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Background: The frequency of the most observed cancer, Non Hodgkin Lymphoma (NHL), is further rising. Diffuse large B-cell lymphoma (DLBCL) is the most common of the NHLs. There are two subgroups of DLBCL with different gene expression patterns: ABC (“Activated B-like DLBCL”) and GCB (“Germinal Center B-like DLBCL”). Without therapy the patients often die within a few months, the ABC type exhibits the more aggressive behaviour. A further B-cell lymphoma is the Mantle cell lymphoma (MCL). It is rare and shows very poor prognosis. There is no cure yet. Methods: In this project these B-cell lymphomas were examined with methods from bioinformatics, to find new characteristics or undiscovered events on the molecular level. This would improve understanding and therapy of lymphomas. For this purpose we used survival, gene expression and comparative genomic hybridization (CGH) data. In some clinical studies, you get large data sets, from which one can reveal yet unknown trends. Results (MCL): The published proliferation signature correlates directly with survival. Exploratory analyses of gene expression and CGH data of MCL samples (n=71) revealed a valid grouping according to the median of the proliferation signature values. The second axis of correspondence analysis distinguishes between good and bad prognosis. Statistical testing (moderate t-test, Wilcoxon rank-sum test) showed differences in the cell cycle and delivered a network of kinases, which are responsible for the difference between good and bad prognosis. A set of seven genes (CENPE, CDC20, HPRT1, CDC2, BIRC5, ASPM, IGF2BP3) predicted, similarly well, survival patterns as proliferation signature with 20 genes. Furthermore, some bands could be associated with prognosis in the explorative analysis (chromosome 9: 9p24, 9p23, 9p22, 9p21, 9q33 and 9q34). Results (DLBCL): New normalization of gene expression data of DLBCL patients revealed better separation of risk groups by the 2002 published signature based predictor. We could achieve, similarly well, a separation with six genes. Exploratory analysis of gene expression data could confirm the subgroups ABC and GCB. We recognized a clear difference in early and late cell cycle stages of cell cycle genes, which can separate ABC and GCB. Classical lymphoma and best separating genes form a network, which can classify and explain the ABC and GCB groups. Together with gene sets which identify ABC and GCB we get a network, which can classify and explain the ABC and GCB groups (ASB13, BCL2, BCL6, BCL7A, CCND2, COL3A1, CTGF, FN1, FOXP1, IGHM, IRF4, LMO2, LRMP, MAPK10, MME, MYBL1, NEIL1 and SH3BP5; Altogether these findings are useful for diagnosis, prognosis and therapy (cytostatic drugs).
There is substantial interest in the identification of genes underlying susceptibility to complex human diseases because of the potential utility of such genes in disease prediction and therapy. The complex age-related macular degeneration (AMD) is a prevalent cause of legal blindness in industrialized countries and predominantly affects the elderly population over 75 years of age. Although vision loss in AMD results from photoreceptor cell death in the central retina, the initial pathogenesis likely involves processes in the retinal pigment epithelium (RPE) (Liang and Godley, 2003). The goal of the current study was to identify and characterize genes specifically or abundantly expressed in the RPE in order to determine more comprehensively the transcriptome of the RPE. In addition, our aim was to assess the role of these genes in AMD pathogenesis. Towards this end, a bovine cDNA library enriched for RPE transcripts was constructed in-house using a PCR-based suppression subtractive hybridization (SSH) technique (Diatchenko et al., 1996, 1999), which normalizes for sequence abundance and achieves high enrichment for differentially expressed genes. CAP3 (Huang and Madan, 1999) was used to assemble the high quality sequences of all the 2379 ESTs into clusters or singletons. 1.2% of the 2379 RPE-ESTs contains vector sequences and was excluded from further analysis. 5% of the RPE-ESTs showed homology to multipe chromosomes and were not included in further assembly process. The rest of the ESTs (2245) were assembled into 175 contigs and 509 singletons, which revealed approximately 684 unique genes in the dataset. Out of the 684, 343 bovine RPE transcripts did not align to their human orthologues. A large fraction of clones were shown to include a considerable 3´untranslated regions of the gene that are not conserved between bovine and human. It is the coding regions that can be conserved between bovine and human and not the 3’ UTR (Sharma et al., 2002). Therefore, more sequencing from the cDNA library with reclustering of those 343 ESTs together with continuous blasting might reveal their human orthologoues. To handle the large volume of data that the RPE cDNA library project has generated a highly efficient and user-friendly RDBMS was designed. Using RDBMS data storage can be managed efficiently and flexibly. The RDBMS allows displaying the results in query-based form and report format with additional annotations, links and search functions. Out of the 341 known and predicted genes identified in this study, 2 were further analyzed. The RPE or/and retina specificity of these two clones were further confirmed by RT-PCR analysis in adult human tissues. Construction of a single nucleotide polymphism (SNP) map was initiated as a first step in future case/control association studies. SNP genotyping was carried out for one of these two clones (RPE01-D2, now known as RDH12). 12 SNPs were identified from direct sequencing of the 23.4-kb region, of which 5 are of high frequency. In a next step, comparison of allele frequencies between AMD patients and healthy controls is required. Completion of the expression analysis for other predicted genes identified during this study is in progress using real time RT-PCR and will provide additional candidate genes for further analyses. This study is expected to contribute to our understanding of the genetic basis of RPE function and to clarify the role of the RPE-expressed genes in the predisposition to AMD. It may also help reveal the mechanisms and pathways that are involved in the development of AMD or other retinal dystrophies.
no abstract available
The flowers of plants of the genus Ludwigia are an important source of food for several species of bees. In the current study, we conducted an experiment with the aim to describe the reproductive biology and phenology of L. nervosa; to identify the species of visiting bees; analyze the foraging behavior of bees; and to investigate whether the reproductive success of the species is related to the foraging activity of bees. We found that the flowers received visits from several native bee species (n = 7), in addition of the exotic honey bees which came to be the dominant species. During visits the majority of the bees foraged in both resources, pollen and nectar. The significantly higher production of fruits in open pollinated pollination experiment compared to artificial cross pollination, suggests honey bees as effective pollinator of this plant species in the study site. Pollen deposition occurs efficiently, given the absence of pollen limitation. Despite massive visitation of honey bees, Ludwigianervosa is attractive to native bees, and therefore it may help to sustain population of both native and exotic pollinators in fragmented humid areas.
Forests are multi-functional system, which have to fulfil different objectives at the same time. The main functions include the production of wood, storage of carbon, the promotion of biological diversity and the provision of recreational space. Yet, global forests are affected by large and intense natural disturbances, like bark beetle infestations. While natural disturbances threaten wood production and are perceived as ‘catastrophe’ diminishing recreational value, biodiversity can benefit from the disturbance-induced changes in forest structures. This trade-off poses a dilemma to managers of bark beetle affected stands, particularly in protected areas designated to both nature conservation and recreation. Forest landscapes need a sustainable management concept aligning these different objectives. In order to support this goal with scientific knowledge, the aim of this work is to analyse ecological and social effects along a gradient of different disturbance severities. In this context, I studied the effects of a disturbance severity gradient on the diversity of different taxonomic groups including vascular plants, mosses, lichens, fungi, arthropods and birds in five national parks in Central Europe. To analyse the recreational value of the landscape I conducted visitor surveys in the same study areas in which the biodiversity surveys were performed. To analyse possible psychological or demographic effects on preferences for certain disturbance intensities, an additional online survey was carried out.
1.
The successional dynamics of forests—from canopy openings to regeneration, maturation, and decay—influence the amount and heterogeneity of resources available for forest-dwelling organisms. Conservation has largely focused only on selected stages of forest succession (e.g., late-seral stages). However, to develop comprehensive conservation strategies and to understand the impact of forest management on biodiversity, a quantitative understanding of how different trophic groups vary over the course of succession is needed.
2.
We classified mixed mountain forests in Central Europe into nine successional stages using airborne LiDAR. We analysed α- and β-diversity of six trophic groups encompassing approximately 3,000 species from three kingdoms. We quantified the effect of successional stage on the number of species with and without controlling for species abundances and tested whether the data fit the more-individuals hypothesis or the habitat heterogeneity hypothesis. Furthermore, we analysed the similarity of assemblages along successional development.
3.
The abundance of producers, first-order consumers, and saprotrophic species showed a U-shaped response to forest succession. The number of species of producer and consumer groups generally followed this U-shaped pattern. In contrast to our expectation, the number of saprotrophic species did not change along succession. When we controlled for the effect of abundance, the number of producer and saproxylic beetle species increased linearly with forest succession, whereas the U-shaped response of the number of consumer species persisted. The analysis of assemblages indicated a large contribution of succession-mediated β-diversity to regional γ-diversity.
4.
Synthesis and applications. Depending on the species group, our data supported both the more-individuals hypothesis and the habitat heterogeneity hypothesis. Our results highlight the strong influence of forest succession on biodiversity and underline the importance of controlling for successional dynamics when assessing biodiversity change in response to external drivers such as climate change. The successional stages with highest diversity (early and late successional stages) are currently strongly underrepresented in the forests of Central Europe. We thus recommend that conservation strategies aim at a more balanced representation of all successional stages.
The Myb-MuvB (MMB) complex plays an essential role in the time-dependent transcriptional activation of mitotic genes. Recently, our laboratory identified a novel crosstalk between the MMB-complex and YAP, the transcriptional coactivator of the Hippo pathway, to coregulate a subset of mitotic genes (Pattschull et al., 2019). Several genetic studies have shown that the Hippo-YAP pathway is essential to drive cardiomyocyte proliferation during cardiac development (von Gise et al., 2012; Heallen et al., 2011; Xin et al., 2011). However, the exact mechanisms of how YAP activates proliferation of cardiomyocytes is not known. This doctoral thesis addresses the physiological role of the MMB-Hippo crosstalk within the heart and characterizes the YAP-B-MYB interaction with the overall aim to identify a potent inhibitor of YAP.
The results reported in this thesis indicate that complete loss of the MMB scaffold protein LIN9 in heart progenitor cells results in thinning of ventricular walls, reduced cardiomyocyte proliferation and early embryonic lethality. Moreover, genetic experiments using mice deficient in SAV1, a core component of the Hippo pathway, and LIN9-deficient mice revealed that the correct function of the MMB complex is critical for proliferation of cardiomyocytes due to Hippo-deficiency. Whole genome transcriptome profiling as well as genome wide binding studies identified a subset of Hippo-regulated cell cycle genes as direct targets of MMB. By proximity ligation assay (PLA), YAP and B-MYB were discovered to interact in embryonal cardiomyocytes. Biochemical approaches, such as co-immunoprecipitation assays, GST-pulldown assays, and µSPOT-based peptide arrays were employed to characterize the YAP-B-MYB interaction. Here, a PY motif within the N-terminus of B-MYB was found to directly interact with the YAP WW-domains. Consequently, the YAP WW-domains were important for the ability of YAP to drive proliferation in cardiomyocytes and to activate MMB target genes in differentiated C2C12 cells. The biochemical information obtained from the interaction studies was utilized to develop a novel competitive inhibitor of YAP called MY-COMP (Myb-YAP competition). In MY-COMP, the protein fragment of B-MYB containing the YAP binding domain is fused to a nuclear localization signal. Co-immunoprecipitation studies as well as PLA revealed that the YAP-B-MYB interaction is robustly blocked by expression of MY-COMP. Adenoviral overexpression of MY-COMP in embryonal cardiomyocytes suppressed entry into mitosis and blocked the pro-proliferative function of YAP. Strikingly, characterization of the cellular phenotype showed that ectopic expression of MY-COMP led to growth defects, nuclear abnormalities and polyploidization in HeLa cells.
Taken together, the results of this thesis reveal the mechanism of the crosstalk between the Hippo signaling pathway and the MMB complex in the heart and form the basis for interference with the oncogenic activity of the Hippo coactivator YAP.
GAS2L3 was identified recently as a target gene of the DREAM complex (Reichert et al., 2010; Wolter et al., 2012). It was shown that GAS2L3 is expressed in a cell cycle specific manner and that depletion of the protein leads to defects in cytokinesis and genomic instability (Wolter et al., 2012).
Major aim of this thesis was, to further characterize the biochemical properties and physiological function of GAS2L3.
By in vitro co-sedimentation and bundling assays, GAS2L3 was identified as a cytoskeleton associated protein which bundles, binds and crosslinks F-actin and MTs. GST pulldown assays and co-immunoprecipitation experiments revealed that GAS2L3 interacts in vitro and in vivo with the chromosomal passenger complex (CPC), a very important regulator of mitosis and cytokinesis, and that the interaction is mediated by the GAR domain of GAS2L3 and the C-terminal part of Borealin and the N-terminal part of Survivin. Kinase assays showed that GAS2L3 is not a substrate of the CPC but is strongly phosphorylated by CDK1 in vitro. Depletion of GAS2L3 by shRNA influenced protein stability and activity of the CPC. However pharmacological studies showed that the decreased CPC activity is not responsible for the observed cytokinesis defects upon GAS2L3 depletion. Immunofluorescence experiments revealed that GAS2L3 is localized to the constriction zone by the CPC in a GAR dependent manner and that the GAR domain is important for proper protein function.
New interacting proteins of GAS2L3 were identified by stable isotope labelling by amino acids in cell culture (SILAC) in combination with tandem affinity purification and subsequent mass spectrometrical analysis. Co-immunoprecipitation experiments further confirmed the obtained mass spectrometrical data.
To address the physiological function of GAS2L3 in vivo, a conditional and a non-conditional knockout mouse strain was established. The non-conditional mouse strain showed a highly increased mortality rate before weaning age probably due to heart failure. The physiological function of GAS2L3 in vivo as well as the exact reason for the observed heart phenotype is not known at the moment.
Biochemical and molecular characterization of an original master sex determining gene in Salmonids
(2016)
Sexual development is a fundamental and versatile process that shapes animal morphology, physiology and behavior. The underlying developmental process is composed of the sex determination and the sex differentiation. Sex determination mechanisms are extremely labile among taxa. The initial triggers of the sex determination process are often genetics called sex determining genes. These genes are expressed in the bipotential gonad and tilt the balance to a developmental program allowing the differentiation of either a testis or an ovary. Fish represent a large and fascinating vertebrate group to study both sex determination and sex differentiation mechanisms. To date, among the known sex determining genes, three gene families namely sox, dmrt and TGF-β factors govern this developmental program. As exception to this rule, sdY “sexually dimorphic on the Y” does not belong to one of these families as it comes from the duplication / evolution of an ancestor gene related to immunity, i.e., the interferon related factor 9, irf9. sdY is the master sex determining gene in salmonids, a group of fishes that include species such as rainbow trout and Atlantic salmon. The present study was aimed to firstly characterize the features of SdY protein. Results indicate that SdY is predominantly localized in the cytoplasm tested in various fish and mammalian cell lines and confirmed by different methods. Predictive in silico analysis revealed that SdY is composed of a β-sandwich core surrounded by three α-helices as well specific characteristics conferring a putative protein-protein interaction site. Secondly, the study was aimed to understand how SdY could trigger testicular differentiation. SdY is a truncated divergent version of Irf9 that has a conserved protein-protein domain but lost the DNA interaction domain of its ancestor gene. It was then hypothesized that SdY could initiate testicular differentiation by protein-protein interactions. To evaluate this we first conducted a yeast-two-hybrid screen that revealed a high proportion of transcription factors including fox proteins. Using various biochemical and cellular methods we confirm an interaction between SdY and Foxl2, a major transcription factor involved in ovarian differentiation and identity maintenance. Interestingly, the interaction of SdY with Foxl2 leads to nuclear translocation of SdY from the cytoplasm. Furthermore, this SdY translocation mechanism was found to be specific to fish Foxl2 and to a lesser extend Foxl3 and not other Fox proteins or mammalian FoxL2. In addition, we found that this interaction allows the stabilization of SdY and prevents its degradation. Finally, to better decipher SdY action we used as a model a mutated version of SdY that was identified in XY females of Chinook salmon natural population. Results show that this mutation induces a local conformation defect obviously leading to a misfolded protein and a quick degradation. Moreover, the mutated version compromised the interaction with Foxl2 defining a minimal threshold to induce testicular differentiation. Altogether results from my thesis propose that SdY would trigger testicular differentiation in salmonids by preventing Foxl2 to promote ovarian differentiation. Further research should be now carried out on how this interaction of SdY and Foxl2 acts in-vivo.
(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses.
MYC is one of the most frequently overexpressed oncogenes in human cancer, and even modestly deregulated MYC can initiate ectopic proliferation in many postmitotic cell types in vivo. Sensitization of cells to apoptosis limits MYC's oncogenic potential. However, the mechanism through which MYC induces apoptosis is controversial. Some studies implicate p19ARF-mediated stabilization of p53, followed by induction of proapoptotic BH3 proteins NOXA and PUMA, whereas others argue for direct regulation of BH3 proteins, especially BIM. Here, we use a single experimental system to systematically evaluate the roles of p19ARF and BIM during MYC-induced apoptosis, in vitro, in vivo, and in combination with a widely used chemotherapeutic, doxorubicin. We find a common specific requirement for BIM during MYC-induced apoptosis in multiple settings, which does not extend to the p53-responsive BH3 family member PUMA, and find no evidence of a role for p19ARF during MYC-induced apoptosis in the tissues examined.
Background: In most trypanosomes, endo and exocytosis only occur at a unique organelle called the flagellar pocket (FP) and the flagellum exits the cell via the FP. Investigations of essential cytoskeleton-associated structures located at this site have revealed a number of essential proteins. The protein TbBILBO1 is located at the neck of the FP in a structure called the flagellar pocket collar (FPC) and is essential for biogenesis of the FPC and parasite survival. TbMORN1 is a protein that is present on a closely linked structure called the hook complex (HC) and is located anterior to and overlapping the collar. TbMORN1 is essential in the bloodstream form of T. brucei. We now describe the location and function of BHALIN, an essential, new FPC-HC protein. Methodology/Principal Findings: Here, we show that a newly characterised protein, BHALIN (BILBO1 Hook Associated LINker protein), is localised to both the FPC and HC and has a TbBILBO1 binding domain, which was confirmed in vitro. Knockdown of BHALIN by RNAi in the bloodstream form parasites led to cell death, indicating an essential role in cell viability. Conclusions/Significance: Our results demonstrate the essential role of a newly characterised hook complex protein, BHALIN, that influences flagellar pocket organisation and function in bloodstream form T. brucei parasites.
In recent years, zebrafish, and to a lesser extent medaka, have become widely used small animal models for human diseases. These organisms have convincingly demonstrated the usefulness of fish for improving our understanding of the molecular and cellular mechanisms leading to pathological conditions, and for the development of new diagnostic and therapeutic tools. Despite the usefulness of zebrafish and medaka in the investigation of a wide spectrum of traits, there is evidence to suggest that other fish species could be better suited for more targeted questions. With the emergence of new, improved sequencing technologies that enable genomic resources to be generated with increasing efficiency and speed, the potential of non-mainstream fish species as disease models can now be explored. A key feature of these fish species is that the pathological condition that they model is often related to specific evolutionary adaptations. By exploring these adaptations, new disease-causing and disease-modifier genes might be identified; thus, diverse fish species could be exploited to better understand the complexity of disease processes. In addition, non-mainstream fish models could allow us to study the impact of environmental factors, as well as genetic variation, on complex disease phenotypes. This Review will discuss the opportunities that such fish models offer for current and future biomedical research.
