2005).
We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.
Mining is one of the major pollution sources worldwide, causing huge disturbances to the environment. Industrial and artisanal mining activities are widespread in Mexico, a major global producer of various metals. This study aimed to assess the ecological impairments resulting from mining activities using aquatic macroinvertebrates assemblages (MA). A multiple co-inertia analysis was applied to determine the relationships between environmental factors, habitat quality, heavy metals, and aquatic macroinvertebrates in 15 study sites in two different seasons (dry and wet) along two rivers running across the Central Plateau of Mexico. The results revealed three contrasting environmental conditions associated with different MAs. High concentrations of heavy metals, nutrients, and salinity limit the presence of several families of seemingly sensitive macroinvertebrates. These factors were found to influence structural changes in MAs, showing that not only mining activities, but also agriculture and presence of villages in the basin, exert adverse effects on macroinvertebrate assemblages. Diversity indices showed that the lowest diversity matched both the most polluted and the most saline rivers. The rivers studied displayed high alkalinity and hardness levels, which can reduce the availability of metals and cause adverse effects on periphyton by inhibiting photosynthesis and damaging MAs. Aquatic biomonitoring in rivers, impacted by mining and other human activities, is critical for detecting the effect of metals and other pollutants to improve management and conservation strategies. This study supports the design of cost-effective and accurate water quality biomonitoring protocols in developing countries.
Ischemic injuries of the cardiovascular system are still the leading cause of death worldwide. They are often accompanied by loss of cardiomyocytes (CM) and their replacement by non-functional heart tissue. Cardiac fibroblasts (CF) play a major role in the recovery after ischemic injury and in the scar formation. In the last few years researchers were able to reprogram fibroblasts into CM in vitro and in murine models of myocardial infarction using various protocols including a cocktail of microRNAs (miRs). These miRs can target hundreds of messenger RNAs and inhibit their translation into proteins, potentially regulating multiple cellular signaling pathways. Because of this, there has been a rising interest in the use of miRs for therapeutic purposes. However, as different miRs have different effects in different cells, there is the danger of causing serious side effects. These could be alleviated by enacting a cell-specific transport of miRs, for example by using aptamers. Aptamers are usually short strands of DNA or RNA, which can fold into a specific three-dimensional confirmation which allows them to bind specifically to target molecules. Aptamers are commonly selected from a large library for their ability to bind to target molecules using a procedure called SELEX. Aptamers have already been used to transport miRs into cancer cells.
In this thesis, we first established the transport of miRs into cells of the cardiovascular system using aptamers. MiR-126 is an important part of the signaling in endothelial cells (EC), protects from atherosclerosis and supports angiogenesis, which is why we chose it as a candidate to transport into the vasculature. We first tested two aptamers for their ability to internalize into EC and fibroblasts. Both the aptamer for the ubiquitously expressed transferrin receptor (TRA) and a general internalizing RNA motif, but not a control construct, could internalize efficiently into all cell types tested. We then designed three chimeras (Ch) using different strategies to connect TRA to miR-126. While all chimeras could internalize efficiently, only Ch3, which connects TRA to Pre-miR-126 using a sticky bridge structure, had functional effects in EC. Ch3 reduced the protein expression of VCAM-1 in EC and increased the VEGF induced sprouting of EC in a spheroid-sprouting assay. Treatment of breast cancer cells with Ch3 emulated the effects of treatment with classical miR-126-3p and miR-126-5p mimics. In the SK-BR3 cell line Ch3 and miR-126-3p reduce the viability of the cells while they reduce recruitment of EC by the MCF7 cell line. miR-126-5p had no apparent effect in the SK-BR3 line, but increased viability of MCF7 cells, as did Ch3. This implies that Ch3 can be processed to both functional miR-126-3p and miR-126-5p in treated cells.
We were unable to achieve a reprogramming of adult murine cardiac fibroblasts into cells resembling CM using the cocktail of 4 miRs. This indicates that the miR-mediated transdifferentiation is only possible in neonatal fibroblasts. The effects in mice after an AMI might possibly be caused by an enhanced plasticity of fibroblasts in and close to the infarcted area.
We also screened to find aptamers specifically binding to cells of the cardiovascular system. We used two oligonucleotide libraries in a cell-SELEX to select candidates which bind to CF, but not EC. We observed that only the library which contains two randomized regions of 26 bases showed an enrichment of species binding to fibroblasts. We then sequenced rounds 5-7 of the SELEX and analyzed the data bioinfomatically to select 10 candidate aptamers. All candidates showed a strong binding not only to CF, but also EC. This indicates that the selection pressure against species binding to EC was not high enough and would have to be increased to find true CF-aptamers. Four promising candidates were also analyzed for their potential to be internalized and we surprisingly found that all of them were internalized by EC and CF more efficiently than TRA. The similar behavior of the candidates implies that they possibly share a ligand, which is expressed both by EC and CF, but more prominently by the latter.
This work demonstrates the possibility of using aptamers to transport miRs into cells of the cardiovascular system. It also shows that it is possible to select aptamers for non-cancerous mammalian cells, which has not been done before. It is reasonable to assume that a refinement of the cell-SELEX will allow selection of cell-specific aptamers. Due to the failure of reprogramming of adult fibroblasts into induced cardiomyocytes we were unable to test whether a miR-mediated reprogramming might be inducible using aptamer transported-miRs. Ultimately, aptamer mediated transport of miRs is a feasible and promising therapeutic option for the treatment of cardiovascular diseases and other disorders like cancer.
The amyloid precursor protein (APP) was discovered in the 1980s as the precursor protein of the amyloid A4 peptide. The amyloid A4 peptide, also known as A-beta (Aβ), is the main constituent of senile plaques implicated in Alzheimer’s disease (AD). In association with the amyloid deposits, increasing impairments in learning and memory as well as the degeneration of neurons especially in the hippocampus formation are hallmarks of the pathogenesis of AD. Within the last decades much effort has been expended into understanding the pathogenesis of AD. However, little is known about the physiological role of APP within the central nervous system (CNS). Allocating APP to the proteome of the highly dynamic presynaptic active zone (PAZ) identified APP as a novel player within this neuronal communication and signaling network. The analysis of the hippocampal PAZ proteome derived from APP-mutant mice demonstrates that APP is tightly embedded in the underlying protein network. Strikingly, APP deletion accounts for major dysregulation within the PAZ proteome network. Ca2+-homeostasis, neurotransmitter release and mitochondrial function are affected and resemble the outcome during the pathogenesis of AD. The observed changes in protein abundance that occur in the absence of APP as well as in AD suggest that APP is a structural and functional regulator within the hippocampal PAZ proteome. Within this review article, we intend to introduce APP as an important player within the hippocampal PAZ proteome and to outline the impact of APP deletion on individual PAZ proteome subcommunities.
In the deep-sea, the interaction between benthic fauna and substrate mainly occurs through bioturbational processes which can be preserved as traces (i.e., lebensspuren). Lebensspuren are common features of deep seafloor landscapes and usually more abundant than the organism that produce them (i.e., tracemakers), rendering them promising proxies to infer biodiversity. The density and diversity relationships between lebensspuren and benthic fauna are to the present day unclear and contradicting hypotheses have been proposed suggesting negative, positive, or even null correlations. To test these hypotheses, in this study lebensspuren, tracemakers (specific epibenthic fauna that produce these traces), degrading fauna (benthic fauna that can erase lebensspuren), and fauna in general were characterized taxonomically at eight deep-sea stations in the Kuril Kamchatka Trench area. No general correlation (over-all study area) could be observed between diversities of lebensspuren, tracemakers, degrading fauna and fauna. However, a diversity correlation was observed between specific stations, showing both negative and positive correlations depending on: 1) the number of unknown tracemakers (especially significant for dwelling lebensspuren); and 2) the lebensspuren with multiple origins; and 3) tracemakers that can produce different lebensspuren. Lebensspuren and faunal density were not correlated. However, lebensspuren density was either positively or negatively correlated with tracemaker densities, depending on the lebensspuren morphotypes. A positive correlation was observed for resting lebensspuren (e.g., ophiuroid impressions, Actinaria circular impressions), while negative correlations were observed for locomotion-feeding lebensspuren (e.g., echinoid trails). In conclusion, lebensspuren diversity may be a good proxy for tracemaker biodiversity when the lebensspuren-tracemaker tandem can be reliable characterized; and lebensspuren-density correlations vary depending the specific lebensspuren residence time, tracemaker density and associated behaviour (rate of movement), but on a global scale abiotic and other biotic 42 factors may also play an important role.
Research in cell and developmental biology requires the application of three-dimensional model systems that reproduce the natural environment of cells. Processes in developmental biology are therefore studied in entire systems like insects or plants. In cell biology, three-dimensional cell cultures (e.g. spheroids or organoids) model the physiology and pathology of cells, tissues or organs. In all systems, the cellular neighborhood and interactions, but also physicochemical influences, are realistically presented. The production and handling of these model systems is rather simple and allows for reproducible characterization.