Habitat fragmentation and destruction due to anthropogenic land use are the major causes of the increasing extinction risk of many species and have a detrimental impact on animal populations in numerous ways. The long-term survival and stability of spatially structured populations in fragmented landscapes largely depends on the colonisation of habitat patches and the exchange of individuals and genes between patches. The degree of inter-patch dispersal, in turn, depends on the dispersal ability of a species (i.e. the combination of physiological and morphological factors that facilitate dispersal) and the landscape structure (i.e. the nature of the landscape matrix or the spatial configuration of habitat patches). As fragmentation of landscapes is increasing and the number of species is continuously declining, a thorough understanding of the causes and consequences of dispersal is essential for managing natural populations and developing effective conservation strategies.
In the context of animal dispersal, movement behaviour is intensively investigated with capture-mark-recapture studies. For the analysis of such experiments, the influence of marking technique, handling and translocation of marked animals on movement pattern is of crucial importance since it may mask the effects of the main research question. Chapter 2 of this thesis presents a capture-mark-recapture study investigating the effect of translocation on the movement behaviour of the blue-winged grasshopper Oedipoda caerulescens. Transferring individuals of this grasshopper species to suitable but unfamilliar sites has a significant influence on their movement behaviour. Translocated individuals moved longer distances, showed smaller daily turning angles, and thus their movements were more directed than those of resident individuals. The effect of translocation was most pronounced on the first day of the experiment, but may persist for longer. On average, daily moved distances of translocated individuals were about 50 % longer than that of resident individuals because they have been transferred to an unfamiliar habitat patch. Depending on experiment duration, this leads to considerable differences in net displacement between translocated and resident individuals. In summary, the results presented in chapter 2 clearly point out that translocation effects should not be disregarded in future studies on arthropod movement, respectively dispersal. Studies not controlling for possible translocation effects may result in false predictions of dispersal behaviour, habitat detection capability or habitat preferences.
Beside direct field observations via capture-mark-recapture methods, genetic markers can be used to investigate animal dispersal. Chapter 3 presents data on the genetic structure of populations of Metrioptera bicolor, a wing-dimorphic bush cricket, in a spatially structured landscape with patches of suitable habitat distributed within a diverse matrix of different habitat types. Using microsatellite markers, the effects of geographic distance and different matrix types on the genetic differentiation among 24 local populations was assessed. The results of this study clearly indicate that for M. bicolor the isolation of local populations severely depends on the type of surrounding matrix. The presence of forest and a river running through the study area was positively correlated with the extent of genetic differentiation between populations. This indicates that both matrix types severely impede gene flow and the exchange of individuals between local populations of this bush cricket. In addition, for a subsample of populations which were separated only by arable land or settlements, a significant positive correlation between pairwise genetic and geographic distances exists. For the complete data set, this correlation could not be found. This is most probably due to the adverse effect of forest and river on gene flow which dominates the effect of geographic distance in the limited set of patches investigated in this study. The analyses in chapter 3 clearly emphasize the differential resistance of different habitat types on dispersal and the importance of a more detailed view on matrix ‘quality’ in metapopulation studies. Studies that focus on the specific dispersal resistance of different matrix types may provide much more detailed information on the dispersal capacity of species than a mere analysis of isolation by distance. Such information is needed to improve landscape oriented models for species conservation.
In addition to direct effects on realised dispersal (see chapter 3), landscape structure on its own is known to act as an evolutionary selection agent because it determines the costs and benefits of dispersal. Both morphological and behavioural traits of individuals and the degree to which a certain genotype responds to environmental variation have heritable components, and are therefore expected to be able to respond to selection pressures. Chapter 4 analyses the influence of patch size, patch connectivity (isolation of populations) and sand dynamics (stability of habitat) on thorax- and wing length as proxies for dispersal ability of O. caerulescens in coastal grey dunes. This study revealed clear and sex-specific effects of landscape dynamics and patch configuration on dispersal-related morphology. Males of this grasshopper species were smaller and had shorter wings if patches were larger and less connected. In addition, both sexes were larger in habitat patches with high sand dynamics compared to those in patches with lower dynamics. The investments in wing length were only larger in connected populations when sand dynamics were low, indicating that both landscape and patch-related environmental factors are of importance. These results are congruent with theoretical predictions on the evolution of dispersal in metapopulations. They add to the evidence that dispersal-related morphology varies and is selected upon in recently structured populations even at small spatial scales.
Dispersal involves different individual fitness costs like increased predation risk, energy expenditure, costs of developing dispersal-related traits, failure to find new suitable habitat as well as reproductive costs. Therefore, the decision to disperse should not be random but depend on the developmental stage or the physiological condition of an individual just as on actual environmental conditions (context-dependent dispersal, e.g. sex- and wing morph-biased dispersal). Biased dispersal is often investigated by comparing the morphology, physiology and behaviour of females and males or sedentary and dispersive individuals. Studies of biased dispersal in terms of capture-mark-recapture experiments, investigating real dispersal and not routine movements, and genetic proofs of biased dispersal are still rare for certain taxa, especially for orthopterans. However, information on biased dispersal is of great importance as for example, undetected biased dispersal may lead to false conclusions from genetic data. In chapter 5 of this thesis, a combined approach of morphological and genetic analyses was used to investigate biased dispersal of M. bicolor. The presented results not only show that macropterous individuals are predestined for dispersal due to their morphology, the genetic data also indicate that macropters are more dispersive than micropters. Furthermore, even within the group of macropterous individuals, males are supposed to be more dispersive than females. To get an idea of the flight ability of M. bicolor, the morphological data were compared with that of Locusta migratoria and Schistocerca gregaria, which are proved to be very good flyers. Based on the morphological data presented here, one can assume a good flight ability for macropters of M. bicolor, although flying individuals of this species are seldom observed in natural populations.
Immunofluorescence is a common method to localise proteins within their cellular context via fluorophore labelled antibodies and for some applications without alternative. However, some protein targets evade detection due to low protein abundance or accessibility issues. In addition, some imaging methods require a massive reduction in antigen density thus impeding detection of even medium-abundant proteins.Here, we show that the fusion of the target protein to TurboID, a biotin ligase labelling lysine residues in close proximity, and subsequent detection of biotinylation by fluorescent streptavidin offers an “all in one” solution to the above-mentioned restrictions. For a wide range of target proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the imaging sensitivity in expansion microscopy and correlative light and electron microscopy, with no loss in resolution. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus or RNA granules, were readily detected with streptavidin, while most antibodies fail to label proteins in these environments. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can be used to map antibody-accessibility to certain cellular regions. As a proof of principle, we mapped antibody access to all trypanosome nuclear pore proteins (NUPs) and found restricted antibody labelling of all FG NUPs of the central channel that are known to be phase-separated, while most non-FG Nups could be labelled. Lastly, we show that streptavidin imaging can resolve dynamic, temporally and spatially distinct sub-complexes and, in specific cases, reveal a history of dynamic protein interaction.In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, can provide information on protein interactions and biophysical environment.
Background:
Ketogenic diets (KDs) or short-term fasting are popular trends amongst supportive approaches for cancer patients. Beta-hydroxybutyrate (3-OHB) is the main physiological ketone body, whose concentration can reach plasma levels of 2–6 mM during KDs or fasting. The impact of 3-OHB on the biology of tumor cells described so far is contradictory. Therefore, we investigated the effect of a physiological concentration of 3 mM 3-OHB on metabolism, proliferation, and viability of breast cancer (BC) cells in vitro.
Methods:
Seven different human BC cell lines (BT20, BT474, HBL100, MCF-7, MDA-MB 231, MDA-MB 468, and T47D) were cultured in medium with 5 mM glucose in the presence of 3 mM 3-OHB at mild hypoxia (5% oxygen) or normoxia (21% oxygen). Metabolic profiling was performed by quantification of the turnover of glucose, lactate, and 3-OHB and by Seahorse metabolic flux analysis. Expression of key enzymes of ketolysis as well as the main monocarboxylic acid transporter MCT2 and the glucose-transporter GLUT1 was analyzed by RT-qPCR and Western blotting. The effect of 3-OHB on short- and long-term cell proliferation as well as chemo- and radiosensitivity were also analyzed.
Results:
3-OHB significantly changed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in BT20 cells resulting in a more oxidative energetic phenotype. MCF-7 and MDA-MB 468 cells had increased ECAR only in response to 3-OHB, while the other three cell types remained uninfluenced. All cells expressed MCT2 and GLUT1, thus being able to uptake the metabolites. The consumption of 3-OHB was not strongly linked to mRNA overexpression of key enzymes of ketolysis and did not correlate with lactate production and glucose consumption. Neither 3-OHB nor acetoacetate did interfere with proliferation. Further, 3-OHB incubation did not modify the response of the tested BC cell lines to chemotherapy or radiation.
Conclusions:
We found that a physiological level of 3-OHB can change the energetic profile of some BC cell lines. However, 3-OHB failed to influence different biologic processes in these cells, e.g., cell proliferation and the response to common breast cancer chemotherapy and radiotherapy. Thus, we have no evidence that 3-OHB generally influences the biology of breast cancer cells in vitro.
The recently observed consistent loss of β-diversity across ecosystems indicates increasingly homogeneous communities in patches of landscapes, mainly caused by increasing land-use intensity. Biodiversity is related to numerous ecosystem functions and stability. Therefore, decreasing β-diversity is also expected to reduce multifunctionality. To assess the impact of homogenization and to develop guidelines to reverse its potentially negative effects, we combine expertise from forest science, ecology, remote sensing, chemical ecology and statistics in a collaborative and experimental β-diversity approach. Specifically, we will address the question whether the Enhancement of Structural Beta Complexity (ESBC) in forests by silviculture or natural disturbances will increase biodiversity and multifunctionality in formerly homogeneously structured production forests. Our approach will identify potential mechanisms behind observed homogenization-diversity-relationships and show how these translate into effects on multifunctionality. At eleven forest sites throughout Germany, we selected two districts as two types of small ‘forest landscapes’. In one of these two districts, we established ESBC treatments (nine differently treated 50x50 m patches with a focus on canopy cover and deadwood features). In the second, the control district, we will establish nine patches without ESBC. By a comprehensive sampling, we will monitor 18 taxonomic groups and measure 21 ecosystem functions, including key functions in temperate forests, on all patches. The statistical framework will allow a comprehensive biodiversity assessment by quantifying the different aspects of multitrophic biodiversity (taxonomical, functional and phylogenetic diversity) on different levels of biodiversity (α-, β-, γ-diversity). To combine overall diversity, we will apply the concept of multidiversity across the 18 taxa. We will use and develop new approaches for quantification and partitioning of multifunctionality at α- and β- scales. Overall, our study will herald a new research avenue, namely by experimentally describing the link between β-diversity and multifunctionality. Furthermore, we will help to develop guidelines for improved silvicultural concepts and concepts for management of natural disturbances in temperate forests reversing past homogenization effects.
Preventing malnutrition through consuming nutritionally appropriate resources represents a challenge for foraging animals. This is due to often high variation in the nutritional quality of available resources. Foragers consequently need to evaluate different food sources. However, even the same food source can provide a plethora of nutritional and non‐nutritional cues, which could serve for quality assessment. We show that bumblebees, Bombus terrestris , overcome this challenge by relying on lipids as nutritional cue when selecting pollen. The bees ‘prioritised’ lipid perception in learning experiments and avoided lipid consumption in feeding experiments, which supported survival and reproduction. In contrast, survival and reproduction were severely reduced by increased lipid contents. Our study highlights the importance of fat regulation for pollen foraging bumblebees. It also reveals that nutrient perception, nutrient regulation and reproductive fitness can be linked, which represents an effective strategy enabling quick foraging decisions that prevent malnutrition and maximise fitness.
The Western Honeybee (Apis mellifera) is among the most versatile species in the world. Its adaptability is rooted in thousands of the differently specialized individuals acting jointly together. Thus, bees that are able to handle a certain task or condition well can back up other individuals less capable to do so on the colony level. Vice versa, the latter individuals might perform better in other situations. This evolutionary recipe for success ensures the survival of colonies despite challenging habitat conditions. In this context, the ectoparasitic mite Varroa destructor reflects the most pronounced biotic challenge to honeybees worldwide. Without proper treatment, infested colonies rapidly dwindle and ultimately die. Nevertheless, resistance behaviours against this parasite have evolved in some populations through natural selection, enabling colonies to survive untreated. In this, different behaviours appear to be adapted to the respective habitat conditions and may complement each other. Yet, the why and how of this behavioural response to the mite remains largely unknown. My thesis focuses on the biological background of Varroa-resistance traits in honeybees and presents important findings for the comprehension of this complex host-parasite interaction. Based on this, I draw implications for both, applied bee breeding and scientific investigations in the field of Varroa-resistance. Specifically, I focus on two traits commonly found in resistant and, to a lower degree, also mite-susceptible colonies: decreased mite reproduction and the uncapping and subsequent recapping of sealed brood cells. Examining failures in the reproductive success of mites as a primary mechanism of Varroa-resistance, I was able to link them to specific bee behaviours and external factors. Since mite reproduction and the brood rearing of bees are inevitably connected, I first investigated the effects of brood interruption on the reproductive success of mites. Brood interruption decreased the reproductive success of mites both immediately and in the long term. By examining the causes of reproductive failure, I could show that this was mainly due to an increased share of infertile mites. Furthermore, I proved that interruption in brood rearing significantly increased the expression of recapping behaviour. These findings consequently showed a dynamic modulation of mite reproduction and recapping, as well as a direct effect of brood interruption on both traits. To further elucidate the plasticity in the expression of both traits, I studied mite reproduction, recapping behaviour and infestation levels over the course of three years. The resulting extensive dataset unveiled a significant seasonal variation in mite reproduction and recapping. In addition, I show that recapping decreases the reproductive success of mites by increasing delayed developing female offspring and cells lacking male offspring. By establishing a novel picture-based brood investigation method, I could furthermore show that both the removal of brood cells and recapping activity specifically target brood ages in which mite offspring would be expected. Recapping, however, did not cause infertility of mites. Considering the findings of my first study, this points towards complementary mechanisms.
This underlines the importance of increased recapping behaviour and decreased mite reproduction as resistance traits, while at the same time emphasising the challenges of reliable data acquisition. To pave the way for a practical application of these findings in breeding, we then investigated the heritability (i.e., the share of genotypic variation on the observed phenotypic variation) of the accounted traits. By elaborating comparable test protocols and compiling data from over 4,000 colonies, we could, for the first time, demonstrate that recapping of infested cells and decreased reproductive success of mites are heritable (and thus selectable) traits in managed honeybee populations.
My thesis proves the importance of recapping and decreased mite reproduction as resistance traits and therefore valuable goals for breeding efforts. In this regard, I shed light on the underlying mechanisms of both traits, and present clear evidence for their interaction and heritability.
Behavioural Analyses of Quinine Processing in Choice, Feeding and Learning of Larval Drosophila
(2012)
Gustatory stimuli can support both immediate reflexive behaviour, such as choice and feeding, and can drive internal reinforcement in associative learning. For larval Drosophila, we here provide a first systematic behavioural analysis of these functions with respect to quinine as a study case of a substance which humans report as "tasting bitter". We describe the dose-effect functions for these different kinds of behaviour and find that a half-maximal effect of quinine to suppress feeding needs substantially higher quinine concentrations (2.0 mM) than is the case for internal reinforcement (0.6 mM). Interestingly, in previous studies (Niewalda et al. 2008, Schipanski et al 2008) we had found the reverse for sodium chloride and fructose/sucrose, such that dose-effect functions for those tastants were shifted towards lower concentrations for feeding as compared to reinforcement, arguing that the differences in dose-effect function between these behaviours do not reflect artefacts of the types of assay used. The current results regarding quinine thus provide a starting point to investigate how the gustatory system is organized on the cellular and/or molecular level to result in different behavioural tuning curves towards a bitter tastant.
A fundamental problem in deciding between mutually exclusive options is that the decision needs to be categorical although the properties of the options often differ but in grade. We developed an experimental handle to study this aspect of behavior organization. Larval Drosophila were trained such that in one set of animals odor A was rewarded, but odor B was not (A+/B), whereas a second set of animals was trained reciprocally (A/B+). We then measured the preference of the larvae either for A, or for B, or for “morphed” mixtures of A and B, that is for mixtures differing in the ratio of the two components. As expected, the larvae showed higher preference when only the previously rewarded odor was presented than when only the previously unrewarded odor was presented. For mixtures of A and B that differed in the ratio of the two components, the major component dominated preference behavior—but it dominated less than expected from a linear relationship between mixture ratio and preference behavior. This suggests that a minor component can have an enhanced impact in a mixture, relative to such a linear expectation. The current paradigm may prove useful in understanding how nervous systems generate discrete outputs in the face of inputs that differ only gradually.
The term ‘urban beekeeping’ connotes a host of meanings—sociopolitical, commercial, ecological and personal—beyond the mere description of where bees and beekeepers happen to coincide. Yet, these meanings are seldom articulated explicitly or brought into critical engagement with the relevant fields of urban ecology and political ecology.
Beginning with a brief account of the history of urban beekeeping in the United States, we draw upon urban ecological theory to construct a conceptual model of urban beekeeping that distinguishes beekeeping in, of and for the city. In our model, beekeeping in the city describes the mere importation of the traditionally rural practice of beekeeping into urban spaces for the private reasons of the individual beekeeper, whereas beekeeping of the city describes beekeeping that is consciously tailored to the urban context, often accompanied by (semi)professionalization of beekeepers and the formation of local expert communities (i.e. beekeeping associations). Beekeeping for the city describes a shift in mindset in which beekeeping is directed to civic ends beyond the boundaries of the beekeeping community per se.
Using this framework, we identify and discuss specific socioecological assets and liabilities of urban beekeeping, and how these relate to beekeeping in, of and for the city. We then formulate actionable guidelines for maturing the practice of urban beekeeping into a beneficent and self‐critical form of urban ecological citizenship; these include fostering self‐regulation within the beekeeping community, harnessing beekeeping as a ‘gateway’ experience for a broader rapprochement between urban residents and nature, and recognizing the political‐ecological context of beekeeping with respect to matters of socioecological justice.
Bee pollination increases yield quantity and quality of cash crops in Burkina Faso, West Africa
(2017)
Mutualistic biotic interactions as among flowering plants and their animal pollinators are a key component of biodiversity. Pollination, especially by insects, is a key element in ecosystem functioning, and hence constitutes an ecosystem service of global importance. Not only sexual reproduction of plants is ensured, but also yields are stabilized and genetic variability of crops is maintained, counteracting inbreeding depression and facilitating system resilience. While experiencing rapid environmental change, there is an increased demand for food and income security, especially in sub-Saharan communities, which are highly dependent on small scale agriculture. By combining exclusion experiments, pollinator surveys and field manipulations, this study for the first time quantifies the contribution of bee pollinators to smallholders’ production of the major cash crops, cotton and sesame, in Burkina Faso. Pollination by honeybees and wild bees significantly increased yield quantity and quality on average up to 62%, while exclusion of pollinators caused an average yield gap of 37% in cotton and 59% in sesame. Self-pollination revealed inbreeding depression effects on fruit set and low germination rates in the F1-generation. Our results highlight potential negative consequences of any pollinator decline, provoking risks to agriculture and compromising crop yields in sub-Saharan West Africa.
Pollination improves the yield of most crop species and contributes to one-third of global crop production, but comprehensive benefits including crop quality are still unknown. Hence, pollination is underestimated by international policies, which is particularly alarming in times of agricultural intensification and diminishing pollination services. In this study, exclusion experiments with strawberries showed bee pollination to improve fruit quality, quantity and market value compared with wind and self-pollination. Bee-pollinated fruits were heavier, had less malformations and reached higher commercial grades. They had increased redness and reduced sugar–acid–ratios and were firmer, thus improving the commercially important shelf life. Longer shelf life reduced fruit loss by at least 11%. This is accounting for 0.32 billion US$ of the 1.44 billion US$ provided by bee pollination to the total value of 2.90 billion US$ made with strawberry selling in the European Union 2009. The fruit quality and yield effects are driven by the pollination-mediated production of hormonal growth regulators, which occur in several pollination-dependent crops. Thus, our comprehensive findings should be transferable to a wide range of crops and demonstrate bee pollination to be a hitherto underestimated but vital and economically important determinant of fruit quality.
My dissertation comprises three studies: (1) an assessment of honey bee colony losses in the USA between 2014 and 2015, (2) an exploration of the potential of reclaimed sand mines as bee habitat, and (3) an evaluation of native and non-native pollinator friendly plants in regard to their attraction to bees. While the first study focuses on honey bees, the latter two studies primarily take wild bees or entire bee communities in focus.
The study on honey bee colony losses was conducted within the framework of the Bee Informed Partnership (BIP, beeinformed.org) and aligns with the annual colony loss surveys which have been conducted in the USA since the winter of 2006/2007. It was the fourth year for which summer and annual losses were calculated in addition to winter losses. Among participants, backyard beekeepers were the largest group (n = 5690), although sideline (n = 169) and commercial (n = 78) beekeepers managed the majority (91.7 %) of the 414 267 surveyed colonies. Overall, 15.1 % of the estimated 2.74 million managed colonies in the USA were included in the study. Total honey bee colony losses (based on the entirety of included colonies) were higher in summer (25.3 %) than in winter (22.3 %) and amounted to 40.6 % for the entire 2014/2015 beekeeping year. Average colony losses per beekeeper or operation were higher in winter (43.7 %) than in summer (14.7 %) and amounted to 49 % for the entire 2014/2015 beekeeping year. Due to the dominance of backyard beekeepers among participants, average losses per operation (or unweighted loss) stronger reflected this smaller type of beekeeper. Backyard beekeepers mainly named colony management issues (e.g., starvation, weak colony in the fall) as causes for mortality, while sideline and commercial beekeepers stronger emphasized parasites or factors outside their control (e.g., varroa, nosema, queen failure).
The second study took place at reclaimed sand mines. Sand mines represent anthropogenically impacted habitats found worldwide, which bear potential for bee conservation. Although floral resources can be limited at these habitats, vegetation free patches of open sandy soils and embankments may offer good nesting possibilities for sand restricted and other bees. We compared bee communities as found in three reclaimed sand mines and at adjacent roadside meadows in Maryland, USA, over two years. Both sand mines and roadsides hosted diverse bee communities with 111 and 88 bee species, respectively. Bee abundances as well as richness and Shannon diversity of bee species were higher in sand mines than at roadsides and negatively correlated with the percentage of vegetational ground cover. Species composition also differed significantly between habitats. Sand mines hosted a higher proportion of ground nesters, more uncommon and more ‘sand loving’ bees similar to natural sandy areas of Maryland. Despite the destruction of the original pre-mining habitat, sand mines thus appear to represent a unique habitat for wild bees, particularly when natural vegetation and open sand spots are encouraged. Considering habitat loss, the lack of natural disturbance regimes, and ongoing declines of wild bees, sand mines could add promising opportunities for bee conservation which has hitherto mainly focused on agricultural and urban habitats.
The third study was an experimental field study on pollinator friendly plants. Bees rely on the pollen and nectar of plants as their food source. Therefore, pollinator friendly plantings are often used for habitat enhancements in bee conservation. Non-native pollinator friendly plants may aid in bee conservation efforts, but have not been tested and compared with native pollinator friendly plants in a common garden experiment. In this study, we seeded mixes of 20 native and 20 non-native pollinator friendly plants in two separate plots at three sites in Maryland, USA. For two years, we recorded flower visitors to the plants throughout the blooming period and additionally sampled bees with pan traps. A total of 3744 bees (120 species) were sampled in the study. Of these, 1708 bees (72 species) were hand netted directly from flowers for comparisons between native and non-native plants. Depending on the season, bee abundance and species richness was either similar or lower (early season and for richness also late season) at native plots compared to non-native plots. Additionally, the overall bee community composition differed significantly between native and non-native plots. Furthermore, native plants were associated with more specialized plant-bee visitation networks compared to non-native plants. In general, visitation networks were more specialized in the early season than the later seasons. Four species (Bombus impatiens, Halictus poeyi/ligatus, Lasioglossum pilosum, and Xylocopa virginica) out of the five most abundant bee species (also including Apis mellifera) foraged more specialized on native than non-native plants. Our study showed that non-native plants were well accepted by a diverse bee community and had a similar to higher attraction for bees compared to native plants. However, we also demonstrated alterations in foraging behavior, bee community assemblage, and visitation networks. As long as used with caution, non-native plants can be a useful addition to native pollinator friendly plantings. This study gives a first example of a direct comparison between native and non-native pollinator friendly plants.
Background: The carpenter ant Camponotus floridanus harbors obligate intracellular mutualistic bacteria (Blochmannia floridanus) in specialized cells, the bacteriocytes, intercalated in their midgut tissue. The diffuse distribution of bacteriocytes over the midgut tissue is in contrast to many other insects carrying endosymbionts in specialized tissues which are often connected to the midgut but form a distinct organ, the bacteriome. C.floridanus is a holometabolous insect which undergoes a complete metamorphosis. During pupal stages a complete restructuring of the inner organs including the digestive tract takes place. So far, nothing was known about maintenance of endosymbionts during this life stage of a holometabolous insect. It was shown previously that the number of Blochmannia increases strongly during metamorphosis. This implicates an important function of Blochmannia in this developmental phase during which the animals are metabolically very active but do not have access to external food resources. Previous experiments have shown a nutritional contribution of the bacteria to host metabolism by production of essential amino acids and urease-mediated nitrogen recycling. In adult hosts the symbiosis appears to degenerate with increasing age of the animals. Results: We investigated the distribution and dynamics of endosymbiotic bacteria and bacteriocytes at different stages during development of the animals from larva to imago by confocal laser scanning microscopy. The number of bacteriocytes in relation to symbiont-free midgut cells varied strongly over different developmental stages. Especially during metamorphosis the relative number of bacteria-filled bacteriocytes increased strongly when the larval midgut epithelium is shed. During this developmental stage the midgut itself became a huge symbiotic organ consisting almost exclusively of cells harboring bacteria. In fact, during this phase some bacteria were also found in midgut cells other than bacteriocytes indicating a cell-invasive capacity of Blochmannia. In adult animals the number of bacteriocytes generally decreased. Conclusions: During the life cycle of the animals the distribution of bacteriocytes and of Blochmannia endosymbionts is remarkably dynamic. Our data show how the endosymbiont is retained within the midgut tissue during metamorphosis thereby ensuring the maintenance of the intracellular endosymbiosis despite a massive reorganization of the midgut tissue. The transformation of the entire midgut into a symbiotic organ during pupal stages underscores the important role of Blochmannia for its host in particular during metamorphosis.
Deadwood represents an important structural component of forest ecosystems, where it provides diverse niches for saproxylic biota. Although wood-inhabiting prokaryotes are involved in its degradation, knowledge about their diversity and the drivers of community structure is scarce. To explore the effect of deadwood substrate on microbial distribution, the present study focuses on the microbial communities of deadwood logs from 13 different tree species investigated using an amplicon based deep-sequencing analysis. Sapwood and heartwood communities were analysed separately and linked to various relevant wood physico-chemical parameters. Overall, Proteobacteria, Acidobacteria and Actinobacteria represented the most dominant phyla. Microbial OTU richness and community structure differed significantly between tree species and between sapwood and heartwood. These differences were more pronounced for heartwood than for sapwood. The pH value and water content were the most important drivers in both wood compartments. Overall, investigating numerous tree species and two compartments provided a remarkably comprehensive view of microbial diversity in deadwood.
b-Type cytochromes
(1980)
Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.
Plants initially accepted by foraging leaf-cutting ants are later avoided if they prove unsuitable for their symbiotic fungus. Plant avoidance is mediated by the waste produced in the fungus garden soon after the incorporation of the unsuitable leaves, as foragers can learn plant odors and cues from the damaged fungus that are both present in the recently produced waste particles. We asked whether avoidance learning of plants unsuitable for the symbiotic fungus can take place entirely at the colony dump. In order to investigate whether cues available in the waste chamber induce plant avoidance in naïve subcolonies, we exchanged the waste produced by subcolonies fed either fungicide-treated privet leaves or untreated leaves and measured the acceptance of untreated privet leaves before and after the exchange of waste. Second, we evaluated whether foragers could perceive the avoidance cues directly at the dump by quantifying the visits of labeled foragers to the waste chamber. Finally, we asked whether foragers learn to specifically avoid untreated leaves of a plant after a confinement over 3 hours in the dump of subcolonies that were previously fed fungicide-treated leaves of that species. After the exchange of the waste chambers, workers from subcolonies that had access to waste from fungicide-treated privet leaves learned to avoid that plant. One-third of the labeled foragers visited the dump. Furthermore, naïve foragers learned to avoid a specific, previously unsuitable plant if exposed solely to cues of the dump during confinement. We suggest that cues at the dump enable foragers to predict the unsuitable effects of plants even if they had never been experienced in the fungus garden.
Auxin is a molecule, which controls many aspects of plant development through both transcriptional and non-transcriptional signaling responses. AUXIN BINDING PROTEIN1 (ABP1) is a putative receptor for rapid non-transcriptional auxin-induced changes in plasma membrane depolarization and endocytosis rates. However, the mechanism of ABP1-mediated signaling is poorly understood. Here we show that membrane depolarization and endocytosis inhibition are ABP1-independent responses and that auxin-induced plasma membrane depolarization is instead dependent on the auxin influx carrier AUX1. AUX1 was itself not involved in the regulation of endocytosis. Auxin-dependent depolarization of the plasma membrane was also modulated by the auxin efflux carrier PIN2. These data establish a new connection between auxin transport and non-transcriptional auxin signaling.
Macroautophagy (hereafter referred to as autophagy) is a homeostatic process that preserves cellular integrity. In mice, autophagy regulates pancreatic ductal adenocarcinoma (PDAC) development in a manner dependent on the status of the tumor suppressor gene Trp53. Studies published so far have investigated the impact of autophagy blockage in tumors arising from Trp53-hemizygous or -homozygous tissue. In contrast, in human PDACs the tumor suppressor gene TP53 is mutated rather than allelically lost, and TP53 mutants retain pathobiological functions that differ from complete allelic loss. In order to better represent the patient situation, we have investigated PDAC development in a well-characterized genetically engineered mouse model (GEMM) of PDAC with mutant Trp53 (Trp53\(^{R172H}\)) and deletion of the essential autophagy gene Atg7. Autophagy blockage reduced PDAC incidence but had no impact on survival time in the subset of animals that formed a tumor. In the absence of Atg7, non-tumor-bearing mice reached a similar age as animals with malignant disease. However, the architecture of autophagy-deficient, tumor-free pancreata was effaced, normal acinar tissue was largely replaced with low-grade pancreatic intraepithelial neoplasias (PanINs) and insulin expressing islet β-cells were reduced. Our data add further complexity to the interplay between Atg7 inhibition and Trp53 status in tumorigenesis.
Synaptic vesicles (SVs) are a key component of neuronal signaling and fulfil different roles depending on their composition. In electron micrograms of neurites, two types of vesicles can be distinguished by morphological criteria, the classical “clear core” vesicles (CCV) and the typically larger “dense core” vesicles (DCV), with differences in electron density due to their diverse cargos. Compared to CCVs, the precise function of DCVs is less defined. DCVs are known to store neuropeptides, which function as neuronal messengers and modulators [1]. In C. elegans, they play a role in locomotion, dauer formation, egg-laying, and mechano- and chemosensation [2]. Another type of DCVs, also referred to as granulated vesicles, are known to transport Bassoon, Piccolo and further constituents of the presynaptic density in the center of the active zone (AZ), and therefore are important for synaptogenesis [3].
To better understand the role of different types of SVs, we present here a new automated approach to classify vesicles. We combine machine learning with an extension of our previously developed vesicle segmentation workflow, the ImageJ macro 3D ART VeSElecT. With that we reliably distinguish CCVs and DCVs in electron tomograms of C. elegans NMJs using image-based features. Analysis of the underlying ground truth data shows an increased fraction of DCVs as well as a higher mean distance between DCVs and AZs in dauer larvae compared to young adult hermaphrodites. Our machine learning based tools are adaptable and can be applied to study properties of different synaptic vesicle pools in electron tomograms of diverse model organisms.
The melanoma·inducing gene of Xiphophorus fish encodes the Xmrk receptor tyrosine kinase. U sing a highly specific antiserum p~oduced against the recombinant receptor expressed with a baculovirus, it is shown that Xmrk is the most abundant phosphotyrosine protein in fish melanoma and thus highly activated in the tumors. Studies on a melanoma cellline revealed that these cells produce an activity that considerably stimulates receptor autophosphorylation. The stimulating activity induces receptor down-regulation and can be depleted from the melanoma cellsupernatant by the immobilized recombinant receptor protein. The fish melanoma cells can thus be considered autocrine tumor cells providing a source for future purification and characterization of the Xmrk ligand.
The FDA approval of targeted therapy with BRAFV600E inhibitors like vemurafenib and dabrafenib in 2011 has been the first major breakthrough in the treatment of metastatic melanoma since almost three decades. Despite increased progression free survival and elevated overall survival rates, complete responses are scarce due to resistance development approximately six months after the initial drug treatment. It was previously shown in our group that melanoma cells under vemurafenib pressure in vitro and in vivo exhibit features of drug-induced senescence. It is known that some cell types, which undergo this cell cycle arrest, develop a so-called senescence associated secretome and it has been reported that melanoma cell lines also upregulate the expression of different factors after senescence induction. This work describes the effect of the vemurafenib-induced secretome on cells. Conditioned supernatants of vemurafenib-treated cells increased the viability of naive fibroblast and melanoma cell lines. RNA analysis of donor melanoma cells revealed elevated transcriptional levels of FGF1, MMP2 and CCL2 in the majority of tested cell lines under vemurafenib pressure, and I could confirm the secretion of functional proteins. Similar observations were also done after MEK inhibition as well as in a combined BRAF and MEK inhibitor treatment situation. Interestingly, the transcription of other FGF ligands (FGF7, FGF17) was also elevated after MEK/ERK1/2 inhibition. As FGF receptors are therapeutically relevant, I focused on the analysis of FGFR-dependent processes in response to BRAF inhibition. Recombinant FGF1 increased the survival rate of melanoma cells under vemurafenib pressure, while inhibition of the FGFR pathway diminished the viability of melanoma cells in combination with vemurafenib and blocked the stimulatory effect of vemurafenib conditioned medium. The BRAF inhibitor induced secretome is regulated by active PI3K/AKT signaling, and the joint inhibition of mTor and BRAFV600E led to decreased senescence induction and to a diminished induction of the secretome-associated genes. In parallel, combined inhibition of MEK and PI3K also drastically decreased mRNA levels of the relevant secretome components back to basal levels.
In summary, I could demonstrate that BRAF inhibitor treated melanoma cell lines acquire a specific PI3K/AKT dependent secretome, which is characterized by FGF1, CCL2 and MMP2. This secretome is able to stimulate other cells such as naive melanoma cells and fibroblasts and contributes to a better survival under drug pressure. These data are therapeutically highly relevant, as they imply the usage of novel drug combinations, especially specific FGFR inhibitors, with BRAF inhibitors in the clinic.
Autoantibodies to components of the nucleolus are a unique serological feature of patients with scleroderma. There are autoantibodies of several specificities; one type produces a speckled pattern of nucleolar staining in immunofluorescence. In actinomycin D and 5,6-dichloro-{j-D-ribofuranosylbenzimidazoletreated Vero cells, staining was restricted to the fibrillar and not the granular regions. By double immunofluorescence, specific rabbit anti-RNA polymerase I antibodies stained the same fibrillar structures in drug-segregated nucleoli as scleroderma sera. Scleroderma sera immunoprecipitated 13 polypeptides from (35S)methionine-labeled HeLa cell extract with molecular weights ranging from 210,000 to 14,000. Similar polypeptides were precipitated by rabbit anti-RNA polymerase I antibodies, and their common identities were confirmed in immunoabsorption experiments. Microinjection of purified IgG from a patient with speckled nucleolar staining effectively inhibited ribosomal RNA transcription. Autoantibodies to RNA polymerase I were restricted to certain patients with scleroderma and were not found in other autoimmune diseases.
Several studies report autoantibody signatures in cancer. The majority of these studies analyzed adult tumors and compared the seroreactivity pattern of tumor patients with the pattern in healthy controls. Here, we compared the autoimmune response in patients with neuroblastoma and patients with Wilms tumor representing two different childhood tumors. We were able to differentiate untreated neuroblastoma patients from untreated Wilms tumor patients with an accuracy of 86.8%, a sensitivity of 87.0% and a specificity of 86.7%. The separation of treated neuroblastoma patients from treated Wilms tumor patients' yielded comparable results with an accuracy of 83.8%. We furthermore identified the antigens that contribute most to the differentiation between both tumor types. The analysis of these antigens revealed that neuroblastoma was considerably more immunogenic than Wilms tumor. The reported antigens have not been found to be relevant for comparative analyses between other tumors and controls. In summary, neuroblastoma appears as a highly immunogenic tumor as demonstrated by the extended number of antigens that separate this tumor from Wilms tumor.