Confocal and light sheet-based fluorescence microscopy (LSFM) enable the observation of these systems while maintaining their three-dimensional integrity. LSFM is applicable to imaging live samples at high spatio-temporal resolution over long periods of time. The quality of the acquired datasets enables the extraction of quantitative features about morphology, functionality and dynamics in the context of the complete system. This approach is referred to as image-based systems biology. Exploiting the potential of the generated datasets requires an image analysis pipeline for data management, visualization and the retrieval of biologically meaningful values.
The goal of this thesis was to identify, develop and optimize modules of the image analysis pipeline. The modules cover data management and reduction, visualization, reconstruction of multiview image datasets, the segmentation and tracking of cell nuclei and the extraction of quantitative features. The modules were developed in an application-driven manner to test and ensure their applicability to real datasets from three-dimensional fluorescence microscopy. The underlying datasets were taken from research projects in developmental biology in insects and plants, as well as from cell biology.
The datasets acquired in fluorescence microscopy are typically complex and require common image processing steps in order to manage, visualize, and analyze the datasets. The first module accomplishes automatic structuring of large image datasets, reduces the data amount by image cropping and compression and computes maximum projection images along different spatial directions. The second module corrects for intensity variations in the generated maximum projection images that occur as a function of time. The program was published as a part of an article in Nature Protocols. Another developed module named BugCube provides a web-based platform to visualize and share the processed image datasets.
In LSFM, samples can be rotated in-between two acquisitions enabling the generation of multiview image datasets. Prior to my work, Frederic Strobl and Alexander Ross acquired the complete embryogenesis of the red flour beetle, Tribolium castaneum, and the field cricket, Gryllus bimaculatus, with LSFM. I evaluated a plugin for the software FIJI as a module for the reconstruction of such datasets. The plugin was optimized for automation and efficiency. We obtained the first high quality three-dimensional reconstructions of Tribolium and Gryllus datasets.
Optical clearing increases the penetration depth into samples, thus providing endpoint images of entire three-dimensional objects with cellular detail. This work contributes a quantitative characterization module that was applied to endpoint images of optically cleared spheroids. A program for the generation of ground truth datasets was developed in order to evaluate the cell nuclei segmentation performance. The program was part of a paper that was published in BMC Bioinformatics. Using the program, I could show that the cell nuclei segmentation is robust and accurate. Approaches from computational topology and graph theory complete the segmentation of cell nuclei. Thus, the developed module provides a comprehensive quantitative characterization of spheroids on the level of the individual cell, the cell neighborhood and the whole cell aggregate. The module was employed in four applications to analyze the influence of different stress conditions on the morphology and cellular arrangement of cells in spheroids. The module was accepted for publication in Scientific Reports along with the results for one application. The cell nuclei segmentation further provided a data source for simulation models that used correlation functions to identify structural zones in spheroids. These results were published in Royal Society Interface.
The final part of this work presents a module for cell tracking and lineage reconstruction. In collaboration with Dr. Alexis Maizel, Dr. Jens Fangerau and Dr. Daniel von Wangenheim, I developed a module to track the positions of all cells involved in lateral root formation in Arabidopsis thaliana and used the extracted positions for extensive data analysis. We reconstructed the cell lineages and established the first atlas of all founder cells that contribute to the formation. The analysis of the retrieved data allowed us to study conserved and individual patterns in lateral root formation. The atlas and parts of the analysis presented in this thesis were published in Current Biology.
In this thesis, I developed modules for an image analysis pipeline in three-dimensional fluorescence microscopy and applied them in interdisciplinary research projects. The modules enabled the organization, processing, visualization and analysis of the datasets. The perspective of the image analysis pipeline is not restricted to image-based systems biology. With ongoing development of the image analysis pipeline, it can also be a valuable tool for medical diagnostics or industrial high-throughput approaches.
The DNA damage response (DDR) is a vast network of molecules that preserves genome integrity and allow the faithful transmission of genetic information in human cells. While the usual response to the detection of DNA lesions in cells involves the control of cell-cycle checkpoints, repair proteins or apoptosis, alterations of the repair processes can lead to cellular dysfunction, diseases, or cancer. Besides, cancer patients with DDR alterations often show poor survival and chemoresistance. Despite the progress made in recent years in identifying genes and proteins involved in DDR and their roles in cellular physiology and pathology, the question of the involvement of DDR in metabolism remains unclear. It remains to study the metabolites associated with specific repair pathways or alterations and to investigate whether differences exist depending on cellular origin. The identification of DDR-related metabolic pathways and of the pathways that cause metabolic reprogramming in DDR-deficient cells may produce new targets for the development of new therapies.
In this thesis, nuclear magnetic resonance spectroscopy (NMR) was used to assess the metabolic consequence of the loss of two central DNA repair proteins with importance in diseases context, ATM and RNase H2, in haematological cells. An increase in intracellular taurine was found in RNase H2- and ATM-deficient cells compared to wild-type cells for these genes and in cells after exposition to a source of DNA damage. The rise in taurine does not appear to result from an increase in its biosynthesis from cysteine, but more likely from other cellular processes such as degradation pathways.
Overall, evidence for metabolic reprogramming in haematological cells with faults in DNA repair resulting from ATM or RNase H2 deficiencies or upon exposition to a source of DNA damage is presented in this study.
White stork (Ciconia ciconia) nestlings can provide quantitative information on the quality of the surrounding environment by indicating the presence of pollutants, as they depend on locally foraged food. This study represents the first comparison of biomarkers in two fractions of white stork nestling blood: plasma and S9 (the post-mitochondrial fraction). The aim of this study was to evaluate acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as to establish a novel fluorescence-based method for glutathione (GSH) and reactive oxygen species (ROS) detection in plasma and S9. Considering the enzymatic biomarkers, lower variability in plasma was detected only for AChE, as CES, GST, and GR had lower variability in S9. Enzyme activity was higher in plasma for AChE, CES, and GST, while GR had higher activity in S9. Regarding the fluorescence-based method, lower variability was detected in plasma for GSH and ROS, although higher GSH detection was reported in S9, and higher ROS was detected in plasma. The present study indicated valuable differences by successfully establishing protocols for biomarker measurement in plasma and S9 based on variability, enzyme activity, and fluorescence. For a better understanding of the environmental effects on nestlings’ physiological condition, biomarkers can be measured in plasma and S9.
In an ideal world, extraction of machine-readable data and knowledge from natural-language biodiversity literature would be done automatically, but not so currently. The BIOfid project has developed some tools that can help with important parts of this highly demanding task, while certain parts of the workflow cannot be automated yet. BIOfid focuses on the 20th century legacy literature, a large part of which is only available in printed form. In this workshop, we will present the current state of the art in mobilisation of data from our corpus, as well as some challenges ahead of us. Together with the participants, we will exercise or explain the following tasks (some of which can be performed by the participants themselves, while other tasks currently require execution by our specialists with special equipment): Preparation of text files as an input; pre-processing with TextImager/TextAnnotator; semiautomated annotation and linking of named entities; generation of output in various formats; evaluation of the output. The workshop will also provide an outlook for further developments regarding extraction of statements from natural-language literature, with the long-term aim to produce machine-readable data from literature that can extend biodiversity databases and knowledge graphs.
Application of a developed tool to visualize newly synthesized AMPA receptor components in situ
(2018)
The information flow between neurons happens at contact points, the synapses. One underlying mechanism of learning and memory is the change in the strength of information flow in selected synapses. In order to match the huge demand in membranes and proteins to build and maintain the neurites' complex architecture, neurons use decentralized protein synthesis. Many candidate proteins for local synthesis are known, and the need of de novo synthesis for memory formation is well established. The underlying mechanisms of how somatic versus dendritic synthesis is regulated are yet to be elucidated. Which proteins are newly synthesized in order to allow learning?
In this thesis protein synthesis is studied in hippocampal neurons. The fractional distribution of somatic and dendritic synthesis for candidate proteins and their subsequent transport to their destination are investigated using a newly developed technique. In the first part of this study we describe the development of this technique and use it in the second part to answer biological questions.
We focus here on AMPA receptor subunits, the key players in fast excitatory transmission. AMPA receptors contain multiple subunits with diverse functions. It remains to be understood, when and where in a neuron these subunits come together to form a protein complex and how the choice of subunits is regulated.
The investigation of the subunits' site of synthesis and redistribution kinetics in this study will help us to understand how neurons are able to change their synaptic strength in an input specific manner which eventually allows learning and memory.
Key questions which are addressed in this study:
How can specific newly synthesized endogenous proteins be visualized in situ? What are the neuron's abilities to locally synthesize and fully assemble AMPA receptor complexes?
How fast do different AMPA receptor subunits redistribute within neurons after synthesis?