Neuroblastoma is the most abundant, solid, extracranial tumor in early childhood and the leading cause of cancer-related childhood deaths worldwide. Patients with high-risk neuroblastoma often show MYCN-amplification and elevated levels of Aurora-A. They have a low overall survival and despite multimodal therapy options a poor therapeutic prognosis. MYCN-amplified neuroblastoma cells depend on Aurora-A functionality. Aurora-A stabilizes MYCN and prevents it from proteasomal degradation by competing with the E3 ligase SCFFBXW7. Interaction between Aurora-A and MYCN can be observed only in S phase of the cell cycle and activation of Aurora-A can be induced by MYCN in vitro. These findings suggest the existence of a profound interconnection between Aurora-A and MYCN in S phase. Nevertheless, the details remain elusive and were investigated in this study.
Fractionation experiments show that Aurora-A is recruited to chromatin in S phase in a MYCN-dependent manner. Albeit being unphosphorylated on the activating T288 residue, Aurora-A kinase activity was still present in S phase and several putative, novel targets were identified by phosphoproteomic analysis. Particularly, eight phosphosites dependent on MYCN-activated Aurora-A were identified. Additionally, phosphorylation of serine 10 on histone 3 was verified as a target of this complex in S phase. ChIP-sequencing experiments reveal that Aurora-A regulates transcription elongation as well as histone H3.3 variant incorporation in S phase. 4sU-sequencing as well as immunoblotting demonstrated that Aurora-A activity impacts splicing. PLA measurements between the transcription and replication machinery revealed that Aurora-A prevents the formation of transcription-replication conflicts, which activate of kinase ATR.
Aurora-A inhibitors are already used to treat neuroblastoma but display dose-limiting toxicity. To further improve Aurora-A based therapies, we investigated whether low doses of Aurora-A inhibitor combined with ATR inhibitor could increase the efficacy of the treatment albeit reducing toxicity. The study shows that the combination of both drugs leads to a reduction in cell growth as well as an increase in apoptosis in MYCN-amplified neuroblastoma cells, which is not observable in MYCN non-amplified neuroblastoma cells. This new approach was also tested by a collaboration partner in vivo resulting in a decrease in tumor burden, an increase in overall survival and a cure of 25% of TH-MYCN mice. These findings indicate indeed a therapeutic window for targeting MYCN-amplified neuroblastoma.
RNA polymerase I preparations purified from a rat hepatoma contained DNA topoisomerase activity. The DNA topoisomerase associated with the polymerase had an Mr of 110000, required Mg2+ but not ATP, and was recognized by anti-topoisomerase I antibodies. When added to RNA polymerase I preparations containing topoisomerase activity, anti-topoisomerase I antibodies were able to inhibit the DNA relaxing activity of the preparation as well as RNA synthesis in vitro. RNA polymerase II prepared by analogous procedures did not contain topoisomerase activity and was not recognized by the antibodies. The topoisomerase I: polymerase I complex was reversibly dissociated by column chromatography on Sephacryl S200 in the presence of 0.25 M (NH4hS04. Topoisomerase I was immunolocalized in the transcriptionally active ribosomal gene complex containing RNA polymerase I in situ. These data indicate that topoisomerase I and RNA polymerase I are tightly complexed both in vivo and in vitro, and suggest a role for DNA topoisomerase I in the transcription of ribosomal genes.
HRAS belongs to the RAS genes superfamily. RAS genes are important players in several human tumors and the single-nucleotide polymorphism rs12628 has been shown to contribute to the risk of bladder, colon, gastrointestinal, oral, and thyroid carcinoma. We hypothesized that this SNP may affect the risk of cutaneous melanoma as well. HRAS gene contains a polymorphic region (rs112587690), a repeated hexanucleotide -GGGCCT- located in intron 1. Three alleles of this region, P1, P2, and P3, have been identified that contain two, three, and four repeats of the hexanucleotide, respectively. We investigated the clinical impact of these polymorphisms in a case–control study. A total of 141 melanoma patients and 118 healthy donors from the North America Caucasian population were screened for rs12628 and rs112587690 polymorphisms. Genotypes were assessed by capillary sequencing or fragment analysis, respectively, and rs12628 CC and rs112587690 P1P1 genotypes significantly associated with increased melanoma risk (OR = 3.83, p = 0.003; OR = 11.3, p = 0.033, respectively), while rs112587690 P1P3 frequency resulted significantly higher in the control group (OR = 0.5, p = 0.017). These results suggest that rs12628 C homozygosis may be considered a potential risk factor for melanoma development in the North American population possibly through the linkage to rs112587690.
Cellular responses to outer stimuli are the basis for all biological processes. Signal integration is achieved by protein cascades, recognizing and processing molecules from the environment. Factors released by pathogens or inflammation usually induce an inflammatory response, a signal often transduced by Tumour Necrosis Factor alpha (TNF). TNFα receptors TNF-R1 and TNF-R2 can in turn lead to apoptosis or proliferation via NF-B. These processes are closely regulated by membrane compartimentalization, protein interactions and trafficking. Fluorescence microscopy offers a reliable and non-invasive method to probe these cellular events. However, some processes on a native membrane are not resolvable, as they are well below the diffraction limit of microscopy. The recent development of super-resolution fluorescence microscopy methods enables the observation of these cellular players well below this limit: by localizing, tracking and counting molecules with high spatial and temporal resolution, these new fluorescence microscopy methods offer a previously unknown insight into protein interactions at the near-molecular level. Direct stochastic optical reconstruction microscopy (dSTORM) utilizes the reversible, stochastic blinking events of small commercially available fluorescent dyes, while photoactivated localization microscopy (PALM) utilizes phototransformation of genetically encoded fluorescent proteins. By photoactivating only a small fraction of the present fluorophores in each observation interval, single emitters can be localized with high precision and a super-resolved image can be reconstructed. Quantum Dot Triexciton imaging (QDTI) utilizes the three-photon absorption (triexcitonic) properties of quantum dots (QD) and to achieve a twofold resolution increase using conventional confocal microscopes. In this thesis, experimental approaches were implemented to achieve super-resolution microscopy in fixed and live-cells to study the spatial and temporal dynamics of TNF and other cellular signaling events. We introduce QDTI to study the three-dimensional cellular distribution of biological targets, offering an easy method to achieve resolution enhancement in combination with optical sectioning, allowing the preliminary quantification of labeled proteins. As QDs are electron dense, QDTI can be used for correlative fluorescence and transmission electron microscopy, proving the versatility of QD probes. Utilizing the phototransformation properties of fluorescent proteins, single-receptor tracking on live cells was achieved, applying the concept of single particle tracking PALM (sptPALM) to track the dynamics of a TNF-R1-tdEos chimera on the membrane. Lateral receptor dynamics can be tracked with high precision and the influences of ligand addition or lipid disruption on TNF-R1 mobility was observed. The results reveal complex receptor dynamics, implying internalization processes in response to TNFα stimulation and a role for membrane domains with reduced fluidity, so-called lipid raft domains, in TNF-R1 compartimentalization prior or post ligand induction. Comparisons with previously published FCS data show a good accordance, but stressing the increased data depth available in sptPALM experiments. Additionally, the active transport of NF-κB-tdEos fusions was observed in live neurons under chemical stimulation and/or inhibition. Contrary to phototransformable proteins that need no special buffers to exhibit photoconversion or photoactivation, dSTORM has previously been unsuitable for in vivo applications, as organic dyes relied on introducing the probes via immunostaining in concert with a reductive, oxygen-free medium for proper photoswitching behaviour. ATTO655 had been previously shown to be suitable for live-cell applications, as its switching behavior can be catalyzed by the reductive environment of the cytoplasm. By introducing the cell-permeant organic dye via a chemical tag system, a high specificity and low background was achieved. Here, the labeled histone H2B complex and thus single nucleosome movements in a live cell can be observed over long time periods and with ~20 nm resolution. Implementing these new approaches for imaging biological processes with high temporal and spatial resolution provides new insights into the dynamics and spatial heterogeneities of proteins, further elucidating their function in the organism and revealing properties that are usually only detectable in vitro.
Nucleoli provide the fascinating possibility of linking morphologically distinct structures such as those seen in the electron microscope with biochemical f eatures of the formation and step wise maturation of ribosomes. Localization of proteins by immunocytochemistry and of rRNA genes and their transcripts by in situ hybridization has greatly improved our understanding of the structural-functional relationships of the nucleolus. The present review describes some recent results obtained by electron microscopic in situ hybridization and argues that this approach has the potential to correlate each step of the complex pre-rRNA maturation pathway with nucleolar structures. Evidence is accumulating that the nucleolus-specific U3 snRNPs (small nuclear ribonucleoprotein particles) participate in rRNA processing events, similar to the role played by the nucleoplasmic snRNPs in mRNA maturation. The intranucleolar distribution of U3 snRNA is consistent with the view that it is involved in both early and late stages of pre-rRNA processing.
Aspergillus is an important fungal genus containing economically important species, as well as pathogenic species of animals and plants. Using eighteen fungal species of the genus Aspergillus, we conducted a comprehensive investigation of conserved genes and their evolution. This also allows us to investigate the selection pressure driving the adaptive evolution in the pathogenic species A. fumigatus. Among single-copy orthologs (SCOs) for A. fumigatus and the closely related species A. fischeri, we identified 122 versus 50 positively selected genes (PSGs), respectively. Moreover, twenty conserved genes of unknown function were established to be positively selected and thus important for adaption. A. fumigatus PSGs interacting with human host proteins show over-representation of adaptive, symbiosis-related, immunomodulatory and virulence-related pathways, such as the TGF-β pathway, insulin receptor signaling, IL1 pathway and interfering with phagosomal GTPase signaling. Additionally, among the virulence factor coding genes, secretory and membrane protein-coding genes in multi-copy gene families, 212 genes underwent positive selection and also suggest increased adaptation, such as fungal immune evasion mechanisms (aspf2), siderophore biosynthesis (sidD), fumarylalanine production (sidE), stress tolerance (atfA) and thermotolerance (sodA). These genes presumably contribute to host adaptation strategies. Genes for the biosynthesis of gliotoxin are shared among all the close relatives of A. fumigatus as an ancient defense mechanism. Positive selection plays a crucial role in the adaptive evolution of A. fumigatus. The genome-wide profile of PSGs provides valuable targets for further research on the mechanisms of immune evasion, antimycotic targeting and understanding fundamental virulence processes.
Dendritic cells (DCs) are antigen presenting cells which serve as a passage between the innate and the acquired immunity. Aspergillosis is a major lethal condition in immunocompromised patients caused by the adaptable saprophytic fungus Aspergillus fumigatus. The healthy human immune system is capable to ward off A. fumigatus infections however immune-deficient patients are highly vulnerable to invasive aspergillosis. A. fumigatus can persist during infection due to its ability to survive the immune response of human DCs. Therefore, the study of the metabolism specific to the context of infection may allow us to gain insight into the adaptation strategies of both the pathogen and the immune cells. We established a metabolic model of A. fumigatus central metabolism during infection of DCs and calculated the metabolic pathway (elementary modes; EMs). Transcriptome data were used to identify pathways activated when A. fumigatus is challenged with DCs. In particular, amino acid metabolic pathways, alternative carbon metabolic pathways and stress regulating enzymes were found to be active. Metabolic flux modeling identified further active enzymes such as alcohol dehydrogenase, inositol oxygenase and GTP cyclohydrolase participating in different stress responses in A. fumigatus. These were further validated by qRT-PCR from RNA extracted under these different conditions. For DCs, we outlined the activation of metabolic pathways in response to the confrontation with A. fumigatus. We found the fatty acid metabolism plays a crucial role, along with other metabolic changes. The gene expression data and their analysis illuminate additional regulatory pathways activated in the DCs apart from interleukin regulation. In particular, Toll-like receptor signaling, NOD-like receptor signaling and RIG-I-like receptor signaling were active pathways. Moreover, we identified subnetworks and several novel key regulators such as UBC, EGFR, and CUL3 of DCs to be activated in response to A. fumigatus. In conclusion, we analyze the metabolic and regulatory responses of A. fumigatus and DCs when confronted with each other.
Bispecific T cell engager (BiTE) display a novel design among the class of bispecific antibodies and hold great promise to fight diverse cancers. BiTE molecules consist of two different binding entities derived from two human IgG antibodies connected by a short peptide linker. Their binding arms are directed against the CD3e chain of the T cell receptor on T cells and against an antigen that is specific for (e.g., CD19 for lymphoma in MT103) or over-expressed on (e.g., EpCAM for epithelial cancer in MT110) tumor cells. Without requirement for pre- or co-stimulation, BiTE molecules efficiently redirect CD3+ T cells towards tumor cells expressing the relevant target antigen. Only a BiTE molecule simultaneously bound to both tumor cell and T cell activates the T cell to exert its cytolytic function resulting in tumor cell death. In T cells stimulated with both BiTE and target cells, elevated levels of caspase activation and increased expression of cytotoxic and signaling proteins are observed. These include cytolytic proteins granzyme B and perforin, activation markers CD69 and CD25 and adhesion molecules CD2 and LFA-1. Activated T cells secrete the usual mix of cytokines, among them pro-inflammatory cytokines IFN-g and TNF-a. The membrane of tumor cells expressing the relevant target antigen is perforated during the attack of BiTE-stimulated effector cells as can be concluded from adenylate kinase release from the cytosol of tumor cells. Ca2+-chelator EGTA completely blocked BiTE-mediated activation of caspases and tumor cell lysis. As perforin is strictly Ca2+-dependent, a major role for this pore-forming protein is assumed for the elimination of tumor cells via BiTE-stimulated T cells. Granzyme B and caspases are main players in BiTE-mediated elimination of tumor cells. Inhibitors of granzyme B or caspases reduce or block, respectively the activation of caspases. However, other signals of apoptosis (cleavage of PARP and fragmentation of DNA) were only reduced by granzyme B inhibitor or caspase inhibitor. Most interestingly, the lytic capacity of BiTE molecules was not impaired by granzyme B inhibitor or caspase inhibitor. It seems that there is no requirement for granzyme B and caspases to be present simultaneously. Instead the data presented provide evidence that they can be replaced one at a time by related proteins. Pre-incubation of effector cells with the glucocorticoids dexamethasone or methylprednisolone resulted in markedly decreased secretion of cytokines by T cells yet only a small reduction in the expression of activation markers and adhesion molecules on T cells and specific lysis of tumor cells upon BiTE stimulation. Soluble factors secreted in an undirected manner by BiTE-stimulated T cells do not mediate tumor cell death by themselves. Bystander cells negative for the antigen that is recognized by the BiTE molecule will not be compromised by BiTE activity. The cytokine TGF-b reduced proliferation as well as granzyme B and perforin expression of BiTE-stimulated T cells. Redirected lysis by BiTE-activated T cells was also decreased under the influence of TGF-b, however lysis was still performed at a reasonable rate (72 % of target cells). TGF-b does not exert a deleterious effect on lytic potential of BiTE-stimulated T cells. The minimal anticipated biological effect level for the BiTE MT110 was determined for the entry of MT110 into phase I clinical studies. Experiments analyzing redirected lysis of tumor cells, expression of activation marker CD25 and cytokine release by T cells revealed a MABEL value of 50 pg/ml for MT110.
Cytokines are hormones that carry information from ceJI to ceH. This information is read from their surface upon binding to transmembrane receptors and by the subsequent initiation of receptor oligomerization. An inftuence on this process through mutagenesis on the hormone surface is highly desirab)e for medical reasons. However, an understanding of hormone-receptor interactions requires insight into the structural changes introduced by the mutations. In this line structural studies on human TL-4 and the medically important IL-4 antagonists YI24D and Y124G are presented. The site a.round YI24 is an important epitope responsible for the a.bility of 11-4 t.o ca.use a signal in the target cells. It is shown that the local main-chain structure around residue 124 in the variants remains unchanged. A strategy is presented here which allows the study of these types of proteins and their variants by NMR which does not require carbon Iabeiied sa.mples.
Past experience contributes to behavioural organization mainly via learning: Animals learn otherwise ordinary cues as predictors for biologically significant events. This thesis studies such predictive, associative learning, using the fruit fly Drosophila melanogaster. I ask two main questions, which complement each other: One deals with the processing of those cues that are to be learned as predictors for an important event; the other one deals with the processing of the important event itself, which is to be predicted. Do fruit flies learn about combinations of olfactory and visual cues? I probe larval as well as adult fruit flies for the learning about combinations of olfactory and visual cues, using a so called ‘biconditional discrimination’ task: During training, one odour is paired with reinforcement only in light, but not in darkness; the other odour in turn is reinforced only in darkness, but not in light. Thus, neither the odours nor the visual conditions alone predict reinforcement, only combinations of both do. I find no evidence that either larval or adult fruit flies were to solve such task, speaking against a cross-talk between olfactory and visual modalities. Previous studies however suggest such cross-talk. To reconcile these results, I suggest classifying different kinds of interaction between sensory modalities, according to their site along the sensory-motor continuum: I consider an interaction ‘truly’ cross-modal, if it is between the specific features of the stimuli. I consider an interaction ’amodal’ if it instead engages the behavioural tendencies or ‘values’ elicited by each stimulus. Such reasoning brings me to conclude that different behavioural tasks require different kinds of interaction between sensory modalities; whether a given kind of interaction will be found depends on the neuronal infrastructure, which is a function of the species and the developmental stage. Predictive learning of pain-relief in fruit flies Fruit flies build two opposing kinds of memory, based on an experience with electric shock: Those odours that precede shock during training are learned as predictors for punishment and are subsequently avoided; those odours that follow shock during training on the other hand are learned as signals for relief and are subsequently approached. I focus on such relief learning. I start with a detailed parametric analysis of relief learning, testing for reproducibility as well as effects of gender, repetition of training, odour identity, odour concentration and shock intensity. I also characterize how relief memories, once formed, decay. In addition, concerning the psychological mechanisms of relief learning, first, I show that relief learning establishes genuinely associative conditioned approach behaviour and second, I report that it is most likely not mediated by context associations. These results enable the following neurobiological analysis of relief learning; further, they will form in the future the basis for a mathematical model; finally, they will guide the researchers aiming at uncovering relief learning in other experimental systems. Next, I embark upon neurogenetic analysis of relief learning. First, I report that fruit flies mutant for the so called white gene build overall more ‘negative’ memories about an experience with electric shock. That is, in the white mutants, learning about the painful onset of shock is enhanced, whereas learning about the relieving offset of shock is diminished. As they are coherently affected, these two kinds of learning should be in a balance. The molecular mechanism of the effect of white on this balance remains unresolved. Finally, as a first step towards a neuronal circuit analysis of relief learning, I compare it to reward learning and punishment learning. I find that relief learning is distinct from both in terms of the requirement for biogenic amine signaling: Reward and punishment are respectively signalled by octopamine and dopamine, for relief learning, either of these seem dispensible. Further, I find no evidence for roles for two other biogenic amines, tyramine and serotonin in relief learning. Based on these findings I give directions for further research.
Division of labor represents a major advantage of social insect communities that accounts for their enormous ecological success. In colonies of the honeybee, Apis mellifera, division of labor comprises different tasks of fertile queens and drones (males) and, in general, sterile female workers. Division of labor also occurs among workers in form of an age-related polyethism. This helps them to deal with the great variety of tasks within the colony. After adult eclosion, workers spend around three weeks with various duties inside the hive such as tending the brood or cleaning and building cells. After this period workers switch to outdoor tasks and become foragers collecting nectar, pollen and water. With this behavioral transition, workers face tremendous changes in their sensory environment. In particular, visual sensory stimuli become important, but also the olfactory world changes. Foragers have to perform a completely new behavioral repertoire ranging from long distance navigation based on landmark orientation and polarized-skylight information to learning and memory tasks associated with finding profitable food sources. However, behavioral maturation is not a purely age-related internal program associated with a change, for example, in juvenile hormone titers. External factors such as primer pheromones like the brood pheromone or queen mandibular pheromone can modulate the timing of this transition. In this way colonies are able to flexibly adjust their work force distribution between indoor and outdoor tasks depending on the actual needs of the colony. Besides certain physiological changes, mainly affecting glandular tissue, the transition from indoor to outdoor tasks requires significant adaptations in sensory and higher-order integration centers of the brain.
The mushroom bodies integrate olfactory, visual, gustatory and mechanosensory information. Furthermore, they play important roles in learning and memory processes. It is therefore not surprising that the mushroom bodies, in particular their main input region, the calyx, undergo volumetric neuronal plasticity. Similar to behavioral maturation, plastic changes of the mushroom bodies are associated with age, but are also to be affected by modulating factors such as task and experience.
In my thesis, I analyzed in detail the neuronal processes underlying volumetric plasticity in the mushroom body. Immunohistochemical labeling of synaptic proteins combined with quantitative 3D confocal imaging revealed that the volume increase of the mushroom body calyx is largely caused by the growth of the Kenyon cell dendritic network. This outgrowth is accompanied by changes in the synaptic architecture of the mushroom body calyx, which is organized in a distinct pattern of synaptic complexes, so called microglomeruli. During the first week of natural adult maturation microglomeruli remain constant in total number. With subsequent behavioral transition from indoor duties to foraging, microglomeruli are pruned while the Kenyon cell dendritic network is still growing. As a result of these processes, the mushroom body calyx neuropil volume enlarges while the total number of microgloumeruli becomes reduced in foragers compared to indoor workers. In the visual subcompartments (calyx collar) this process is induced by visual sensory stimuli as the beginning of pruning correlates with the time window when workers start their first orientation flights. The high level of analysis of cellular and subcellular process underlying structural plasticity of the mushroom body calyx during natural maturation will serve as a framework for future investigations of behavioral plasticity in the honeybee.
The transition to foraging is not purely age-dependent, but gets modulated, for example, by the presence of foragers. Ethyl oleate, a primer pheromone that is present only in foragers, was shown to delay the onset of foraging in nurse bees. Using artificial application of additional ethyl oleate in triple cohort colonies, I tested whether it directly affects adult neuronal plasticity in the visual input region of the mushroom body calyx. As the pheromonal treatment failed to induce a clear behavioral phenotype (delayed onset of foraging) it was not possible to show a direct link between the exposure to additional ethyl oleate and neuronal plasticity in mushroom body calyx. However, the general results on synaptic maturation confirmed my data of natural maturation processes in the mushroom body calyx.
Given the result that dendritic plasticity is a major contributor to neuronal plasticity in the mushroom body calyx associated with division of labor, the question arose which proteins could be involved in mediating these effects. Calcium/calmodulin-dependent protein kinase II (CaMKII) especially in mammals, but also in insects (Drosophila, Cockroach), was shown to be involved in facilitating learning and memory processes like long-term synaptic potentiation. In addition to presynaptic effects, the protein was also revealed to directly interact with cytoskeleton elements in the postsynapse. It therefore is a likely candidate to mediate structural synaptic plasticity. As part of my thesis, the presence and distribution of CaMKII was analyzed, and the results showed that the protein is highly concentrated in a distinct subpopulation of the mushroom body intrinsic neurons, the noncompact Kenyon cells. The dendritic network of this population arborizes in two calyx subregions: one receiving mainly olfactory input – the lip – and the collar receiving visual input. This distribution pattern did not change with age or task. The high concentration of CaMKII in dendritic spines and its overlap with f-actin indicates that CaMKII could be a key player inducing structural neuronal plasticity associated with learning and memory formation and/or behavioral transitions related to division of labor. Interestingly CaMKII immunoreactivity was absent in the basal ring, another subregion of the mushroom body calyx formed almost exclusively by the inner compact Kenyon cells and known to receive combined visual and olfactory input. This indicates differences of this mushroom body subregion regarding the molecular mechanisms controlling plastic changes in corresponding Kenyon cells.
How is timing of behavioral and neuronal plasticity regulated? The primer pheromone ethyl oleate was found in high concentrations on foragers and was shown to influence behavioral maturation by delaying the onset of foraging when artificially applied in elevated concentrations. But how is ethyl oleate transferred and how does it shift the work force distribution between indoor and outdoor tasks? Previous work showed that ethyl oleate concentrations are highest in the honeycrop of foragers and suggested that it is transferred and communicated inside the colony via trophallaxis. The results of this thesis however clearly show, that ethyl oleate was not present inside the honey crop or the regurgitate, but rather in the surrounding tissue of the honey crop. As additionally the second highest concentration of ethyl oleate was measured on the surface of the cuticle of forgers, trophallaxis was ruled out as a mode of transmission. Neurophysiological measurements at the level of the antennae (electroantennogram recordings) and the first olfactory neuropil (calcium imaging of activity in the antennal lobe) revealed that the primer pheromone ethyl oleate is received and processed as an olfactory stimulus. Appetitive olfactory conditioning using the proboscis extension response as a behavioral paradigm showed that ethyl oleate can be associated with a sugar reward. This indicates that workers are able to perceive, learn and memorize the presence of this pheromone. As ethyl oleate had to be presented by a heated stimulation device at close range, it can be concluded that this primer pheromone acts via close range/contact chemoreception through the olfactory system. This is also supported by previous behavioral observations.
Taken together, the findings presented in this thesis revealed structural changes in the synaptic architecture of the mushroom body calyx associated with division of labor. For the primer pheromone ethyl oleate, which modulates the transition from nursing to foraging, the results clearly showed that it is received via the olfactory system and presumably acts via this pathway. However, manipulation experiments did not indicate a direct effect of ethyl oleate on synaptic plasticity. At the molecular level, CaMKII is a prime candidate to mediate structural synaptic plasticity in the mushroom body calyx. Future combined structural and functional experiments are needed to finally link the activity of primer pheromones like ethyl oleate to the molecular pathways mediating behavioral and synaptic plasticity associated with division of labor in Apis mellifera. The here identified underlying processes will serve as excellent models for a general understanding of fundamental mechanisms promoting behavioral plasticity.
Quantifying the spatio-temporal distribution of arthropods in tropical rainforests represents a first step towards scrutinizing the global distribution of biodiversity on Earth. To date most studies have focused on narrow taxonomic groups or lack a design that allows partitioning of the components of diversity. Here, we consider an exceptionally large dataset (113,952 individuals representing 5,858 species), obtained from the San Lorenzo forest in Panama, where the phylogenetic breadth of arthropod taxa was surveyed using 14 protocols targeting the soil, litter, understory, lower and upper canopy habitats, replicated across seasons in 2003 and 2004. This dataset is used to explore the relative influence of horizontal, vertical and seasonal drivers of arthropod distribution in this forest. We considered arthropod abundance, observed and estimated species richness, additive decomposition of species richness, multiplicative partitioning of species diversity, variation in species composition, species turnover and guild structure as components of diversity. At the scale of our study (2km of distance, 40m in height and 400 days), the effects related to the vertical and seasonal dimensions were most important. Most adult arthropods were collected from the soil/litter or the upper canopy and species richness was highest in the canopy. We compared the distribution of arthropods and trees within our study system. Effects related to the seasonal dimension were stronger for arthropods than for trees. We conclude that: (1) models of beta diversity developed for tropical trees are unlikely to be applicable to tropical arthropods; (2) it is imperative that estimates of global biodiversity derived from mass collecting of arthropods in tropical rainforests embrace the strong vertical and seasonal partitioning observed here; and (3) given the high species turnover observed between seasons, global climate change may have severe consequences for rainforest arthropods.
Arthropod dark taxa provide new insights into diversity responses to bark beetle infestations
(2022)
Natural disturbances are increasing around the globe, also impacting protected areas. Although previous studies have indicated that natural disturbances result in mainly positive effects on biodiversity, these analyses mostly focused on a few well established taxonomic groups, and thus uncertainty remains regarding the comprehensive impact of natural disturbances on biodiversity. Using Malaise traps and meta‐barcoding, we studied a broad range of arthropod taxa, including dark and cryptic taxa, along a gradient of bark beetle disturbance severities in five European national parks. We identified order‐level community thresholds of disturbance severity and classified barcode index numbers (BINs; a cluster system for DNA sequences, where each cluster corresponds to a species) as negative or positive disturbance indicators. Negative indicator BINs decreased above thresholds of low to medium disturbance severity (20%–30% of trees killed), whereas positive indicator BINs benefited from high disturbance severity (76%–98%). BINs allocated to a species name contained nearly as many positive as negative disturbance indicators, but dark and cryptic taxa, particularly Diptera and Hymenoptera in our data, contained higher numbers of negative disturbance indicator BINs. Analyses of changes in the richness of BINs showed variable responses of arthropods to disturbance severity at lower taxonomic levels, whereas no significant signal was detected at the order level due to the compensatory responses of the underlying taxa. We conclude that the analyses of dark taxa can offer new insights into biodiversity responses to disturbances. Our results suggest considerable potential for forest management to foster arthropod diversity, for example by maintaining both closed‐canopy forests (>70% cover) and open forests (<30% cover) on the landscape.
Cristae architecture is important for the function of mitochondria, the organelles that play the central role in many cellular processes. The mitochondrial contact site and cristae organizing system (MICOS) together with the sorting and assembly machinery (SAM) forms the mitochondrial intermembrane space bridging complex (MIB), a large protein complex present in mammalian mitochondria that partakes in the formation and maintenance of cristae. We report here a new subunit of the mammalian MICOS/MIB complex, an armadillo repeat-containing protein 1 (ArmC1). ArmC1 localizes both to cytosol and mitochondria, where it associates with the outer mitochondrial membrane through its carboxy-terminus. ArmC1 interacts with other constituents of the MICOS/MIB complex and its amounts are reduced upon MICOS/MIB complex depletion. Mitochondria lacking ArmC1 do not show defects in cristae structure, respiration or protein content, but appear fragmented and with reduced motility. ArmC1 represents therefore a peripheral MICOS/MIB component that appears to play a role in mitochondrial distribution in the cell.
Arboreal spiders in deciduous and coniferous trees were investigated on their distribution and diversity. Insecticidal knock-down was used to comprehensively sample spiders from 175 trees from 2001 to 2003 in the Białowieża forest and three remote forests in Poland. We identified 140 species from 9273 adult spiders. Spider communities were distinguished between deciduous and coniferous trees. The richest fauna was collected from Quercus where beta diversity was also highest. A tree-species-specific pattern was clearly observed for Alnus, Carpinus, Picea and Pinus trees and also for those tree species that were fogged in only four or three replicates, namely Betula and Populus. This hitherto unrecognised association was mainly due to the community composition of common species identified in a Dufrene-Legendre indicator species analysis. It was not caused by spatial or temporal autocorrelation. Explaining tree-species specificity for generalist predators like spiders is difficult and has to involve physical and ecological tree parameters like linkage with the abundance of prey species. However, neither did we find a consistent correlation of prey group abundances with spiders nor could differences in spider guild composition explain the observed pattern. Our results hint towards the importance of deterministic mechanisms structuring communities of generalist canopy spiders although the casual relationship is not yet understood.
Cognition refers to the ability to of animals to acquire, process, store and use vital information from the environment. Cognitive processes are necessary to predict the future and reduce the uncertainty of the ever-changing environment. Classically, research on animal cognition focuses on decisive cognitive tests to determine the capacity of a species by the testing the ability of a few individuals. This approach views variability between these tested key individuals as unwanted noise and is thus often neglected. However, inter-individual variability provides important insights to behavioral plasticity, cognitive specialization and brain modularity. Honey bees Apis mellifera are a robust and traditional model for the study of learning, memory and cognition due to their impressive capabilities and rich behavioral repertoire. In this thesis I have applied a novel view on the learning abilities of honey bees by looking explicitly at individual differences in a variety of learning tasks. Are some individual bees consistently smarter than some of her sisters? If so, will a smart individual always perform good independent of the time, the context and the cognitive requirements or do bees show distinct isolated ‘cognitive modules’?
My thesis presents the first comprehensive investigation of consistent individual differences in the cognitive abilities of honey bees. To speak of an individual as behaving consistently, a crucial step is to test the individual multiple times to examine the repeatability of a behavior. I show that free-flying bees remain consistent in a visual discrimination task for three consecutive days. Successively, I explored individual consistency in cognitive proficiency across tasks involving different sensory modalities, contexts and cognitive requirements. I found that free-flying bees show a cognitive specialization between visual and olfactory learning but remained consistent across a simple discrimination task and a complex concept learning task. I wished to further explore individual consistency with respect to tasks of different cognitive complexity, a question that has never been tackled before in an insect. I thus performed a series of four experiments using either visual or olfactory stimuli and a different training context (free-flying and restrained) and tested bees in a discrimination task, reversal learning and negative patterning. Intriguingly, across all these experiments I evidenced the same results: The bees’ performances were consistent across the discrimination task and reversal learning and negative patterning respectively. No association was evidenced between reversal learning and negative patterning. After establishing the existence of consistent individual differences in the cognitive proficiency of honey bees I wished to determine factors which could underlie these differences. Since genetic components are known to underlie inter-individual variability in learning abilities, I studied the effects of genetics on consistency in cognitive proficiency by contrasting bees originating from either from a hive with a single patriline (low genetic diversity) or with multiple patrilines (high genetic diversity). These two groups of bees showed differences in the patterns of individually correlated performances, indicating a genetic component accounts for consistent cognitive individuality. Another major factor underlying variability in learning performances is the individual responsiveness to sucrose solution and to visual stimuli, as evidenced by many studies on restrained bees showing a positive correlation between responsiveness to task relevant stimuli and learning performances. I thus tested whether these relationships between sucrose/visual responsiveness and learning performances are applicable for free-flying bees. Free-flying bees were again subjected to reversal learning and negative patterning and subsequently tested in the laboratory for their responsiveness to sucrose and to light. There was no evidence of a positive relationship between sucrose/visual responsiveness and neither performances of free-flying bees in an elemental discrimination, reversal learning and negative patterning. These findings indicate that relationships established between responsiveness to task relevant stimuli and learning proficiency established in the laboratory with restrained bees might not hold true for a completely different behavioral context i.e. for free-flying bees in their natural environment.
These results show that the honey bee is an excellent insect model to study consistency in cognitive proficiency and to identify the underlying factors. I mainly discuss the results with respect to the question of brain modularity in insects and the adaptive significance of individuality in cognitive abilities for honey bee colonies. I also provide a proposition of research questions which tie in this theme of consistent cognitive proficiency and could provide fruitful areas for future research.
Bees rely on floral pollen and nectar for food. Therefore, pollinator friendly plantings are often used to enrich habitats in bee conservation efforts. As part of these plantings, non‐native plants may provide valuable floral resources, but their effects on native bee communities have not been assessed in direct comparison with native pollinator friendly plantings. In this study, we performed a common garden experiment by seeding mixes of 20 native and 20 non‐native pollinator friendly plant species at separate neighboring plots at three sites in Maryland, USA, and recorded flower visitors for 2 years. A total of 3,744 bees (120 species) were collected. Bee abundance and species richness were either similar across plant types (midseason and for abundance also late season) or lower at native than at non‐native plots (early season and for richness also late season). The overall bee community composition differed significantly between native and non‐native plots, with 11 and 23 bee species being found exclusively at one plot type or the other, respectively. Additionally, some species were more abundant at native plant plots, while others were more abundant at non‐natives. Native plants hosted more specialized plant–bee visitation networks than non‐native plants. Three species out of the five most abundant bee species were more specialized when foraging on native plants than on non‐native plants. Overall, visitation networks were more specialized in the early season than in late seasons. Our findings suggest that non‐native plants can benefit native pollinators, but may alter foraging patterns, bee community assemblage, and bee–plant network structures.
Natural genetic variation makes it possible to discover evolutionary changes that have been maintained in a population because they are advantageous. To understand genotype–phenotype relationships and to investigate trait architecture, the existence of both high-resolution genotypic and phenotypic data is necessary. Arabidopsis thaliana is a prime model for these purposes. This herb naturally occurs across much of the Eurasian continent and North America. Thus, it is exposed to a wide range of environmental factors and has been subject to natural selection under distinct conditions. Full genome sequencing data for more than 1000 different natural inbred lines are available, and this has encouraged the distributed generation of many types of phenotypic data. To leverage these data for meta analyses, AraPheno (https://arapheno.1001genomes.org) provide a central repository of population-scale phenotypes for A. thaliana inbred lines. AraPheno includes various features to easily access, download and visualize the phenotypic data. This will facilitate a comparative analysis of the many different types of phenotypic data, which is the base to further enhance our understanding of the genotype–phenotype map.
Applying microarray‐based techniques to study gene expression patterns: a bio‐computational approach
(2010)
The regulation and maintenance of iron homeostasis is critical to human health. As a constituent of hemoglobin, iron is essential for oxygen transport and significant iron deficiency leads to anemia. Eukaryotic cells require iron for survival and proliferation. Iron is part of hemoproteins, iron-sulfur (Fe-S) proteins, and other proteins with functional groups that require iron as a cofactor. At the cellular level, iron uptake, utilization, storage, and export are regulated at different molecular levels (transcriptional, mRNA stability, translational, and posttranslational). Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5’- or 3‘- untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC). To identify novel IRE-containing mRNAs, we integrated biochemical, biocomputational, and microarray-based experimental approaches. Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Methods In this project response to the iron treatment was examined under different conditions using bioinformatical methods. This would improve our understanding of an iron regulatory network. For these purposes we used microarray gene expression data. To identify novel IRE-containing mRNAs biochemical, biocomputational, and microarray-based experimental approaches were integrated. IRP/IRE messenger ribonucleoproteins were immunoselected and their mRNA composition was analysed using an IronChip microarray enriched for genes predicted computationally to contain IRE-like motifs. Analysis of IronChip microarray data requires specialized tool which can use all advantages of a customized microarray platform. Novel decision-tree based algorithm was implemented using Perl in IronChip Evaluation Package (ICEP). Results IRE-like motifs were identified from genomic nucleic acid databases by an algorithm combining primary nucleic acid sequence and RNA structural criteria. Depending on the choice of constraining criteria, such computational screens tend to generate a large number of false positives. To refine the search and reduce the number of false positive hits, additional constraints were introduced. The refined screen yielded 15 IRE-like motifs. A second approach made use of a reported list of 230 IRE-like sequences obtained from screening UTR databases. We selected 6 out of these 230 entries based on the ability of the lower IRE stem to form at least 6 out of 7 bp. Corresponding ESTs were spotted onto the human or mouse versions of the IronChip and the results were analysed using ICEP. Our data show that the immunoselection/microarray strategy is a feasible approach for screening bioinformatically predicted IRE genes and the detection of novel IRE-containing mRNAs. In addition, we identified a novel IRE-containing gene CDC14A (Sanchez M, et al. 2006). The IronChip Evaluation Package (ICEP) is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip, but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls (Vainshtein Y, et al., 2010).