Antimicrobial resistance became a serious threat to the worldwide public health in this century. A better understanding of the mechanisms, by which bacteria infect host cells and how the host counteracts against the invading pathogens, is an important subject of current research. Intracellular bacteria of the Salmonella genus have been frequently used as a model system for bacterial infections. Salmonella are ingested by contaminated food or water and cause gastroenteritis and typhoid fever in animals and humans. Once inside the gastrointestinal tract, Salmonella can invade intestinal epithelial cells. The host cell can fight against intracellular pathogens by a process called xenophagy. For complex systems, such as processes involved in the bacterial infection of cells, computational systems biology provides approaches to describe mathematically how these intertwined mechanisms in the cell function. Computational systems biology allows the analysis of biological systems at different levels of abstraction. Functional dependencies as well as dynamic behavior can be studied. In this thesis, we used the Petri net formalism to gain a better insight into bacterial infections and host defense mechanisms and to predict cellular behavior that can be tested experimentally. We also focused on the development of new computational methods.
In this work, the first realization of a mathematical model of the xenophagic capturing of Salmonella enterica serovar Typhimurium in epithelial cells was developed. The mathematical model expressed in the Petri net formalism was constructed in an iterative way of modeling and analyses. For the model verification, we analyzed the Petri net, including a computational performance of knockout experiments named in silico knockouts, which was established in this work. The in silico knockouts of the proposed Petri net are consistent with the published experimental perturbation studies and, thus, ensures the biological credibility of the Petri net. In silico knockouts that have not been experimentally investigated yet provide hypotheses for future investigations of the pathway.
To study the dynamic behavior of an epithelial cell infected with Salmonella enterica serovar Typhimurium, a stochastic Petri net was constructed. In experimental research, a decision like "Which incubation time is needed to infect half of the epithelial cells with Salmonella?" is based on experience or practicability. A mathematical model can help to answer these questions and improve experimental design. The stochastic Petri net models the cell at different stages of the Salmonella infection. We parameterized the model by a set of experimental data derived from different literature sources. The kinetic parameters of the stochastic Petri net determine the time evolution of the bacterial infection of a cell. The model captures the stochastic variation and heterogeneity of the intracellular Salmonella population of a single cell over time. The stochastic Petri net is a valuable tool to examine the dynamics of Salmonella infections in epithelial cells and generate valuable information for experimental design.
In the last part of this thesis, a novel theoretical method was introduced to perform knockout experiments in silico. The new concept of in silico knockouts is based on the computation of signal flows at steady state and allows the determination of knockout behavior that is comparable to experimental perturbation behavior. In this context, we established the concept of Manatee invariants and demonstrated the suitability of their application for in silico knockouts by reflecting biological dependencies from the signal initiation to the response. As a proof of principle, we applied the proposed concept of in silico knockouts to the Petri net of the xenophagic recognition of Salmonella. To enable the application of in silico knockouts for the scientific community, we implemented the novel method in the software isiKnock. isiKnock allows the automatized performance and visualization of in silico knockouts in signaling pathways expressed in the Petri net formalism. In conclusion, the knockout analysis provides a valuable method to verify computational models of signaling pathways, to detect inconsistencies in the current knowledge of a pathway, and to predict unknown pathway behavior.
In summary, the main contributions of this thesis are the Petri net of the xenophagic capturing of Salmonella enterica serovar Typhimurium in epithelial cells to study the knockout behavior and the stochastic Petri net of an epithelial cell infected with Salmonella enterica serovar Typhimurium to analyze the infection dynamics. Moreover, we established a new method for in silico knockouts, including the concept of Manatee invariants and the software isiKnock. The results of these studies are useful to a better understanding of bacterial infections and provide valuable model analysis techniques for the field of computational systems biology.
The turnover time of terrestrial ecosystem carbon is an emergent ecosystem property that quantifies the strength of land surface on the global carbon cycle–climate feedback. However, observation- and modeling-based estimates of carbon turnover and its response to climate are still characterized by large uncertainties. In this study, by assessing the apparent whole ecosystem carbon turnover times (τ) as the ratio between carbon stocks and fluxes, we provide an update of this ecosystem level diagnostic and its associated uncertainties in high spatial resolution (0.083∘) using multiple, state-of-the-art, observation-based datasets of soil organic carbon stock (Csoil), vegetation biomass (Cveg) and gross primary productivity (GPP). Using this new ensemble of data, we estimated the global median τ to be 43+7−7 yr (median+difference to percentile 75−difference to percentile 25) when the full soil is considered, in contrast to limiting it to 1 m depth. Only considering the top 1 m of soil carbon in circumpolar regions (assuming maximum active layer depth is up to 1 m) yields a global median τ of 37+3−6 yr, which is longer than the previous estimates of 23+7−4 yr (Carvalhais et al., 2014). We show that the difference is mostly attributed to changes in global Csoil estimates. Csoil accounts for approximately 84 % of the total uncertainty in global τ estimates; GPP also contributes significantly (15 %), whereas Cveg contributes only marginally (less than 1 %) to the total uncertainty. The high uncertainty in Csoil is reflected in the large range across state-of-the-art data products, in which full-depth Csoil spans between 3362 and 4792 PgC. The uncertainty is especially high in circumpolar regions with an uncertainty of 50 % and a low spatial correlation between the different datasets (0.2<r<0.5) when compared to other regions (0.6<r<0.8). These uncertainties cast a shadow on current global estimates of τ in circumpolar regions, for which further geographical representativeness and clarification on variations in Csoil with soil depth are needed. Different GPP estimates contribute significantly to the uncertainties of τ mainly in semiarid and arid regions, whereas Cveg causes the uncertainties of τ in the subtropics and tropics. In spite of the large uncertainties, our findings reveal that the latitudinal gradients of τ are consistent across different datasets and soil depths. The current results show a strong ensemble agreement on the negative correlation between τ and temperature along latitude that is stronger in temperate zones (30–60∘ N) than in the subtropical and tropical zones (30∘ S–30∘ N). Additionally, while the strength of the τ–precipitation correlation was dependent on the Csoil data source, the latitudinal gradients also agree among different ensemble members. Overall, and despite the large variation in τ, we identified robust features in the spatial patterns of τ that emerge beyond the differences stemming from the data-driven estimates of Csoil, Cveg and GPP. These robust patterns, and associated uncertainties, can be used to infer τ–climate relationships and for constraining contemporaneous behavior of Earth system models (ESMs), which could contribute to uncertainty reductions in future projections of the carbon cycle–climate feedback. The dataset of τ is openly available at https://doi.org/10.17871/bgitau.201911 (Fan et al., 2019).
The turnover time of terrestrial carbon (τ) controls the global carbon cycle – climate feedback and, yet, is poorly simulated by the current Earth System Models (ESMs). In this study, by assessing apparent carbon turnover time as the ratio between carbon stocks and fluxes, we provide a new, updated ensemble of diagnostic terrestrial carbon turnover times and associated uncertainties on a global scale using multiple, state-of-the-art, observation-based datasets of soil organic carbon stock (Csoil), vegetation biomass (Cveg) and gross primary productivity (GPP). Using this new ensemble, we estimated the global average τ to be 42$% &' years when the full soil depth is considered, longer than the previous estimates of 23$) &* years. Only considering the top 1 m (assuming maximum active layer depth is up to 1 meter) of soil carbon in circumpolar regions yields a global τ of 35$) &' years. Csoil in circumpolar regions account for two thirds of the total uncertainty in global τ estimates, whereas Csoil in non-circumpolar contributes merely 9.38%. GPP (2.25%) and Cveg (0.05%) contribute even less to the total uncertainty. Therefore, the high uncertainty in Csoil is the main factor behind the uncertainty in global τ, as reflected in the larger range of full-depth Csoil (3152-4372 PgC). The uncertainty is especially high in circumpolar regions with a behaviour of ESMs which could contribute to uncertainty reductions in future projections of the carbon cycle - climate feedback. The dataset of the terrestrial turnover time ensemble (DOI: 10.17871/bgitau.201911) is openly available from the data portal: https://doi.org/10.17871/bgitau.201911 (Fan et al., 2019) uncertainty of 50% and the spatial correlations among different datasets are also low compared to other regions. Overall, we argue that current global datasets do not support robust estimates of τ globally, for which we need clarification on variations of Csoil with soil depth and stronger estimates of Csoil in circumpolar regions. Despite the large variation in both magnitude and spatial patterns of τ, we identified robust features in the spatial patterns of τ that emerge regardless of soil depth and differences in data sources of Csoil, Cveg and GPP. Our findings show that the latitudinal gradients of τ are consistent across different datasets and soil depth. Furthermore, there is a strong consensus on the negative correlation between τ and temperature along latitude that is stronger in temperate zones (30ºN-60ºN) than in subtropical and tropical zones (30ºS30ºN). The identified robust patterns can be used to infer the response of τ to climate and for constraining contemporaneous behaviour of ESMs which could contribute to uncertainty reductions in future projections of the carbon cycle - climate feedback. The dataset of the terrestrial turnover time ensemble (DOI:10.17871/bgitau.201911) is openly available from the data portal: https://doi.org/10.17871/bgitau.201911 (Fan et al., 2019).