In the work here presented four distinctly different problems were investigated. The first problem was an investigation into the degradation of Dichloroethylene (DCE) and 1,1-bis (p-Chlorophenyl)-2-dichloroethylene (DDE) utilising pure bacterial cultures. The second investigation dealt with the degradation of DDE and polychlorinated Biphenyl’s (PCB’s) utilising anaerobic sediments and soils from New Zealand. The third investigation worked on the Granulation of anaerobic River-sediments in Upflow Anaerobic Sludge Blanket (UASB) Reactors. The last investigation describes the commissioning of an industrial aerobic Wastewater Treatment Plant and the Implementation of biological Nitrogen- and Phosphate removal in this Wastewater Treatment Plant. Since the chemical Structure of DCE and DDE have certain similarities, Bacteria that were capable of degrading DCE, were tested here, whether they would also be able to degrade DDE utilising a co-metabolic pathway. In the experiments the aerobic bacteria Methylosinus trichosporium and Mycobacterium vaccae and the anaerobic bacteria Acetobacterium woodii and Clostridium butyricum were used. Approximately 60% of the added DCE was degraded by M. vaccae, while M. trichosporium degraded approximately 50%. A. woodii and C. butyricum degraded 40% and 30% respectively of the added DCE. Further experiments with these cultures and DDE lead to a microbial degradation of DDE to an extent of 34.6% for M. vaccae, 14.1% for C. butyricum, 2.2% for A. woodii and 10.5% for M. trichosporium. Additional experiments, utilising [14C]-DDE, showed that the DDE had not been degraded but were attached to the bacterial cells. The second investigation utilised anaerobic soils and sediments from New Zealand to study the anaerobic co-metabolic degradation of DDE and PCB’s. The soils and sediments originated from the River Waikato, from Wastewater Ponds in Kinleith, Marine-Sediments from Mapua, and a variety of soils comtaminated with Pentachlorophenyl (PCP). The cultures from these soils and sediments were raised on a variety of Carbon- and Energy-sources. Beside DDE, Aroclor 1260, and a mix of four pure PCB-Congeneres (one Tetra-, one Hexa, one Hepta- and one Deca-Chlorobiphenyl) were used to test for the reductive dechlorination. The cultivation process of the baceria lasted six months. Samples of the cultures were taken after zero, three and six months. These samples were tested for the increase of cell-protein, the degradation of carbon- and energy-sources, and the removal of the added polychlorinated chemicals. The organochlorines were analysed using reversed phase HPLC and FID-GC. When a change in the Chromatogram was detected the respective cultures were further analysed using ECD-GC and GC-MS. The results showed that the culutres grew under these conditions, but no degradation of DDE and the PCB-Mix could be detected, and only small changes in the composition/chromatograms of Aroclor 1260 were found. The third investigation worked on the Granulation of River-Sediments in UASB-Reactors. Sediments from the River Waikato in New Zealand and the River Saale in Germany were used. In both cases the Granulation process was successful, which was demonstrated by microscopic comparisons of the Sediments and the resulting Granules. The two main bacterial cultures detected were Methanosarcina- and Methanothrix-like cultures. The main carbon- and energy-source was Lactic Acid, which was used at a concentration of 21,8 g COD/L. The Granulation-Process was a combination of using high a COD-Concentration combined with a low Volumetric Loading-Rate. Comparisons of the specific degradation-rates of a variety of carbon- and energy-sources between the Sediments and the Granules, showed no increased degradation rates in regard to the same cell-mass, but the increased bio-mass in the Granules allowed for higher degradation-rates within the UASB-reactors. The fourth investigation describes the commissioning of an industrial Wastewater Treatment Plant for a Dairy-Site in Edendale, Southland, New Zealand. This Plant consists of a DAF-Unit (Dissolved Air Flotation), two Extended Aeration Lagoons with Activated Sludge and two Clarifiers, one for the Activated Sludge and the second for the dosing of Aluminium-Sulphate and the removal of Phosphat-Sulphate. Biological processes for the removal of carbon- and energy-sources were optimised and biological processes for the reduction of Nitrogen- and Phosphate-Concentrations within the wastewater were implemented and optimised. Bilogical removal rates for COD of 95% and above, for Nitrogen of 85-92% and Phosphate of 64-83% were achieved.
Cell culture and protein target-based compound screening strategies, though broadly utilized in selecting candidate compounds, often fail to eliminate candidate compounds with non-target effects and/or safety concerns until late in the drug developmental process. Phenotype screening using intact research animals is attractive because it can help identify small molecule candidate compounds that have a high probability of proceeding to clinical use. Most FDA approved, first-in-class small molecules were identified from phenotypic screening. However, phenotypic screening using rodent models is labor intensive, low-throughput, and very expensive. As a novel alternative for small molecule screening, we have been developing gene expression disease profiles, termed the Transcriptional Disease Signature (TDS), as readout of small molecule screens for therapeutic molecules. In this concept, compounds that can reverse, or otherwise affect known disease-associated gene expression patterns in whole animals may be rapidly identified for more detailed downstream direct testing of their efficacy and mode of action. To establish proof of concept for this screening strategy, we employed a transgenic strain of a small aquarium fish, medaka (Oryzias latipes), that overexpresses the malignant melanoma driver gene xmrk, a mutant egfr gene, that is driven by a pigment cell-specific mitf promoter. In this model, melanoma develops with 100% penetrance. Using the transgenic medaka malignant melanoma model, we established a screening system that employs the NanoString nCounter platform to quantify gene expression within custom sets of TDS gene targets that we had previously shown to exhibit differential transcription among xmrk-transgenic and wild-type medaka. Compound-modulated gene expression was identified using an internet-accessible custom-built data processing pipeline. The effect of a given drug on the entire TDS profile was estimated by comparing compound-modulated genes in the TDS using an activation Z-score and Kolmogorov-Smirnov statistics. TDS gene probes were designed that target common signaling pathways that include proliferation, development, toxicity, immune function, metabolism and detoxification. These pathways may be utilized to evaluate candidate compounds for potential favorable, or unfavorable, effects on melanoma-associated gene expression. Here we present the logistics of using medaka to screen compounds, as well as, the development of a user-friendly NanoString data analysis pipeline to support feasibility of this novel TDS drug-screening strategy.
Neoplasms of the skin represent the most frequent tumors worldwide; fortunately, most of them are benign or semi-malignant and well treatable. However, the two most aggressive and deadly forms of malignant skin-neoplasms are melanoma and Merkel cell carcinoma (MCC), being responsible for more than 90% of skin-cancer related deaths. The last decade has yielded enormous progress in melanoma therapy with the advent of targeted therapies, like BRAF or MEK inhibitors, and immune-stimulating therapies, using checkpoint antibodies targeting CTLA- 4, PD-1 or PD-L1. Very recent studies suggest that also MCC patients benefit from a treatment with checkpoint antibodies. Nevertheless, in an advanced metastatic stage, a cure for both of these aggressive malignancies is still hard to achieve: while only a subset of patients experience durable benefit from the immune-based therapies, the widely applicable targeted therapies struggle with development of resistances that inevitably occur in most patients, and finally lead to their death. The four articles included in this thesis addressed current questions concerning therapy and carcinogenesis of melanoma and MCC. Moreover, they are discussed in the light of the up-to-date research regarding targeted and immune-based therapies. In article I we demonstrated that besides apoptosis, MAPK pathway inhibition in BRAF-mutated melanoma cells also induces senescence, a permanent cell cycle arrest. These cells may provide a source for relapse, as even permanently arrested cancer cells can contribute to a pro-tumorigenic milieu. To identify molecular factors determining the differential response, we established M14 melanoma cell line derived single cell clones that either undergo cell death or arrest when treated with BRAF/MEK inhibitors. Using these single cell clones, we demonstrated in article IV that downregulation of the pro-apoptotic BH3-only protein BIK via epigenetic silencing is involved in apoptosis deficiency, which can be overcome by HDAC inhibitors. These observations provide a possible explanation for the lack of a complete and durable response to MAPK inhibitor treatment in melanoma patients, and suggest the application of HDAC inhibitors as a complimentary therapy to MAPK pathway inhibition. Concerning MCC, we scrutinized the interactions between the Merkel cell polyomavirus’ (MCV) T antigens (TA) and the tumor suppressors p53 and Rb in article II and III, respectively. In article III, we demonstrated that the cell cycle master regulator Rb is the crucial target of MCV large T (LT), while it - in contrast to other polyomavirus LTs - exhibits much lower affinity to the related proteins p107 and p130. Knockdown of MCV LT led to proliferation arrest in MCC cells, which can be rescued by knockdown of Rb, but not by knockdown of p107 and p130. Contrary to Rb, restriction of p53 in MCC seems to be independent of the MCV TAs, as we demonstrated in article II. In conclusion, the presented thesis has revealed new molecular details, regarding the response of melanoma cells towards an important treatment modality and the mechanisms of viral carcinogenesis in MCC.
Predation on pest organisms is an essential ecosystem function supporting yields in modern agriculture. However, assessing predation rates is intricate, and they can rarely be linked directly to predator densities or functions. We tested whether sentinel prey aphid cards are useful tools to assess predation rates in the field. Therefore, we looked at aphid cards of different sizes on the ground level as well as within the vegetation. Additionally, by trapping ground‐dwelling predators, we examined whether obtained predation rates could be linked to predator densities and traits. Predation rates recorded with aphid cards were independent of aphid card size. However, predation rates on the ground level were three times higher than within the vegetation. We found both predatory carabid activity densities as well as community weighted mean body size to be good predictors for predation rates. Predation rates obtained from aphid cards are stable over card type and related to predator assemblages. Aphid cards, therefore, are a useful, efficient method for rapidly assessing the ecosystem function predation. Their use might especially be recommended for assessments on the ground level and when time and resource limitations rule out more elaborate sentinel prey methods using exclosures with living prey animals.
Summary Timber harvesting is currently the most common commercial utilisation activity in tropical forests. Assessing the effects of logging on different aspects of biodiversity and general ecosystem properties is hence of prime importance if the few remaining areas of intact tropical forest are to be protected effectively and efficiently. Tropical amphibian communities are an appropriate model system for studies on the impacts of human-induced environmental changes on the dynamics of complex biological systems. This thesis elaborates on patterns of diversity changes in tropical forest amphibian communities facing habitat alterations associated with selective logging in two globally important eco-regions (Côte d’Ivoire, Upper Guinea, West Africa and Guyana, the Guiana Shield, northern South America). The thesis is organised along two main themes. After a general introduction, a section on general methodology and an introduction to the model systems studied, the first theme moves from general patterns to underlying processes. A second theme running through both chapters carries from undisturbed systems to disturbed systems. A final section integrates findings and addresses implications for conservation management of anthropogenically altered tropical forests. Several case studies at the species- population and community level are being presented and data on the direct and indirect impacts of anthropogenic habitat alteration on respective organizational levels are provided. A key statement that is stressed on throughout the studies is the fact that common measures of diversity, such as species richness and species-diversity only inadequately reflect processes of diversity change following anthropogenic disturbance. They also fail to describe actual impacts on the dynamics of complex biological systems. It is argued that commonly used measures produce an incoherent and insufficient picture of diversity patterns and the underlying processes that shape these patterns. Thus, an understanding of higher levels of diversity, such as β-diversity and functional diversity (and hence compositional patterns) appears to be the key to effectively mitigating the impacts of human-induced disturbance on amphibian communities. It is shown that the predictability of amphibian community composition depends on the respective level of anthropogenic disturbance imposed on a particular habitat. Hence, human activities that lead to changes in the structure of a forest, such as logging, not only alter simple system descriptors, such as the number of species in a given community, but rather alter the dynamics of the entire system. In this context, functional diversity is shown to be an important aspect underlying the actual mechanism that leads to the observed change of predictability patterns. Functional differences between species, rather than number of species per se appear to be the decisive factor in sustaining desirable ecosystem states and thus in maintaining important ecosystem services. Because biological diversity appears to play a substantial role in ecosystem resilience required to safeguard essential ecosystem functions in the face of environmental change, the thesis calls for a critical revision of common diversity assessments approaches. The studies advocate the reconsideration of the uncritical use of widespread measures and descriptors of biodiversity on grounds of inconsistent patterns found throughout numerous studies, including those presented herein.
Background
Fermented wheat germ extract (FWGE) sold under the trade name Avemar exhibits anticancer activity in vitro and in vivo. Its mechanisms of action are divided into antiproliferative and antimetabolic effects. Its influcence on cancer cell metabolism needs further investigation. One objective of this study, therefore, was to further elucidate the antimetabolic action of FWGE. The anticancer compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ) is the major bioactive compound in FWGE and is probably responsible for its anticancer activity. The second objective of this study was to compare the antiproliferative properties in vitro of FWGE and the DMBQ compound.
Methods
The IC\(_{50}\) values of FWGE were determined for nine human cancer cell lines after 24 h of culture. The DMBQ compound was used at a concentration of 24 μmol/l, which is equal to the molar concentration of DMBQ in FWGE. Cell viability, cell cycle, cellular redox state, glucose consumption, lactic acid production, cellular ATP levels, and the NADH/NAD\(^+\) ratio were measured.
Results
The mean IC\(_{50}\) value of FWGE for the nine human cancer cell lines tested was 10 mg/ml. Both FWGE (10 mg/ml) and the DMBQ compound (24 μmol/l) induced massive cell damage within 24 h after starting treatment, with changes in the cellular redox state secondary to formation of intracellular reactive oxygen species. Unlike the DMBQ compound, which was only cytotoxic, FWGE exhibited cytostatic and growth delay effects in addition to cytotoxicity. Both cytostatic and growth delay effects were linked to impaired glucose utilization which influenced the cell cycle, cellular ATP levels, and the NADH/NAD\(^+\) ratio. The growth delay effect in response to FWGE treatment led to induction of autophagy.
Conclusions
FWGE and the DMBQ compound both induced oxidative stress-promoted cytotoxicity. In addition, FWGE exhibited cytostatic and growth delay effects associated with impaired glucose utilization which led to autophagy, a possible previously unknown mechanism behind the influence of FWGE on cancer cell metabolism.
The eukaryotic parasite Trypanosoma brucei has evolved sophisticated strategies to persist within its mammalian host. Trypanosomes evade the hosts' immune system by antigenic variation of their surface coat, consisting of variant surface glycoproteins (VSGs). Out of a repertoire of thousands of VSG genes, only one is expressed at any given time from one of the 15 telomeric expression sites (ES). The VSG is stochastically exchanged either by a transcriptional switch of the active ES (in situ switch) or by a recombinational exchange of the VSG within the active ES. However, for infections to persist, the parasite burden has to be limited. The slender (sl) bloodstream form secretes the stumpy induction factor (SIF), which accumulates with rising parasitemia. SIF induces the irreversible developmental transition from the proliferative sl to the cell cycle-arrested but fly-infective stumpy (st) stage once a concentration threshold is reached. Thus, antigenic variation and st development ensure persistent infections and transmissibility. A previous study in monomorphic cells indicated that the attenuation of the active ES could be relevant for the development of trypanosomes. The present thesis investigated this hypothesis using the inducible overexpression of an ectopic VSG in pleomorphic trypanosomes, which possess full developmental competence. These studies revealed a surprising phenotypic plasticity: while the endogenous VSG was always down-regulated upon induction, the ESactivity determined whether the VSG overexpressors arrested in growth or kept proliferating. Full ES-attenuation induced the differentiation of bona fide st parasites independent of the cell density and thus represents the sole natural SIF-independent differentiation trigger to date. A milder decrease of the ES-activity did not induce phenotypic changes, but appeared to prime the parasites for SIF-induced differentiation. These results demonstrate that antigenic variation and development are linked and indicated that the ES and the VSG are independently regulated. Therefore, I investigated in the second part of my thesis how ES-attenuation and VSG-silencing can be mediated. Integration of reporters with a functional or defective VSG 3'UTR into different genomic loci showed that the maintenance of the active state of the ES depends on a conserved motif within the VSG 3'UTR. In situ switching was only triggered when the telomere-proximal motif was partially deleted, suggesting that it serves as a DNA-binding motif for a telomere-associated protein. The VSG levels seem to be additionally regulated in trans based on the VSG 3'UTR independent of the genomic context, which was reinforced by the regulation of a constitutively expressed reporter with VSG 3' UTR upon ectopic VSG overexpression.
Antibodies directed against RNA polymerase II (B) from Drosophila melanogaster were obtained from rabbit sera and, as monoclonal immunoglobulins, from mouse hybridomas and shown to cross-react with the amphibian enzyme protein. Localization by indirect immunofluorescence microscopy revealed the association of this enzyme with chromatin of interphase nuclei of amphibian cells and its absence in nucleoli. Purified immunoglobulins were microinjected in to nuclei ofliving vitellogenic oocytes of Ple1lrodeles waltlii and X enopus laevis and their effects on transcriptional processes were monitored by biochemical and light and electron microscopic stud ies. RNA polymerase II antibodies from rabbit sera caused a rapid and almost complete release of nascent transcripts from the chromatin axis of the loops of lampbrush chromosomes, followed by collapse of the loops and their retraction on the main chromosome axis. Monoclonal murine antibodies to the Iarge RNA polymerase II subunits also inhibited transcription in chromosome Ioops but appeared to inhibit initiation rather than elongation events. Activities of class land III RNA polymerases were not significantly affected by injection of antibodies to polymerase II, indicating immunological differences between the three RNA polymerases. The potential value of the in vitro test system described , as a very sensitive assay for detecting proteins involved in transcription in living cells, is discussed. 1
Circadian clocks prepare the organism to cyclic environmental changes in light, temperature, or food availability. Here, we characterized the master clock in the brain of a strongly photoperiodic insect, the aphid Acyrthosiphon pisum, immunohistochemically with antibodies against A. pisum Period (PER), Drosophila melanogaster Cryptochrome (CRY1), and crab Pigment-Dispersing Hormone (PDH). The latter antibody detects all so far known PDHs and PDFs (Pigment-Dispersing Factors), which play a dominant role in the circadian system of many arthropods. We found that, under long days, PER and CRY are expressed in a rhythmic manner in three regions of the brain: the dorsal and lateral protocerebrum and the lamina. No staining was detected with anti-PDH, suggesting that aphids lack PDF. All the CRY1-positive cells co-expressed PER and showed daily PER/CRY1 oscillations of high amplitude, while the PER oscillations of the CRY1-negative PER neurons were of considerable lower amplitude. The CRY1 oscillations were highly synchronous in all neurons, suggesting that aphid CRY1, similarly to Drosophila CRY1, is light sensitive and its oscillations are synchronized by light-dark cycles. Nevertheless, in contrast to Drosophila CRY1, aphid CRY1 was not degraded by light, but steadily increased during the day and decreased during the night. PER was always located in the nuclei of the clock neurons, while CRY was predominantly cytoplasmic and revealed the projections of the PER/CRY1-positive neurons. We traced the PER/CRY1-positive neurons through the aphid protocerebrum discovering striking similarities with the circadian clock of D. melanogaster: The CRY1 fibers innervate the dorsal and lateral protocerebrum and putatively connect the different PER-positive neurons with each other. They also run toward the pars intercerebralis, which controls hormone release via the neurohemal organ, the corpora cardiaca. In contrast to Drosophila, the CRY1-positive fibers additionally travel directly toward the corpora cardiaca and the close-by endocrine gland, corpora allata. This suggests a direct link between the circadian clock and the photoperiodic control of hormone release that can be studied in the future.
Plant performance is correlated with element concentrations in plant tissue, which may be impacted by adverse chemical soil conditions. Antibiotics of veterinary origin can adversely affect plant performance. They are released to agricultural fields via grazing animals or manure, taken up by plants and may be stored, transformed or sequestered by plant metabolic processes. We studied the potential effects of three antibiotics (penicillin, sulfadiazine, and tetracycline) on plant element contents (macro- and microelements). Plant species included two herb species (Brassica napus and Capsella bursa-pastoris) and two grass species (Triticum aestivum and Apera spica-venti), representing two crop species and two noncrop species commonly found in field margins, respectively. Antibiotic concentrations were chosen as to reflect in vivo situations, that is, relatively low concentrations similar to those detected in soils. In a greenhouse experiment, plants were raised in soil spiked with antibiotics. After harvest, macro- and microelements in plant leaves, stems, and roots were determined (mg/g). Results indicate that antibiotics can affect element contents in plants. Penicillin exerted the greatest effect both on element contents and on scaling relationships of elements between plant organs. Roots responded strongest to antibiotics compared to stems and leaves. We conclude that antibiotics in the soil, even in low concentrations, lead to low-element homeostasis, altering the scaling relationships between roots and other plant organs, which may affect metabolic processes and ultimately the performance of a plant.
The development of all honey bee castes proceeds through three different life stages all of which encounter microbial infections to a various extent. We have examined the immune strength of honey bees across all developmental stages with emphasis on the temporal expression of cellular and humoral immune responses upon artificial challenge with viable Escherichia coli bacteria. We employed a broad array of methods to investigate defence strategies of infected individuals: (a) fate of bacteria in the haemocoel; (b) nodule formation and (c) induction of antimicrobial peptides (AMPs). Newly emerged adult worker bees and drones were able to activate efficiently all examined immune reactions. The number of viable bacteria circulating in the haemocoel of infected bees declined rapidly by more than two orders of magnitude within the first 4–6 h post-injection (p.i.), coinciding with the occurrence of melanised nodules. Antimicrobial activity, on the other hand, became detectable only after the initial bacterial clearance. These two temporal patterns of defence reactions very likely represent the constitutive cellular and the induced humoral immune response. A unique feature of honey bees is that a fraction of worker bees survives the winter season in a cluster mostly engaged in thermoregulation. We show here that the overall immune strength of winter bees matches that of young summer bees although nodulation reactions are not initiated at all. As expected, high doses of injected viable E.coli bacteria caused no mortality in larvae or adults of each age. However, drone and worker pupae succumbed to challenge with E.coli even at low doses, accompanied by a premature darkening of the pupal body. In contrast to larvae and adults, we observed no fast clearance of viable bacteria and no induction of AMPs but a rapid proliferation of E.coli bacteria in the haemocoel of bee pupae ultimately leading to their death.
Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C\(_{6}\) and long-chain C\(_{16}\)-ceramides and azido-functionalized ceramide analogs against pathogenic Neisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C\(_{6}\)-ceramide were active against Neisseria meningitidis and N. gonorrhoeae, whereas they were inactive against Escherichia coli and Staphylococcus aureus. Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω–azido-C\(_{6}\)-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω–azido-C\(_{6}\)-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenic Neisseriae.
Anti-CNTN1 IgG3 induces acute conduction block and motor deficits in a passive transfer rat model
(2019)
Background:
Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown.
Methods:
In the present study, we aimed to model acute autoantibody exposure by intraneural injection of IgG of patients with anti-contacin-1 autoantibodies to Lewis rats. Patient IgG obtained during acute onset of disease (IgG3 predominant) and IgG from the chronic phase of disease (IgG4 predominant) were studied in comparison.
Results:
Conduction blocks were measured in rats injected with the “acute” IgG more often than after injection of “chronic” IgG (83.3% versus 35%) and proved to be reversible within a week after injection. Impaired nerve conduction was accompanied by motor deficits in rats after injection of the “acute” IgG but only minor structural changes of the nodes. Paranodal complement deposition was detected after injection of the “acute IgG”. We did not detect any inflammatory infiltrates, arguing against an inflammatory cascade as cause of damage to the nerve. We also did not observe dispersion of paranodal proteins or sodium channels to the juxtaparanodes as seen in patients after chronic exposure to anti-contactin-1.
Conclusions:
Our data suggest that anti-contactin-1 IgG3 induces an acute conduction block that is most probably mediated by autoantibody binding and subsequent complement deposition and may account for acute onset of disease in these patients. This supports the notion of anti-contactin-1-associated neuropathy as a paranodopathy with the nodes of Ranvier as the site of pathogenesis.
Anthropogenic and environmental drivers shape diversity of naturalized plants across the Pacific
(2021)
Aim
The Pacific exhibits an exceptional number of naturalized plant species, but the drivers of this high diversity and the associated compositional patterns remain largely unknown. Here, we aim to (a) improve our understanding of introduction and establishment processes and (b) evaluate whether this information is sufficient to create scientific conservation tools, such as watchlists.
Location
Islands in the Pacific Ocean, excluding larger islands such as New Zealand, Japan, the Philippines and Indonesia.
Methods
We combined information from the most up‐to‐date data sources to quantify naturalized plant species richness and turnover across island groups and investigate the effects of anthropogenic, biogeographic and climate drivers on these patterns. In total, we found 2,672 naturalized plant species across 481 islands and 50 island groups, with a total of 11,074 records.
Results
Most naturalized species were restricted to few island groups, and most island groups have a low number of naturalized species. Island groups with few naturalized species were characterized by a set of widespread naturalized species. Several plant families that contributed many naturalized species globally also did so in the Pacific, particularly Fabaceae and Poaceae. However, many families were significantly over‐ or under‐represented in the Pacific naturalized flora compared to other regions of the world. Naturalized species richness increased primarily with increased human activity and island altitude/area, whereas similarity between island groups in temperature along with richness differences was most important for beta diversity.
Main conclusions
The distribution and richness of naturalized species can be explained by a small set of drivers. The Pacific region contains many naturalized plant species also naturalized in other regions in the world, but our results highlight key differences such as a stronger role of anthropogenic drivers in shaping diversity patterns. Our results establish a basis for predicting and preventing future naturalizations in a threatened biodiversity hotspot.
Desert ants of the genus Cataglyphis possess remarkable visual navigation capabilities. Although Cataglyphis species lack a trail pheromone system, Cataglyphis fortis employs olfactory cues for detecting nest and food sites. To investigate potential adaptations in primary olfactory centers of the brain of C. fortis, we analyzed olfactory glomeruli (odor processing units) in their antennal lobes and compared them to glomeruli in different Cataglyphis species. Using confocal imaging and 3D reconstruction, we analyzed the number, size and spatial arrangement of olfactory glomeruli in C. fortis, C.albicans, C.bicolor, C.rubra, and C.noda. Workers of all Cataglyphis species have smaller numbers of glomeruli (198–249) compared to those previously found in olfactory-guided ants. Analyses in 2 species of Formica – a genus closely related to Cataglyphis – revealed substantially higher numbers of olfactory glomeruli (c. 370), which is likely to reflect the importance of olfaction in these wood ant species. Comparisons between Cataglyphis species revealed 2 special features in C. fortis. First, with c. 198 C. fortis has the lowest number of glomeruli compared to all other species. Second, a conspicuously enlarged glomerulus is located close to the antennal nerve entrance. Males of C. fortis possess a significantly smaller number of glomeruli (c. 150) compared to female workers and queens. A prominent male-specific macroglomerulus likely to be involved in sex pheromone communication occupies a position different from that of the enlarged glomerulus in females. The behavioral significance of the enlarged glomerulus in female workers remains elusive. The fact that C. fortis inhabits microhabitats (salt pans) that are avoided by all other Cataglyphis species suggests that extreme ecological conditions may not only have resulted in adaptations of visual capabilities, but also in specializations of the olfactory system.
Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.
The occurrence of stacked annulate tamellae is documented for a plant cell system, namely for pollen mother cells and developing pollen grains of Canna generalis. Their structural subarchiteeture and relationship to endoplasmie reticulum (ER) and nuclear envelope cisternae is described in detail. The results demonstrate structural homology between plant and animal annulate lamellae and are compatible with, though do not prove, the view that annulate lamcllar cisternae may originate as a degenerative form of endoplasmic retieulum.
Scorpions, living in North African semideserts are - in spite of disrupting experimental interferences - able to maintain a certain direction in their natural environment in the dark on a plane surface. Under comparable laboratory conditions, excluding the possibility of light or gravity orientation, they can orient themselves if a directed air current passes over the "arena." In most cases the scorpions do not run necessarily with or against the wind, but rather maintain constant angles to the air current for anywhere from minutes to many hours. They are running anemomenotactically (ref. 1). Under identical conditions many species of beetles also orient themselves to air currents (refs. 2 to 4). The main problems to be solved in the study of anemomenotactic orientation are: (1) Which physical qualities of the air current have an influence on the anemomenotaxis? (2) With which sense organs do beetles and scorpions perceive wind directions? (3) Which physiological mechanism is the basis of anemomenotactic orientation? (4) What is the biological significance of anemomenotaxis in beetles and scorpions? With respect to these problems, more study has been done on beetles than on scorpions. Therefore, due to lack of space, I shall discuss mainly some of the results obtained in experiments with dung beetles (Geotrupes silvaticus, G. ,Stercorarius, G. armifrons, G. niger, Scarabaeus variolosus) and tenebrionid beetles (Tenebrio molitor, Pimelia grossa, P. tenuicomis, Scaurus dubius).
Background
The metacestode larva of Echinococcus multilocularis (Cestoda: Taeniidae) develops in the liver of intermediate hosts (typically rodents, or accidentally in humans) as a labyrinth of interconnected cysts that infiltrate the host tissue, causing the disease alveolar echinococcosis. Within the cysts, protoscoleces (the infective stage for the definitive canid host) arise by asexual multiplication. These consist of a scolex similar to that of the adult, invaginated within a small posterior body. Despite the importance of alveolar echinococcosis for human health, relatively little is known about the basic biology, anatomy and development of E. multilocularis larvae, particularly with regard to their nervous system.
Results
We describe the existence of a subtegumental nerve net in the metacestode cysts, which is immunoreactive for acetylated tubulin-α and contains small populations of nerve cells that are labeled by antibodies raised against several invertebrate neuropeptides. However, no evidence was found for the existence of cholinergic or serotoninergic elements in the cyst wall. Muscle fibers occur without any specific arrangement in the subtegumental layer, and accumulate during the invaginations of the cyst wall that form brood capsules, where protoscoleces develop. The nervous system of the protoscolex develops independently of that of the metacestode cyst, with an antero-posterior developmental gradient. The combination of antibodies against several nervous system markers resulted in a detailed description of the protoscolex nervous system, which is remarkably complex and already similar to that of the adult worm.
Conclusions
We provide evidence for the first time of the existence of a nervous system in the metacestode cyst wall, which is remarkable given the lack of motility of this larval stage, and the lack of serotoninergic and cholinergic elements. We propose that it could function as a neuroendocrine system, derived from the nervous system present in the bladder tissue of other taeniids. The detailed description of the development and anatomy of the protoscolex neuromuscular system is a necessary first step toward the understanding of the developmental mechanisms operating in these peculiar larval stages.
BACKGROUND: In the face of growing resistance in malaria parasites to drugs, pharmacological combination therapies are important. There is accumulating evidence that methylene blue (MB) is an effective drug against malaria. Here we explore the biological effects of both MB alone and in combination therapy using modeling and experimental data.
RESULTS: We built a model of the central metabolic pathways in P. falciparum. Metabolic flux modes and their changes under MB were calculated by integrating experimental data (RT-PCR data on mRNAs for redox enzymes) as constraints and results from the YANA software package for metabolic pathway calculations. Several different lines of MB attack on Plasmodium redox defense were identified by analysis of the network effects. Next, chloroquine resistance based on pfmdr/and pfcrt transporters, as well as pyrimethamine/sulfadoxine resistance (by mutations in DHF/DHPS), were modeled in silico. Further modeling shows that MB has a favorable synergism on antimalarial network effects with these commonly used antimalarial drugs.
CONCLUSIONS: Theoretical and experimental results support that methylene blue should, because of its resistance-breaking potential, be further tested as a key component in drug combination therapy efforts in holoendemic areas.
In mammals the melanocortin 4 receptor (Mc4r) signaling system has been mainly associated with the regulation of appetite and energy homeostasis. In fish of the genus Xiphophorus (platyfish and swordtails) puberty onset is genetically determined by a single locus, which encodes the mc4r. Wild populations of Xiphophorus are polymorphic for early and late-maturing individuals. Copy number variation of different mc4r alleles is responsible for the difference in puberty onset. To answer whether this is a special adaptation of the Mc4r signaling system in the lineage of Xiphophorus or a more widely conserved mechanism in teleosts, we studied the role of Mc4r in reproductive biology of medaka (Oryzias latipes), a close relative to Xiphophorus and a well-established model to study gonadal development. To understand the potential role of Mc4r in medaka, we characterized the major features of the Mc4r signaling system (mc4r, mrap2, pomc, agrp1). In medaka, all these genes are expressed before hatching. In adults, they are mainly expressed in the brain. The transcript of the receptor accessory protein mrap2 co-localizes with mc4r in the hypothalamus in adult brains indicating a conserved function of modulating Mc4r signaling. Comparing growth and puberty between wild-type and mc4r knockout medaka revealed that absence of Mc4r does not change puberty timing but significantly delays hatching. Embryonic development of knockout animals is retarded compared to wild-types. In conclusion, the Mc4r system in medaka is involved in regulation of growth rather than puberty.
The MEK5/ ERK5 kinase module is a relatively new discovered mitogen-activated protein kinase (MAPK) signalling pathway with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle a function of the cascade during muscle differentiation was examined. ERK5 becomes activated upon induction of differentiation in mouse myoblasts. The selective activation of the pathway results in promoter activation of differentiation-specific genes, such as the cdk-inhibitor p21 gene, the myosin light chain (MLC1A) gene, or an E-box containing promoter element, where myogenic basic-helix-loop-helix proteins such as MyoD or myogenin bind. Moreover, myogenic differentiation is completely blocked, when ERK5 expression is inhibited by antisense RNA. The effect can be detected also on the expression level of myogenic determination and differentiation markers such as p21, MyoD and myogenin. Another new finding is that stable expression of ERK5 in C2C12 leads to differentiation like phenotype and to increased p21 expression levels under growth conditions. These results provide first evidence that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.
Rhodococcus equi is a Gram-positive intracellular pathogen which can cause severe bronchopneumonia in foals. In recent years, the role of this bacterium as human pathogen has been noted, as R.equi infections in humans have increase in frequency. This increase is associated with the rise in immunosupressed individuals, specially AIDS patients, where infection leads to symptoms and pathology similar to those seen in foals with a high mortality rate. Due to its capability to survive and multiply in murine and equine macrophages, R.equi has been classified as a facultative intracellular bacterium. R.equi is found frequently in macrophages in alveolar infiltrate from infected animals. The pathogenicity of R.equi depends on its ability to exist and multiply inside macrophages and has been associated with the presence of virulence plasmids. It has been observed that, inside foal alveolar macrophages, R.equi-containing vacuoles (RCVs) do not mature into phagolysosomes. However, most of the intracellular events during R.equi infection have not been investigated in detail. The aim of this study was to elucidate the intracellular compartmentation of R.equi and the mechanism by which the bacteria avoid destruction in host macrophages. The importance of the virulence-associated plasmids of R.equi for the establishment of RCVs was also evaluated. Furthermore, the intracellular fate of viable and non-viable R.equi was compared in order to study whether viability of R.equi influeciantes the establishment of RCVs. In this study, the RCV was characterized by using a variety of endocytic markers to follow the path of the bacteria trhough murine macropages. Transmission electron microscopy-base analysis showed that R.equi was found equally frequently in phagosomes with loosely or thightly apposed membranes, and RCV often contains numerous membranous vesicles. Laser scanning microscopy of infected macrophages showed that the majority of phagosomes containing R.equi acquired transiently the early endosomal markers Rab5, Ptlns3P, and EEA-1, suggesting initially undisturbed phagosome maturation. Although the RCV acquired some late endosomal markers, such as Rab7, LAMP-1, and Lamp-2, they did not acquired vATPase, did not interact with pre-labeled lysosomes, and failed to acidify. These data clearly suggest that the RCV is a compartment which has left vacuoles that resemble multivesicular body compartments (MVB), which are transport intermediates between early and late endosomes and display internal vesicles very similar to the ones observed within RCVs. Analyisis of several R.equi strains containing either VapA- or VapB-expressing plasmids or neither demonstrated that the possession of the virulence-associated plasmids does not affect phagosome trafficking over a two hour period of infection. The finding that non-viable R.equi was still able to inhibit phagosome maturation (although not to the same extent as viable R.equi did) suggests that heat-insensitive factors, such as cell periphery lipids, may play a major role in inhibition of phagosome maturation, although heat-sensitive factors may also be involved.
Mushroom bodies (MBs) are multisensory integration centers in the insect brain involved in learning and memory formation. In the honeybee, the main sensory input region (calyx) of MBs is comparatively large and receives input from mainly olfactory and visual senses, but also from gustatory/tactile modalities. Behavioral plasticity following differential brood care, changes in sensory exposure or the formation of associative long-term memory (LTM) was shown to be associated with structural plasticity in synaptic microcircuits (microglomeruli) within olfactory and visual compartments of the MB calyx. In the same line, physiological studies have demonstrated that MB-calyx microcircuits change response properties after associative learning. The aim of this review is to provide an update and synthesis of recent research on the plasticity of microcircuits in the MB calyx of the honeybee, specifically looking at the synaptic connectivity between sensory projection neurons (PNs) and MB intrinsic neurons (Kenyon cells). We focus on the honeybee as a favorable experimental insect for studying neuronal mechanisms underlying complex social behavior, but also compare it with other insect species for certain aspects. This review concludes by highlighting open questions and promising routes for future research aimed at understanding the causal relationships between neuronal and behavioral plasticity in this charismatic social insect.
LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun12/2 meiocytes attached telomeres retained the capacity to form bouquetlike clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun12/2 mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.