Abstract: The hallmarks of Alzheimer’s disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aβ-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network.
Author Summary: More than 20 years ago, the amyloid precursor protein (APP) was identified as the precursor protein of the Aβ peptide, the main component of senile plaques in brains affected by Alzheimer’s disease. However, little is known about the physiological function of amyloid precursor protein. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone highlights APP as a hitherto unknown player within the presynaptic network. The hippocampus is the most prominent brain region for learning and memory consolidation, and a vulnerable target for neurodegenerative disease, e. g. Alzheimer’s disease. Therefore, our experimental design is focused on the hippocampal neurotransmitter release site. Currently, the underlying mechanism of how APP acts within presynaptic networks is still elusive. Within the scope of this research article, we constructed a network of APP within the presynaptic active zone and how deletion of APP affects these individual networks. We combine bioinformatics tools and biochemical approaches to address the dataset provided by proteomics. Furthermore, we could unravel that APP executes regulatory functions within the synaptic vesicle cycle, cytoskeletal rearrangements and Ca2+-homeostasis. Taken together, our findings offer a new perspective on the physiological function of APP in the central nervous system and may provide a molecular link to the pathogenesis of Alzheimer’s disease.
Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.
Mitochondrial cristae morphology is highly variable and altered under numerous pathological conditions. The protein complexes involved are largely unknown or only insufficiently characterized. Using complexome profiling we identified apolipoprotein O (APOO) and apolipoprotein O-like protein (APOOL) as putative components of the Mitofilin/MINOS protein complex which was recently implicated in determining cristae morphology. We show that APOOL is a mitochondrial membrane protein facing the intermembrane space. It specifically binds to cardiolipin in vitro but not to the precursor lipid phosphatidylglycerol. Overexpression of APOOL led to fragmentation of mitochondria, a reduced basal oxygen consumption rate, and altered cristae morphology. Downregulation of APOOL impaired mitochondrial respiration and caused major alterations in cristae morphology. We further show that APOOL physically interacts with several subunits of the MINOS complex, namely Mitofilin, MINOS1, and SAMM50. We conclude that APOOL is a cardiolipin-binding component of the Mitofilin/MINOS protein complex determining cristae morphology in mammalian mitochondria. Our findings further assign an intracellular role to a member of the apolipoprotein family in mammals.
Using apoE phenotyping by immunoblotting and apoE genotyping we identified four heterozygous carriers of a rare apolipoprotein (apo) E2 variant, apoE2 (Arg136 → Cys). ApoE2 (Arg136 → Cys) was not distinct from apoE2 (Arg158 → Cys) by phenotyping, but produced a unique pattern of bands on CfoI restriction typing of a 244 bp apoE gene fragment. Two of the four apoE2 (Arg136 → Cys)/3 heterozygotes had elevated triglycerides, two were normolipidemic. The composition of very low density lipoproteins (VLDL) was normal in each of the four apoE2 (Arg136 → Cys) carriers, regardless of the triglyceride concentrations. None of the apoE2 (Arg136 → Cys) carriers displayed a broad β-band and none revealed β-migrating particles in the VLDL. The two hypertriglyceridemic carriers of apoE2 (Arg136 → Cys) were, therefore, classified as having type IV rather than type III hyperlipoproteinemia. LDL receptor binding activities were studied using recombinant apoE loaded to dimyristoylphosphatidylcholine (DMPC) vesicles and to VLDL and from an apoE-deficient individual. LDL receptor binding of apoE2 (Arg136 → Cys) was 14% of apoE3 and was thus higher than that of apoE2 (Arg158 → Cys). Both apoE2 (Arg136 → Cys) and apoE2 (Arg158 → Cys) displayed substantial heparin binding (61 and 53% of apoE3, respectively). As the dominant apoE variants known so far are characterized by more pronounced reductions of heparin binding, we suggest that apoE2 (Arg136 → Cys) is not associated with dominant expression of type III hyperlipoproteinemia. These findings lend support to the concept that apoE variants predisposing to dominant type III hyperlipoproteinemia differ from recessive mutations by a more severe defect in heparin binding.—März, W., M. M. Hoffmann, H. Scharnagl, E. Fisher, M. Chen, M. Nauck, G. Feussner, and H. Wieland. Apolipoprotein E2 (Arg136 → Cys) mutation in the receptor binding domain of apoE is not associated with dominant type III hyperlipoproteinemia.
Alternative polyadenylation (APA) is a widespread mechanism that contributes to the sophisticated dynamics of gene regulation. Approximately 50% of all protein-coding human genes harbor multiple polyadenylation (PA) sites; their selective and combinatorial use gives rise to transcript variants with differing length of their 3' untranslated region (3'UTR). Shortened variants escape UTR-mediated regulation by microRNAs (miRNAs), especially in cancer, where global 3'UTR shortening accelerates disease progression, dedifferentiation and proliferation. Here we present APADB, a database of vertebrate PA sites determined by 3' end sequencing, using massive analysis of complementary DNA ends. APADB provides (A)PA sites for coding and non-coding transcripts of human, mouse and chicken genes. For human and mouse, several tissue types, including different cancer specimens, are available. APADB records the loss of predicted miRNA binding sites and visualizes next-generation sequencing reads that support each PA site in a genome browser. The database tables can either be browsed according to organism and tissue or alternatively searched for a gene of interest. APADB is the largest database of APA in human, chicken and mouse. The stored information provides experimental evidence for thousands of PA sites and APA events. APADB combines 3' end sequencing data with prediction algorithms of miRNA binding sites, allowing to further improve prediction algorithms. Current databases lack correct information about 3'UTR lengths, especially for chicken, and APADB provides necessary information to close this gap. Database URL: http://tools.genxpro.net/apadb/
Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.
This review summarizes studies of protection against singlet oxygen and radical damage by carotenoids. The main focus is on how substitutions of the carotenoid molecules determine high antioxidant activities such as singlet oxygen quenching and radical scavenging. Applied assays were carried out either in vitro in solvents or with liposomes, and in a few cases with living organisms. In the latter, protection by carotenoids especially of photosynthesis against light- and UV-stress is of major importance, but also heterotrophic organisms suffer from high light and UV exposure which can be alleviated by carotenoids. Carotenoids to be compared include C30, C40 and C50 molecules either acyclic, monocyclic or bicyclic with different substitutions including sugar and fatty acid moieties. Although some studies are difficult to compare, there is a tendency towards mono and bicyclic carotenoids with keto groups at C-4/C-4’ and the longest possible polyene structure functions to act best in singlet oxygen quenching and radical scavenging. Size of the carotenoid and lipophilic substituents such as fatty acids seem to be of minor importance for their activity but hydroxyl groups at an acyclic end and especially glycosylation of these hydroxyl groups enhance carotenoid activity.
Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers
(2014)
Background: HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure.
Results: Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction.
Conclusions: 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.
Phenytoin (PHT), valproic acid, and modern antiepileptic drugs (AEDs), eg, remacemide, loreclezole, and safinamide, are only effective within a maximum of 70%-80% of epileptic patients, and in many cases the clinical use of AEDs is restricted by their side effects. Therefore, a continuous need remains to discover innovative chemical entities for the development of active and safer AEDs. Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. To determine the potential of H3Rs ligands as new AEDs, we recently reported that no anticonvulsant effects were observed for the (S)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propanamide (1). In continuation of our research, we asked whether anticonvulsant differences in activities will be observed for its R-enantiomer, namely, (R)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propaneamide (2) and analogs thereof, in maximum electroshock (MES)-, pentylenetetrazole (PTZ)-, and strychnine (STR)-induced convulsion models in rats having PHT and valproic acid (VPA) as reference AEDs. Unlike the S-enantiomer (1), the results show that animals pretreated intraperitoneally (ip) with the R-enantiomer 2 (10 mg/kg) were moderately protected in MES and STR induced models, whereas proconvulsant effect was observed for the same ligand in PTZ-induced convulsion models. However, animals pretreated with intraperitoneal doses of 5, 10, or 15 mg/kg of structurally bulkier (R)-enantiomer (3), in which 3-piperidinopropan-1-ol in ligand 2 was replaced by (4-(3-(piperidin-1-yl)propoxy)phenyl)methanol, and its (S)-enantiomer (4) significantly and in a dose-dependent manner reduced convulsions or exhibited full protection in MES and PTZ convulsions model, respectively. Interestingly, the protective effects observed for the (R)-enantiomer (3) in MES model were significantly greater than those of the standard H3R inverse agonist/antagonist pitolisant, comparable with those observed for PHT, and reversed when rats were pretreated with the selective H3R agonist R-(α)-methyl-histamine. Comparisons of the observed antagonistic in vitro affinities among the ligands 1-6 revealed profound stereoselectivity at human H3Rs with varying preferences for this receptor subtype. Moreover, the in vivo anticonvulsant effects observed in this study for ligands 1-6 showed stereoselectivity in different convulsion models in male adult rats.