Author summary:
Correct genome haploidization during meiosis requires tightly regulated chromosome movements that follow a highly conserved choreography during prophase I. Errors in these movements cause subsequent meiotic defects, which typically lead to infertility. At the beginning of meiotic prophase, chromosome ends are tethered to the nuclear envelope (NE). This attachment of telomeres appears to be mediated by well-conserved membrane spanning protein complexes within the NE (LINC complexes). In mouse meiosis, the two main LINC components SUN1 and SUN2 were independently described to localize at the sites of telomere attachment. While SUN1 has been demonstrated to be critical for meiotic telomere attachment, the precise role of SUN2 in this context, however, has been discussed controversially in the field. Our current study was targeted to determine the factual capacity of SUN2 in telomere attachment and chromosome movements in SUN1 deficient mice. Remarkably, although telomere attachment is impaired in the absence of SUN1, we could find a yet undescribed SUN1-independent telomere attachment, which presumably is mediated by SUN2 and KASH5. This SUN2 mediated telomere attachment is stable throughout prophase I and functional in moving telomeres within the NE. Thus, our results clearly indicate that SUN1 and SUN2, at least partially, fulfill redundant meiotic functions.
Efficient expression systems are required for analysis of gene regulation and function in teleost fish. To develop such systems, a nurober of inducible or constitutive promoter and enhancer sequences of fish or higher vertebrate origin were tested for activity in a variety of fish celllines andin embryos of the Japanese medaka fish (Oryzias latipes) and Xiphophorus. The activity of the different promoterenhancer combinations were quantitated. Considerable differences were found for some constructs if tested in vitro or in vivo. From the data obtained, a set of expression vectors for basic research as weH as for aquaculture purposes were established.
Epigenetic alterations may contribute to the generation of cancer cells in a multi-step process of tumorigenesis following irradiation of normal body cells. Primary human fibroblasts with intact cell cycle checkpoints were used as a model to test whether X-ray irradiation with 2 and 4 Gray induces direct epigenetic effects (within the first cell cycle) in the exposed cells. ELISA-based fluorometric assays were consistent with slightly reduced global DNA methylation and hydroxymethylation, however the observed between-group differences were usually not significant. Similarly, bisulfite pyrosequencing of interspersed LINE-1 repeats and centromeric α-satellite DNA did not detect significant methylation differences between irradiated and non-irradiated cultures. Methylation of interspersed ALU repeats appeared to be slightly increased (one percentage point; p = 0.01) at 6 h after irradiation with 4 Gy. Single-cell analysis showed comparable variations in repeat methylation among individual cells in both irradiated and control cultures. Radiation-induced changes in global repeat methylation, if any, were much smaller than methylation variation between different fibroblast strains. Interestingly, α-satellite DNA methylation positively correlated with gestational age. Finally, 450K methylation arrays mainly targeting genes and CpG islands were used for global DNA methylation analysis. There were no detectable methylation differences in genic (promoter, 5' UTR, first exon, gene body, 3' UTR) and intergenic regions between irradiated and control fibroblast cultures. Although we cannot exclude minor effects, i.e. on individual CpG sites, collectively our data suggest that global DNA methylation remains rather stable in irradiated normal body cells in the early phase of DNA damage response.
Melanoma formation in Xiphophorus hybrids is mediated by a growth factor receptor tyrosine kinase oncogene encoded by the Tu locus. In the wild-type parental fish no tumors occur due to the activity of a locus that regulates the activity of the melanoma oncogene. Molecu/ar identification of this regulatory locus (R) requires a precise physical map of the chromosomal region. Therefore we studied esterase isozymes in Xiphophorus, two of which have been previously reported to be linked to locus R. We confinn that ES 1 is a distant marker for R ( approx. 30cM), and contrary to earlier studies, we show that this isozyme is present in all species of the genus and at similar activity Ievels in all organs tested. ES4, which has also been reported to be linked to R, was found to be a misclassification of liver ES1. In an attempt to identify markersthat bridge the large distance between ESl and R, we have generated DNA probes which are highly polymorphic. They will be useful in finding Iandmarks on a physical map of the R-containing chromosomal region.
Analysis of \(Trypanosoma\) \(brucei\) motility and the infection process in the tsetse fly vector
(2021)
African trypanosomes are protist pathogens that are infective for a wide spectrum of mammalian hosts. Motility has been shown to be essential for their survival and represents an important virulence factor. Trypanosoma brucei is transmitted by the bite of the bloodsucking tsetse fly, the only vector for these parasites. The voyage through the fly is complex and requires several migration, proliferation and differentiation steps, which take place in a defined order and in specific fly tissues.
The first part of this doctoral thesis deals with the establishment of the trypanosome tsetse system as a new model for microswimmer analysis. There is an increasing interdisciplinary interest in microbial motility, but a lack of accessible model systems. Therefore, this work introduces the first enclosed in vivo host parasite system that is suitable for analysis of diverse microswimmer types in specific microenvironments. Several methods were used and adapted to gain unprecedented insights into trypanosome motion, the fly´s interior architecture and the physical interaction between host and parasite. This work provides a detailed overview on trypanosome motile behavior as a function of development in diverse host surroundings. In additional, the potential use of artificial environments is shown. This can be used to partly abstract the complex fly architecture and analyze trypanosome motion in defined nature inspired geometries.
In the second part of the thesis, the infection of the tsetse fly is under investigation. Two different trypanosome forms exist in the blood: proliferative slender cells and cell cycle arrested stumpy cells. Previous literature states that stumpy cells are pre adapted to survive inside the fly, whereas slender cells die shortly after ingestion. However, infection experiments in our laboratory showed that slender cells were also potentially infective. During this work, infections were set up so as to minimize the possibility of stumpy cells being ingested, corroborating the observation that slender cells are able to infect flies. Using live cell microscopy and fluorescent reporter cell lines, a comparative analysis of the early development following infection with either slender or stumpy cells was performed. The experiments showed, for the first time, the survival of slender trypanosomes and their direct differentiation to the procyclic midgut stage, contradicting the current view in the field of research. Therefore, we can shift perspectives in trypanosome biology by proposing a revised life cycle model of T. brucei, where both bloodstream stages are infective for the vector.
The live sciences currently undergo a paradigm shift to computer aided discoveries. Discoveries in the live sciences were historically made by either direct observation or as a result of chemical assays. Today we see a growing shift toward computer aided analysis and visualization. This gradual process happens in microscopy. Multidimensional laser scanning microscopy can acquire very complex multichannel data from fixed or live specimen. New probes such as visible fluorescent proteins let us observe the expression of genes and track protein localization. Ion sensitive dyes change intensity with the concentration of ions in the cell. The laser scanning confocal allows us to record these processes in three dimensions over time. This work demonstrates the application of software analysis to multidimensional microscopy data. We introduce methods for volume investigation, ion flux analysis and molecular modeling. The visualization methods are based on a multidimensional data model to accommodate complex datasets. The software uses vector processing and multiple processors to accelerate volume rendering and achieve interactive rendering. The algorithms are based on human visual perception and allow the observer a wide range of mixed render modes. The software was used to reconstruct the pituitary development in zebrafish and observe the degeneration of neurons after injury in a mouse model. Calicum indicator dyes have long been used to study calcium fluxes. We optimized the imaging method to minimize impact on the cell. Live cells were imaged continuously for 45 minutes and subjected to increasing does of a drug. We correlated the amplitude of calcium oscillations to increasing doses of a drug and obtain single cell dose response curves. Because this method is very sensitive and measures single cell responses it has potential in drug discovery and characterization. Microtubules form a dynamic cytoskeleton, which is responsible for cell shape, intracellular transport and has an integral role in mitosis. A hallmark of microtubule organization is lateral interactions. Microtubules are bundles by proteins into dense structures. To estimate the contribution of this bundling process, we created a fractal model of microtubule organization. This model demonstrates that morphology of complex microtubule arrays can be explained by bundling alone. In summary we showed that advances in software for visualization, data analysis and modeling lead to new discoveries.
The phase space for the standard model of the basic four forces for n quanta includes all possible ensemble combinations of their quantum states m, a total of n**m states. Neighbor states reach according to transition possibilities (S-matrix) with emergent time from entropic ensemble gradients.
We replace the “big bang” by a condensation event (interacting qubits become decoherent) and inflation by a crystallization event – the crystal unit cell guarantees same symmetries everywhere. Interacting qubits solidify and form a rapidly growing domain where the n**m states become separated ensemble states, rising long-range forces stop ultimately further growth. After that very early events, standard cosmology with the hot fireball model takes over. Our theory agrees well with lack of inflation traces in cosmic background measurements, large-scale structure of voids and filaments, supercluster formation, galaxy formation, dominance of matter and life-friendliness.
We prove qubit interactions to be 1,2,4 or 8 dimensional (agrees with E8 symmetry of our universe). Repulsive forces at ultrashort distances result from quantization, long-range forces limit crystal growth. Crystals come and go in the qubit ocean. This selects for the ability to lay seeds for new crystals, for self-organization and life-friendliness.
We give energy estimates for free qubits vs bound qubits, misplacements in the qubit crystal and entropy increase during qubit decoherence / crystal formation. Scalar fields for color interaction and gravity derive from the permeating qubit-interaction field. Hence, vacuum energy gets low only inside the qubit crystal. Condensed mathematics may advantageously model free / bound qubits in phase space.
Background: Patterns that arise from an ecological process can be driven as much from the landscape over which the process is run as it is by some intrinsic properties of the process itself. The disentanglement of these effects is aided if it possible to run models of the process over artificial landscapes with controllable spatial properties. A number of different methods for the generation of so-called ‘neutral landscapes’ have been developed to provide just such a tool. Of these methods, a particular class that simulate fractional Brownian motion have shown particular promise. The existing methods of simulating fractional Brownian motion suffer from a number of problems however: they are often not easily generalisable to an arbitrary number of dimensions and produce outputs that can exhibit some undesirable artefacts. Methodology: We describe here an updated algorithm for the generation of neutral landscapes by fractional Brownian motion that do not display such undesirable properties. Using Monte Carlo simulation we assess the anisotropic properties of landscapes generated using the new algorithm described in this paper and compare it against a popular benchmark algorithm. Conclusion/Significance: The results show that the existing algorithm creates landscapes with values strongly correlated in the diagonal direction and that the new algorithm presented here corrects this artefact. A number of extensions of the algorithm described here are also highlighted: we describe how the algorithm can be employed to generate landscapes that display different properties in different dimensions and how they can be combined with an environmental gradient to produce landscapes that combine environmental variation at the local and macro scales.
Melanotic melanoma (MM) of Xiphophorus (Teleostei: Poeciliidae) was studied by conventional preparations and freeze-etch preparations for electron microscopy. MM of Xiphophorus exhibits tightly packed pigment cells with prominent dendritic processes and interdigitations of their plasma membranes. The most impressive feature of MM cells is the occurrence of Iarge lobulated nuclei with numerous nuclear pores and some nuclear pockets. Abundant spheroidal or ellipsoidal melanosomes (diameter 200-650 nm) and vesicular structures are distributed throughout the cellular dendrites, whereas the perinucJear cytoplasm is free of melanosomes.
A further characteristic feature of melanoma cells in fish is the occurrence of melanosome complexes (i.e., "compound melanosomes"). These melanosome complexes consist of a few to numerous melanosomes, which are enveloped by a separate rnembrane. Pinocytotic vesicles couJd be demonstrated with distinct differences in frequency and distribution patterns, indicating differences in the metabolic activities of the cells in the same melanoma. Intercellular junctions are lacking in the MM cells.
The conventional TEM technique showed clear advantages in the demonstration of intemal architecture of organelles, whereas FE bad considerable potential in respect to the visualization of membrane surface specializations.
An ordered NotI fragment map containing over 60 loci and encompassing approximately 17 Mb has been constructed for human chromosome band llpl5. Forty-two probes, including 11 NotI-linking cosmids, were subregionaUy mapped to llpl5 using a subset of the Jl-deletion hybrids. These and 23 other probes defining loci previously mapped to 11p15 were hybridized to genomic DNA digested with NotI and 5 other infrequently cleaving restriction enzymes and separated by pulsed-field gel electrophoresis. Thirty-nine distinct NotI fragments were detected encompassing approximately 85% of the estimated length of llp15. The predicted order of the gene loci used is cenMYODI- PTH-CALCA-ST5-RBTNI-HPX-HBB-RRMlTH/ INS!1GF2-H19-CTSD-MUC2-DRD4-HRAS-RNHtel. This map wiu allow higher resolution mapping of new Ilp15 markers, facilitate positional cloning of disease genes, and provide a framework for the physical mapping of llp15 in clone contigs.
Whereas the role of calcium ions (Ca\(^{2+}\)) in plant signaling is well studied, the physiological significance of pH‐changes remains largely undefined.
Here we developed CapHensor, an optimized dual‐reporter for simultaneous Ca\(^{2+}\) and pH ratio‐imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio‐temporal relationships between membrane voltage, Ca\(^{2+}\)‐ and pH‐dynamics revealed interconnections previously not described.
In tobacco PTs, we demonstrated Ca\(^{2+}\)‐dynamics lag behind pH‐dynamics during oscillatory growth, and pH correlates more with growth than Ca\(^{2+}\). In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA‐responses and even opened stomata in the presence of ABA, disclosing an important pH‐dependent GC signaling node. In MCs, a flg22‐induced membrane depolarization preceded Ca2+‐increases and cytosolic acidification by c. 2 min, suggesting a Ca\(^{2+}\)/pH‐independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage‐, Ca\(^{2+}\)‐ and pH‐responses.
We propose close interrelation in Ca\(^{2+}\)‐ and pH‐signaling that is cell type‐ and stimulus‐specific and the pH having crucial roles in regulating PT growth and stomata movement.
Societal Impact Statement
Pollen relates to many aspects of human and environmental health, which protection and improvement are endorsed by the United Nations Sustainable Development Goals. By highlighting these connections in the frame of current challenges in monitoring and research, we discuss the need of more integrative and multidisciplinary pollen research related to societal needs, improving health of humans and our ecosystems for a sustainable future.
Summary
Pollen is at once intimately part of the reproductive cycle of seed plants and simultaneously highly relevant for the environment (pollinators, vector for nutrients, or organisms), people (food safety and health), and climate (cloud condensation nuclei and climate reconstruction). We provide an interdisciplinary perspective on the many and connected roles of pollen to foster a better integration of the currently disparate fields of pollen research, which would benefit from the sharing of general knowledge, technical advancements, or data processing solutions. We propose a more interdisciplinary and holistic research approach that encompasses total environmental pollen diversity (ePD) (wind and animal and occasionally water distributed pollen) at multiple levels of diversity (genotypic, phenotypic, physiological, chemical, and functional) across space and time. This interdisciplinary approach holds the potential to contribute to pressing human issues, including addressing United Nations Sustainable Development Goals, fostering social and political awareness of these tiny yet important and fascinating particles.
Systems biology looks for emergent system effects from large scale assemblies of molecules and data, for instance in the human platelets. However, the computational efforts in all steps before such insights are possible can hardly be under estimated. In practice this involves numerous programming tasks, the establishment of new database systems but as well their maintenance, curation and data validation. Furthermore, network insights are only possible if strong algorithms decipher the interactions, decoding the hidden system effects. This thesis and my work are all about these challenges. To answer this requirement, an integrated platelet network, PlateletWeb, was assembled from different sources and further analyzed for signaling in a systems biological manner including multilevel data integration and visualization. PlateletWeb is an integrated network database and was established by combining the data from recent platelet proteome and transcriptome (SAGE) studies. The information on protein-protein interactions and kinase-substrate relationships extracted from bioinformatical databases as well as published literature were added to this resource. Moreover, the mass spectrometry-based platelet phosphoproteome was combined with site-specific phosphorylation/ dephosphorylation information and then enhanced with data from Phosphosite and complemented by bioinformatical sequence analysis for site-specific kinase predictions. The number of catalogued platelet proteins was increased by over 80% as compared to the previous version. The integration of annotations on kinases, protein domains, transmembrane regions, Gene Ontology, disease associations and drug targets provides ample functional tools for platelet signaling analysis. The PlateletWeb resource provides a novel systems biological workbench for the analysis of platelet signaling in the functional context of protein networks. By comprehensive exploration, over 15000 phosphorylation sites were found, out of which 2500 have the corresponding kinase associations. The network motifs were also investigated in this anucleate cell and characterize signaling modules based on integrated information on phosphorylation and protein-protein interactions. Furthermore, many algorithmic approaches have been introduced, including an exact approach (heinz) based on integer linear programming. At the same time, the concept of semantic similarities between two genes using Gene Ontology (GO) annotations has become an important basis for many analytical approaches in bioinformatics. Assuming that a higher number of semantically similar gene functional annotations reflect biologically more relevant interactions, an edge score was devised for functional network analysis. Bringing these two approaches together, the edge score, based on the GO similarity, and the node score, based on the expression of the proteins in the analyzed cell type (e.g. data from proteomic studies), the functional module as a maximum-scoring sub network in large protein-protein interaction networks was identified. This method was applied to various proteome datasets (different types of blood cells, embryonic stem cells) to identify protein modules that functionally characterize the respective cell type. This scalable method allows a smooth integration of data from various sources and retrieves biologically relevant signaling modules.
The olive tree is a venerable Mediterranean plant and often used in traditional medicine. The main aim of the present study was to evaluate the effect of Olea europaea L. cv. Arbosana leaf extract (OLE) and its encapsulation within a spanlastic dosage form on the improvement of its pro-oxidant and antiproliferative activity against HepG-2, MCF-7, and Caco-2 human cancer cell lines. The LC-HRESIMS-assisted metabolomic profile of OLE putatively annotated 20 major metabolites and showed considerable in vitro antiproliferative activity against HepG-2, MCF-7, and Caco-2 cell lines with IC\(_{50}\) values of 9.2 ± 0.8, 7.1 ± 0.9, and 6.5 ± 0.7 µg/mL, respectively. The encapsulation of OLE within a (spanlastic) nanocarrier system, using a spraying method and Span 40 and Tween 80 (4:1 molar ratio), was successfully carried out (size 41 ± 2.4 nm, zeta potential 13.6 ± 2.5, and EE 61.43 ± 2.03%). OLE showed enhanced thermal stability, and an improved in vitro antiproliferative effect against HepG-2, MCF-7, and Caco-2 (IC\(_{50}\) 3.6 ± 0.2, 2.3 ± 0.1, and 1.8 ± 0.1 µg/mL, respectively) in comparison to the unprocessed extract. Both preparations were found to exhibit pro-oxidant potential inside the cancer cells, through the potential inhibitory activity of OLE against glutathione reductase and superoxide dismutase (IC\(_{50}\) 1.18 ± 0.12 and 2.33 ± 0.19 µg/mL, respectively). These inhibitory activities were proposed via a comprehensive in silico study to be linked to the presence of certain compounds in OLE. Consequently, we assume that formulating such a herbal extract within a suitable nanocarrier would be a promising improvement of its therapeutic potential.