Mast cells are long-lived tissue-resident leukocytes, located most abundantly in the skin and mucosal surfaces. They belong to the first line of defence of the body, protecting against invading pathogens, toxins and allergens. Their secretory granules are densely packed with a plethora of mediators, which can be released immediately upon activation of the cell. Next to their role in IgE-mediated allergic diseases and in promoting inflammation, potential anti-inflammatory functions have been assigned to mast cells, depending on the biological setting. The aim of this thesis was to contribute to a better understanding of the role of mast cells during the resolution of a local inflammation. Therefore, in a first of step a suitable model of a local inflammation had to be identified. Since comparison of the two Toll-like receptor (TLR)-agonists zymosan and lipopolysaccharide (LPS), which are most commonly used to locally induce inflammation, revealed a systemic response after LPS-injection and a local inflammation after zymosan-injection, the TLR2 agonist zymosan was chosen for the subsequent experiments. Multi epitope ligand cartography (MELC) combined with statistical neighbourhood analysis showed that mast cells are located in an anti-inflammatory microenvironment next to M2 macrophages during resolution of inflammation, while neutrophils and M1 macrophages are located in the zymosan-filled core of the inflammation. Furthermore, infiltrating neutrophils during peak inflammation and an increasing population of macrophages phagocytosing neutrophils during resolution of inflammation could be observed. MELC as well as flow cytometry analysis of mast cell-deficient mice revealed a decreased phagocytosing activity of macrophages in the absence of mast cells. As an untargeted approach to identify mast cell-derived mediators induced by zymosan, mRNA sequencing of bone marrow-derived mast cells (BMMCs) was performed. Gene ontology term analysis of the sequencing data revealed the induction of the type I interferon (IFN) pathway as the dominant response. Contradicting previous studies, I could validate the production of IFN-β by mast cells in response to zymosan and LPS in vitro. Furthermore IFN-β expression by mast cells was also detected in vivo. In accordance with previous studies regarding other cell types the release of IFN-β by mast cells depends on endosomal signaling. The potential of IFN-β to enhance the phagocytosing activity of macrophages has been demonstrated recently. Besides IFN-β, various other mediators with reported enhancing effects on macrophage phagocytosis were also induced by zymosan in BMMCs, including Interleukin (IL)-1β, IL-4, IL-13, and Prostaglandin (PG) E2. Thus, either one of these mediators alone or a combination of them could promote macrophage phagocytosis.
In conclusion, I herein present mast cells as a novel source for IFN-β induced by non-viral TLR ligands and demonstrate their enhancing effect on macrophage phagocytosis, thereby contributing to the resolution of inflammation.
The present article analyzes a prominent yet relatively understudied contact space among Native American, New Zealand Maori, and aboriginal Taiwanese literatures: the struggle of indigenous peoples to negotiate optimal relationships between themselves and the natural world, particularly in light of capitalist modernity and globalization. Many indigenous narratives draw sharp distinctions between native peoples and outsiders, predictably portraying the former as protectors and the latter as destroyers of both nature and indigenous local cultures. The Native American Chickasaw writer Linda Hogan's (1947-) novel 'People of the Whale' (2008), the Maori writer Patricia Grace's (1937-) novel 'Patiki' (1986), and the aboriginal Taiwanese writer Topas Tamapima's short story "Zuihou de lieren" are no exception. But these texts also problematize notions of the so-called "ecological native." They do so most conspicuously by revealing the ambiguous relationships those peoples believed closest to nature have with the nonhuman world, that is to say their environmental ambiguity ('ecoambiguity') (Thornber 2012).
Islands are particularly noteworthy for global conservation because of the high number of species they host, the high levels of species endemism, and the large number and proportion of species at risk of extinction. Much of the conservation threat on islands is from invasive species. Whilst biosecurity is an increasing focus of attention for authorities globally, species are continuing to establish in new locations outside of their native ranges. Among invasive species, ants are a prominent taxon, especially on islands. Over the past decade, following the detection of one of the world’s worst invasive ant species, African big-headed ant Pheidole megacephala, the environmental management authority on world-heritage-listed Lord Howe Island has focused attention on invasive ants. This detection influenced the creation of biosecurity measures to prevent further incursions of exotic species, particularly ants. Despite these efforts, over the following decade numerous ant species were collected on the island for the first time, indicating a serious biosecurity problem. Here, we investigate the chronosequence of ant introductions to Lord Howe Island to quantify the extent and nature of the island’s ant biosecurity problem. A total of 45 species have been collected on the island and of these, 12 are considered to be endemic, and a further seven are possibly native. Nineteen of the 26 introduced species (42% of the total fauna and 73% of the introduced fauna) were only found for the first time in the last 15 years. All but two of the species that are not native to Lord Howe Island are native to the Australian mainland, indicating that the biosecurity threat comes from the transport of goods from the Australian mainland. We suggest that the pattern of accelerating ant species accumulation on Lord Howe Island is probably not an isolated phenomenon, and that it is probably occurring on most islands globally that are habitable by ants and visited by people.
Diffusion of inner membrane proteins is a prerequisite for correct functionality of mitochondria. The complicated structure of tubular, vesicular or flat cristae and their small connections to the inner boundary membrane impose constraints on the mobility of proteins making their diffusion a very complicated process. Therefore we investigate the molecular transport along the main mitochondrial axis using highly accurate computational methods. Diffusion is modeled on a curvilinear surface reproducing the shape of mitochondrial inner membrane (IM). Monte Carlo simulations are carried out for topologies resembling both tubular and lamellar cristae, for a range of physiologically viable crista sizes and densities. Geometrical confinement induces up to several-fold reduction in apparent mobility. IM surface curvature per se generates transient anomalous diffusion (TAD), while finite and stable values of projected diffusion coefficients are recovered in a quasi-normal regime for short- and long-time limits. In both these cases, a simple area-scaling law is found sufficient to explain limiting diffusion coefficients for permeable cristae junctions, while asymmetric reduction of the junction permeability leads to strong but predictable variations in molecular motion rate. A geometry-based model is given as an illustration for the time-dependence of diffusivity when IM has tubular topology. Implications for experimental observations of diffusion along mitochondria using methods of optical microscopy are drawn out: a non-homogenous power law is proposed as a suitable approach to TAD. The data demonstrate that if not taken into account appropriately, geometrical effects lead to significant misinterpretation of molecular mobility measurements in cellular curvilinear membranes.
This paper describes work on the morphological and syntactic annotation of Sumerian cuneiform as a model for low resource languages in general. Cuneiform texts are invaluable sources for the study of history, languages, economy, and cultures of Ancient Mesopotamia and its surrounding regions. Assyriology, the discipline dedicated to their study, has vast research potential, but lacks the modern means for computational processing and analysis. Our project, Machine Translation and Automated Analysis of Cuneiform Languages, aims to fill this gap by bringing together corpus data, lexical data, linguistic annotations and object metadata. The project’s main goal is to build a pipeline for machine translation and annotation of Sumerian Ur III administrative texts. The rich and structured data is then to be made accessible in the form of (Linguistic) Linked Open Data (LLOD), which should open them to a larger research community. Our contribution is two-fold: in terms of language technology, our work represents the first attempt to develop an integrative infrastructure for the annotation of morphology and syntax on the basis of RDF technologies and LLOD resources. With respect to Assyriology, we work towards producing the first syntactically annotated corpus of Sumerian.
Annotated world bibliography of host fruits of Bactrocera latifrons (Hendel) (Diptera: Tephritidae)
(2013)
Bactrocera latifrons (Hendel) (Diptera: Tephritidae) infests fruits and vegetables of a number of different plant species, with host plants primarily found in the plant families Solanaceae and Cucurbitaceae. Although B. latifrons is of primarily Asian distribution (e.g., Pakistan, India, Sri Lanka, Burma, China [Fujian, Yunnan, Hong Kong, Hainan], Thailand, Laos, Vietnam, Malaysia, Singapore, Taiwan, and Brunei), its range has expanded through introductions into Hawaii, Okinawa, Tanzania, and Kenya, and poses a threat of introduction into other countries where it does not presently occur. As with other tephritid fruit fly species, establishment of B. latifrons can have significant economic consequences, including damage and loss of food production, as well as requirements for implementation of costly quarantine treatments to permit export of commodities susceptible to infestation by B. latifrons. In order to avoid these adverse economic consequences, one needs to prevent the entry, establishment and spread of B. latifrons into a new habitat. To successfully achieve this, an accurate knowledge of the fly’s host plants is essential. Cognizant of this need, we prepared, and present here, a worldwide list of host plants for B. latifrons, with annotations on reported laboratory and field infestation data. Overall, a total of 59 plant species from 14 plant families are identified as hosts of B. latifrons, based on reported field infestation data.
Animals use the geomagnetic field and astronomical cues to obtain compass information. The magnetic compass is not a uniform mechanism, as several functional modes have been described in different animal groups. The Sun compass requires the internal clock to interpret the position of the Sun. For star compass orientation, night-migrating birds seem to use the star pattern as a whole, without involving the internal clock. Both the astronomical compass mechanisms are based on learning processes to adapt them to the geographic latitude where the animals live and, in long-living animals, to compensate for the seasonal changes. Several mechanisms are used to determine the compass course to a goal. Using information collected during the outward journey is mostly done by path integration: recording the direction with a compass and integrating its twists and turns. Migratory animals have innate programs to guide them to their still unknown goal. Highly mobile animals with large ranges develop a so-called navigational ‘map’, a mental representation of the spatial distribution of navigational factors within their home region and their migration route. The nature of the factors involved is not yet entirely clear; magnetic intensity and inclination are the ones best supported so far.
The brain vascular system is composed of specialized endothelial cells, which regulate the movement of ions, molecules and cells from the blood lumen to the central nervous system (CNS). Endothelial cells in the brain form the blood-brain barrier (BBB) that is essential to maintain the brain homeostasis and protect the CNS from pathogens and toxins for a proper neurological function. Endothelium together with other cellular components such as pericytes, astrocytes and the basement membrane, forms the neurovascular unit (NVU), the structural unit of the BBB. Breakdown of the BBB occurs in various neurological disorders, leading to edema and neuronal damage. Therapeutic strategies focusing on factors that regulate the permeability of the BBB may help to improve neurological disorders and facilitate drug delivery to the brain.
Angiopoietins (Ang) are potential candidates for therapeutic targeting the BBB due to their role in regulating the vascular permeability in periphery. They are key growth factors that control angiogenesis and vessel maturation. Ang-1 and Ang-2 possess similar binding affinities to the Tie2 receptor tyrosine kinase, which is almost exclusively expressed on endothelial cells. Ang-1 is expressed in smooth muscle cells and pericytes, and binds in a paracrine manner to Tie2. This results in phosphorylation of the receptor and induction of downstream signaling pathways leading to vessel maturation via pericyte recruitment and blood vessel stabilization. Ang-2, on the other hand, is stored in Weibel Palade bodies in endothelial cells and is released upon inflammatory or angiogenic stimuli. Therefore, in mature, stabilized blood vessels, Ang-2 expression is low. Increased level of Ang-2 is only observed during development or in pathology such as ischemia, cancer and inflammation. When Ang-2 is released, it acts in an autocrine manner and interferes with Tie2 phosphorylation in a context-dependent way. Antagonizing the receptor results in de-stabilization of the vessels, often accompanied by reduced numbers of pericytes leading to myeloid cell infiltration. In conjunction with the vascular endothelial growth factor (VEGF), Ang-2 contributes to blood vessel sprouting, whereupon in absence of VEGF it promotes vessel regression. ...
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
In the past decades, the use and production of chemicals has been on the rise globally due to increasing industrialization and intensive agriculture; resulting in the occurrence and ecotoxicological risks of chemicals of emerging concern (CECs) in the aquatic compartments. Risks include changes in community structure resulting in the dominance of one species and ecosystem imbalance. When dominant disease-causing organisms are in the environment, the disease transmission is increased. For example, host snails for the schistosomiasis, a human trematode disease, are known to be tolerant to pesticide
exposure compared to the predators. This would therefore result in an increased abundance of snails which consequently increase the disease transmission in the human population.
Kenya, being a low income country faces a lot of challenges with provision of clean water, diseases and sanitation facilities, and increasing population which results in intensive agriculture coupled with pesticide use. Although a lot of research has been carried out on the environmental occurrence and risk of CECs (Chapter 1), most of these studies have been done in developed countries with limited information from Africa. Additionally, research in Africa focused on urban areas with limited number of compounds analyzed and mostly in the water phase, and inadequate information on the effects of CECs on the aquatic organisms. In order to reduce this knowledge gap, this dissertation focused on identification and quantification of CECs present in water, sediment and snails from western Kenya, and the contribution of pesticides to the transmission of schistosomiasis.
Chapter 2 gives a summary of the results and discussion of the dissertation. In Chapter 3, a comprehensive chemical analysis was carried out on 48 water samples to identify compounds, spatial patterns and associated risks for fish, crustacean and algae using toxic unit (TU) approach. A total of 78 compounds were detected with pesticides and biocides being the compounds most frequently detected. Spatial pattern analysis revealed limited compound grouping based on land use. Acute risk for crustaceans and algae were driven by one to three individual compounds. These compounds responsible for toxicity were prioritized as candidate compounds for monitoring and regulation in Kenya.
In Chapter 4, an extension of Chapter 3 was done to cover the CECs present in snails and sediment from the 48 sites. A total of 30 compounds were found in snails and 78 in sediments with 68 additional compounds being found which were not previously detected in water. Higher contaminant concentrations were found in agricultural sites than in areas without anthropogenic activities. The highest acute toxicity (TU 0.99) was determined for crustaceans based on compounds in sediment samples. The risk was driven by diazinon and pirimiphos-methyl. Acute and chronic risks to algae were driven by diuron whereas fish were found to be at low to no acute risk.
In Chapter 5, the effect of pesticide contamination on schistosomiasis transmission was evaluated by applying complimentary laboratory and field studies. In the field studies, the ecological mechanisms through which pesticides and physical chemical parameters affect host snails, predators and competitors were investigated. Pesticide data was obtained from the results in chapter 3. The overall distribution of grazers and predators was not affected by pesticide pollution. However, within the grazers, pesticide pollution increased dominance of host snails. On the contrary, the host-snail competitors were highly sensitive to pesticide exposure. For the laboratory studies, macroinvertebrates including Schistosoma-host snails, competitors and predators were exposed to 6 concentrations levels of imidacloprid and diazinon. Snails showed higher insecticide tolerance compared to competitors and predators. Finally, Chapter 6 summarizes the conclusions of this dissertation, placing it in a broader
context. In this dissertation, a comprehensive chemical characterization and risk assessment of CECs has been carried out in freshwater systems; together with the effects of pesticides on schistosomiasis transmission in rural western Kenya. Results of this dissertation showed that rural areas are contaminated posing a risk to aquatic organisms which contribute to schistosomiasis transmission. This shows the need for regular monitoring and policy formulation to reduce pollutant emissions which contributes negatively to both ecological and human health effects.
A large number of chemicals are constantly introduced to surface water from anthropogenic and natural sources. Although substantial efforts have been made to identify these chemicals (e.g potentially anthropogenic contaminants) in surface waters using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS), a large number of LC-HRMS chemical signals often with high peak intensity are left unidentified. In addition to synthetic chemicals and transformation products, these signals may also represent plant secondary metabolites (PSMs) released from vegetation through various pathways such as leaching, surface run-off and rain sewers or input of litter from vegetation. While this may be considered as a confounding factor in screening of water contaminants, it could also contribute to the cumulative toxic risk of water contamination. However, it is hardly known to what extent these metabolites contribute to the chemical mixture of surface waters. Thus, reducing the number of unknowns in water samples by identifying also PSMs in significant concentrations in surface waters will help to improve monitoring and assessment of water quality potentially impacted by complex mixtures of natural and synthetic compounds. Therefore, the main focus of the present study was to identify the occurrence of PSMs in river waters and explore the link between the presence of vegetation along rivers and detection of their corresponding PSMs in river
water.
In order to achieve the goals of the present thesis, two chemical screening approaches, namely, non-target and target screening using LC-HRMS were implemented. (1) Non-target analysis involving a novel approach has been applied to associate unknown peaks of high intensity in LC-HRMS to PSMs from surrounding vegetation by focusing on peaks overlapping between river water and aqueous plant extracts (Annex A1). (2) LC–HRMS target screening in river waters were performed for about 160 PSMs, which were selected from a large phytotoxin database (Annex A2 and A3) considering their expected abundance in the vegetation, their potential mobility, persistence and toxicity in the water cycle and commercial availability of standards.
In non-target screening (Annex A1), a high number of overlapping peaks has been found in between aqueous plant extracts and water from adjacent location, suggesting a significant impact of vegetation on chemical mixtures detectable in river waters. The chemical structures were assigned for 12 pairs of peaks while several pairs of peaks
whose MS/MS spectra matched but no structure suggestion were made by the implemented software tools for retrieving possible chemical structure. Nevertheless, the pairs of peaks with matching spectra represented the same chemical structure. The identified compound belonged to different compound classes such as coumarins, flavonoids besides others. For the identified PSMs individual concentration up to 5 µg/L were measured. The concentration and the number of detected PSMs per sample were correlated with the rain event and vegetation coverage.
Target screening unraveled the occurrence of 33 out of 160 target compounds in river waters (Annex A2 and A3). The identified compounds belonged to different classes such as alkaloids, coumarins, flavonoids, and other compounds. Individual compound concentrations were up to several thousand ng/L with the toxic alkaloids narciclasine and
lycorine recording highest maximum concentrations. The neurotoxic alkaloid coniine from poison hemlock was detected at concentrations up to 0.4 µg/L while simple coumarins
esculetin and fraxidin occurred at concentrations above 1 µg/L. The occurrence of some PSMs in river water were correlated to the specific vegetation growing along the rivers while the others were linked to a wide range of vegetation. As an example, narciclasine and lycorine was emitted by the dominant plant species from Amaryllidaceae family (e.g. Galanthus nivalis (snow drop), Leucojum vernum and Anemone nemorosa) while intermedine and echimidine were from Symphytum officinale. The ubiquitous occurrence of simple coumarins fraxidin, scopoletin and aesculetin could be linked to their presence in a wide range of vegetation.
Due to lack of aquatic toxicity data for the identified PSMs (in both target and non-target) and extremely scarce exposure data, no reliable risk assessment was possible.
Alternatively, risk estimation was performed using the threshold for toxicological concern (TTC) concept developed for drinking water contaminants. Many of the identified PSMs
exceeded the TTC value (0.1 µg/L) thus caution should be taken when using such surface waters for drinking water abstraction or recreational use.
This thesis provides an overview of the occurrence of PSMs in river water impacted by the massive presence of vegetation. Concentration for many of the identified PSMs are well within the range of those of synthetic environmental contaminants. Thus, this study adds to a series of recent results suggesting that possibly toxic PSMs occur in relevant concentrations in European surface waters and should be considered in monitoring and risk assessment of water resources. Aquatic toxicity data for PSMs are extensively lacking but are required to include these compounds in the assessment of risks to aquatic organisms and for eliminating risks to human health during drinking water production.
This thesis is concerned with protein structures determined by nuclear magnetic resonance (NMR), and the text focuses on their analysis in terms of accuracy, gauged by the correspondence between the structural model and the experimental data it was calculated from, and in terms of precision, i.e. the degree of uncertainty of the atomic positions. Additionally, two protein structure calculation projects are described...
Movement of the Rieske domain of the iron–sulfur protein is essential for intramolecular electron transfer within complex III2 (CIII2) of the respiratory chain as it bridges a gap in the cofactor chain towards the electron acceptor cytochrome c. We present cryo-EM structures of CIII2 from Yarrowia lipolytica at resolutions up to 2.0 Å under different conditions, with different redox states of the cofactors of the high-potential chain. All possible permutations of three primary positions were observed, indicating that the two halves of the dimeric complex act independently. Addition of the substrate analogue decylubiquinone to CIII2 with a reduced high-potential chain increased the occupancy of the Qo site. The extent of Rieske domain interactions through hydrogen bonds to the cytochrome b and cytochrome c1 subunits varied depending on the redox state and substrate. In the absence of quinols, the reduced Rieske domain interacted more closely with cytochrome b and cytochrome c1 than in the oxidized state. Upon addition of the inhibitor antimycin A, the heterogeneity of the cd1-helix and ef-loop increased, which may be indicative of a long-range effect on the Rieske domain.
The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. In spite of the rich knowledge about yeast FAS in structure and function, its assembly remained elusive until recently, when co-translational interaction of the β-subunit with the nascent α-subunit was found to initiate assembly. Here, we characterize the co-translational assembly of yeast FAS at a molecular level. We show that the co-translationally formed interface is sensitive to subtle perturbations, so that the exchange of two amino acids located in the emerging interface can prevent assembly. On the other hand, assembly can also be initiated via the co-translational interaction of the subunits at other sites, which implies that this process is not strictly site or sequence specific. We further highlight additional steps in the biogenesis of yeast FAS, as the formation of a dimeric subunit that orchestrates complex formation and acts as platform for post-translational phosphopantetheinylation. The presented data supports the understanding of the recently discovered prevalence of eukaryotic complexes for co-translational assembly, and is valuable for further harnessing FAS in the biotechnological production of aliphatic compounds.
Due to recent technical developments, it became evident that the mammalian transcriptome is much more complex than originally expected. Alternative splicing(AS) and the transcription of long non-coding RNAs (lncRNAs) are two phenomenas which have been greatly underestimated in their frequency. Nowadays it is accepted that almost every gene has at least one alternative isoform and the number of lncRNAs exceeds the one of protein-coding genes.
We built user-friendly web interfaces which can process Affymetrix GeneChip Exon 1.0 ST Arrays (exon arrays) and GeneChip Gene 1.0 ST Arrays (gene arrays)for the analysis of alternative splicing events. Results are presented with detailed annotation information and graphics to identify splice events and to facilitate biological validations. Based on two studies using exon arrays, we show how our tools were used to profile genome-wide splicing changes under silencing of Jmjd6 and under hypoxic conditions. Since gene arrays are not intended for AS analysis originally, we demonstrated their applicability by profiling alternative splicing events during embryonic heart development.
To measure lncRNAs expressions with exon arrays, we completely re-annotation all probes and built a lncRNA specific annotation. To demonstrate the applicability of exon arrays in combination with our annotation, we profiled the expression of tens of thousands of lncRNAs. Further, our custom annotation allows for a detailed inspection of lncRNAs and to distinguish between isoforms, as we validated by RTPCR.
To allow for a general usage to the research community, we integrated the annotation in an easy-to-use web interface, which provides various helpful features for the analysis of lncRNAs.
RNA modifications are widespread in the RNA world. Nevertheless, their functions remain enigmatic. Recent analysis in tRNAs, mRNAs and rRNAs have revealed that apart from enriching their topological potential, these chemical modifications provide an added significant regulatory level to gene expression...
5‐Lipoxygenase (5‐LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5‐LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter‐interacting proteins, obtained by DNA pulldown and label‐free quantitative mass spectrometry. Protein preparations from myeloid and B‐lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specific ALOX5 promotor binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel‐like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G‐quadruplex structures. As G‐quadruplexes are implicated in gene regulation, spectroscopic and antibody‐based methods were used to confirm their presence within the GC‐rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form DNA G‐quadruplex structures, which may play a functional role in ALOX5 gene regulation.
Analysis of coding principles in the olfactory system and their application in cheminformatics
(2007)
Unser Geruchssinn vermittelt uns die Wahrnehmung der chemischen Welt. Im Laufe der Evolution haben sich in unserem olfaktorischen System Mechanismen entwickelt, die wahrscheinlich optimal auf die Erfüllung dieser Aufgabe angepasst sind. Die Analyse dieser Verarbeitungsstrategien verspricht Einblicke in effiziente Algorithmen für die Kodierung und Verarbeitung chemischer Information, deren Entwicklung und Anwendung dem Kern der Chemieinformatik entspricht. In dieser Arbeit nähern wir uns der Entschlüsselung dieser Mechanismen durch die rechnerische Modellierung von funktionellen Einheiten des olfaktorischen Systems. Hierbei verfolgten wir einen interdisziplinären Ansatz, der die Gebiete der Chemie, der Neurobiologie und des maschinellen Lernens mit einbezieht.
The TATA Box Binding Protein (TBP) is a 20 kD protein that is essential and universally conserved in eucarya and archaea. Especially among archaea, organisms can be found that live below 0°C as well as organisms that grow above 100°C. The archaeal TBPs show a high sequence identity and a similar structure consisting of α-helices and β-sheets that are arranged in a saddle-shape 2-symmetric fold. In previous studies, we have characterized the thermal stability of thermophilic and mesophilic archaeal TBPs by infrared spectroscopy and showed the correlation between the transition temperature (Tm) and the optimal growth temperature (OGT) of the respective donor organism. In this study, a “new” mutant TBP has been constructed, produced, purified and analyzed for a deeper understanding of the molecular mechanisms of thermoadaptation. The β-sheet part of the mutant consists of the TBP from Methanothermobacter thermoautotrophicus (OGT 65°C, MtTBP65) whose α-helices have been exchanged by those of Methanosarcina mazei (OGT 37°C, MmTBP37). The Hybrid-TBP irreversibly aggregates after thermal unfolding just like MmTBP37 and MtTBP65, but the Tm lies between that of MmTBP37 and MtTBP65 indicating that the interaction between the α-helical and β-sheet part of the TBP is crucial for the thermal stability. The temperature stability is probably encoded in the variable α-helices that interact with the highly conserved and DNA binding β-sheets.
Fas Ligand (FasL; CD95L; CD178; TNSF6) is a 40 kDa glycosylated type II transmembrane protein with 279 aa in mice and 281 aa in humans that belongs to the tumor necrosis factor (TNF) family. The extracellular domain (ECD) harbors a TNF homology domain, the receptor binding site, a motif for self assembly and trimerization, and several putative N-glycosylation and a metalloprotease cleavage site/s. The cytoplasmic tail of FasL is the longest of all TNFL family members and contains several conserved signaling motifs, such as a putative tandem Casein kinase I phosphorylation site, a unique proline-rich domain (PRD) and phosphorylatable tyrosine residues (Y7 in mice; Y7, Y9, Y13 in human). The FasL/Fas system is renowned for the potent induction of apoptosis in the receptor-bearing cell and is especially important for immune system functions. It is involved in the killing of target cells by natural killer (NK) and cytotoxic T cells, in the (self) elimination of effector cells following the proliferative phase of an immune response (activation-induced cell death; AICD), in the maintenance of immuneprivileged sites and in the induction and maintenance of peripheral tolerance. Owing to its potent pro-apoptotic signaling capacity and important functions, FasL expression and activity are tightly regulated at transcriptional and posttranscriptional levels and restricted to few cell types, such as immune effector cells and cells of immune-privileged sites. In contrast, Fas is expressed in a variety of tissues including lymphoid tissues, liver, heart, kidney, pancreas, brain and ovary. In addition to its pro-apoptotic function, the FasL/Fas system can also elicit nonapoptotic signals in the receptor-expressing cell. Among others, Fas-signaling exerts co-stimulatory functions in the immune system, e.g. by promoting survival, activation and proliferation of T cells. Besides the capacity to deliver a signal into receptor-bearing cells (‘forward signal’), FasL can receive and transmit signals into the ligand-expressing cell. This phenomenon has been described for several TNF family ligands and is known as ‘reverse signaling’. The first evidence for the existence of reverse signaling into FasL-bearing cells stems from two studies that demonstrated either co-stimulation of murine CD8+ T cell lines by FasL cross-linking or inhibition of activation-induced proliferation of murine CD4+ T cells. In both cases, the observed changes of proliferative behaviour critically depended on the presence of a signaling-competent FasL. Almost certainly, the FasL ICD is functionally involved in signal-transmission: (i) The ICD is highly conserved across species and harbors several signaling motifs, most notably a unique PRD. (ii) Numerous proteins have been identified which interact with the FasL PRD via their SH3 or WW domains and regulate various aspects of FasL biology, such as FasL sorting, storage, cell surface expression and the linkage of FasL to intracellular signaling pathways. (iii) Post-translational modifications of the ICD have been implicated in the sorting of FasL to vesicles and the FasL-dependent activation of Nuclear factor of activated T cells (NFAT). (iv) Proteolytic processing of FasL liberates the ICD and allows its translocation into the nucleus where it might influence gene transcription. (v) It could be shown that overexpression of the FasL ICD is sufficient to initiate reverse signaling upon concomitant T cell receptor (TCR) stimulation and ICD cross-linking. Conflicting data on the consequences of FasL reverse signaling exist, and costimulatory as well as inhibitory functions have been reported. These discrepancies probably reflect the use of artificial experimental systems. Neither the precise molecular mechanism underlying FasL reverse signaling, nor its physiological relevance have been addressed at the endogenous protein level in vivo. Therefore, a ‘knockout/knockin’ mouse model in which wildtype FasL was replaced with a deletion mutant lacking the intracellular portion (FasL Delta Intra) was established in the group of PD Dr. Martin Zörnig. In the present study, FasL Delta Intra mice were phenotypically characterized and were employed to investigate the physiological consequences of FasL reverse signaling at the molecular and cellular level. To ensure that FasL Delta Intra mice represent a suitable model to study the consequences of FasL reverse signaling, we demonstrated that activated lymphocytes from homozygous FasL Delta Intra or wildtype mice express comparable amounts of (truncated) FasL at the cell surface. The truncated protein retains the capacity to induce apoptosis in Fas receptor-positive target cells, as co-culture assays with FasL-expressing activated lymphocytes and Fas-sensitive target cells showed. Additionally, systematic screening of unchallenged mice did not reveal any phenotypic abnormalities. Notably, signs of a lymphoproliferative autoimmune disease associated with FasL-deficiency could not be detected. As several reports have implicated FasL reverse signaling in the regulation of T cell expansion and activation, proliferation of lymphocytes isolated from FasL Delta Intra and wildtype mice in response to antigen receptor stimulation was investigated. Using CFSE dilution assays it could be demonstrated that the proliferative response of CD4+ T cells, CD8+ T cells and of B cells was enhanced in the absence of the FasL ICD. Interestingly, this effect was most pronounced in B cells and could only be detected in CD4+ T cells after depletion of CD4+CD25+ regulatory T cells. To our Summary knowledge, this is the first time that FasL reverse signaling has been demonstrated in B cells. In a series of experiments, the activation of several pathways that are known to play important roles in signal-transmission initiated upon antigen receptor triggering was assessed. As a molecular correlate for the observed enhancement of activation-induced proliferation, Extracellular signal regulated kinase (ERK1/2) phosphorylation was significantly increased in FasL Delta Intra mice following antigen receptor crosslinking. Surprisingly, B cell stimulation lead to a comparable extent of activating phosphorylations on S38 in c-Raf and S218/S222 in MEK1/2 in cells isolated from wildtype and FasL Delta Intra mice, indicating that Mitogen activated protein kinases (MAPKs) upstream of ERK1/2 (Raf-1 and MEK1/2) apparently do not contribute to the differential regulation of ERK1/2. Experiments in which activation-induced Akt phosphorylation (S473) was quantified also did not suggest a participation of Phosphoinositol specific kinase 3 (PI3K)/Akt signals in this process. Instead, further characterization of the upstream pathway revealed an involvement of Phospholipase C gamma (PLC gamma) and Protein kinase C (PKC) signals in FasL-dependent ERK1/2- regulation. Previous studies in our group revealed a Notch-like processing of FasL, resulting in the transcriptional regulation of a reporter gene. Furthermore, an interaction of the FasL ICD with the transcription factor Lymphoid-enhancer binding factor-1 (Lef-1) that affected Lef-1-dependent reporter gene transcription could be demonstrated. Therefore, a molecular analysis of activated lymphocytes was performed to identify FasL reverse signaling target genes. The differential expression of promising candidates was verified by quantitative real-time PCR (qRT-PCR), which showed that the transcription of genes associated with lymphocyte proliferation and activation was increased in FasL Delta Intra mice compared to wildtype mice. Interestingly, an extensive regulation of Lef-1-dependent Wnt/beta-Catenin signalingrelated genes was found. Lef-1 mRNA (RT-PCR) and protein (intracellular FACS staining) could be detected in mature B cells, suggesting the possibility of FasL ICD-mediated inhibition of Lef-1-dependent gene expression in these cells, initiated by Notch-like processing of FasL. To investigate the consequences of FasL reverse signaling in vivo, a potential participation of the FasL ICD in the regulation of immune responses upon various challenges was analyzed. In experiments in which thymocyte proliferation or the expansion of antigen-specific T cells following a challenge with the superantigen Staphylococcus enterotoxin B (SEB), with Lymphocytic choriomeningitis virus (LCMV) or with Listeria monocytogenes were investigated, comparable results were obtained with wildtype and FasL Delta Intra mice. Likewise, the recruitment of neutrophils in a thioglycollate-induced model of peritonitis was not affected by deletion of the FasL ICD. These findings might reflect regulatory mechanisms operating in vivo, such as control exerted by regulatory T cells. Along these lines, proliferative differences in CD4+ T cells could only be detected ex vivo after depletion of CD4+CD25+ regulatory T cells. Furthermore, several in vitro studies indicate that retrograde FasL signals can be observed under conditions of suboptimal lymphocyte stimulation, but not when the TCR is optimally stimulated. Therefore, the potent initiation of antigen receptor signaling by stimuli like SEB or LCMV might have masked inhibitory FasL reverse signaling in these experiments. In agreement with the observed hyperactivation of lymphocytes in the absence of the ICD ex vivo, the increase in germinal center B cells (GCs) following immunization with the hapten 3-hydroxy 4-nitrophenylacetyl (NP) and the number of antibody-secreting PCs was significantly higher in FasL Delta Intra mice. The larger quantity of PCs correlated with increased titers of NP-binding, i.e. antigen-specific, IgM and IgG1 antibodies in the serum of FasL Delta Intra mice after immunization. These data suggest that FasL reverse signaling exerts immunmodulatory functions. Supporting this notion, a model of Ovalbumin-induced allergic airway inflammation revealed an involvement of retrograde FasL-signals in the recruitment of immune effector cells into the lung and in the activation of T cells following exposure of mice to Ovalbumin. Together, our ex vivo and in vivo findings based on endogenous FasL protein levels demonstrate that FasL ICD-mediated reverse signaling is a negative modulator of certain immune responses. It is tempting to speculate that FasL reverse signaling might be a fine-tuning mechanism to prevent autoimmune diseases, a theory which will be tested in adequate mouse models in the future.