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Comparing the appetitive learning performance of six European honeybee subspecies in a common apiary
(2021)
The Western honeybee (Apis mellifera L.) is one of the most widespread insects with numerous subspecies in its native range. How far adaptation to local habitats has affected the cognitive skills of the different subspecies is an intriguing question that we investigate in this study. Naturally mated queens of the following five subspecies from different parts of Europe were transferred to Southern Germany: A. m. iberiensis from Portugal, A. m. mellifera from Belgium, A. m. macedonica from Greece, A. m. ligustica from Italy, and A. m. ruttneri from Malta. We also included the local subspecies A. m. carnica in our study. New colonies were built up in a common apiary where the respective queens were introduced. Worker offspring from the different subspecies were compared in classical olfactory learning performance using the proboscis extension response. Prior to conditioning, we measured individual sucrose responsiveness to investigate whether possible differences in learning performances were due to differential responsiveness to the sugar water reward. Most subspecies did not differ in their appetitive learning performance. However, foragers of the Iberian honeybee, A. m. iberiensis, performed significantly more poorly, despite having a similar sucrose responsiveness. We discuss possible causes for the poor performance of the Iberian honeybees, which may have been shaped by adaptation to the local habitat.
Malignant melanomas (MM) in the fish Xiphophorus and in humans were studied both by transmission electron microscopy (TEM) and freeze-etching (FE). In both fish and human melanomas the cells show interdigitations of the,plasma membranes. The nuclei are large and lobulated and have many nuclear pores. Melanosomes are abundant and melanosome complexes ("compound melanosomes") occur regularly. Pinocytotic vesicles could be demonstrated in fish and human melanomas showing iocal differences in frequency and distribution patterns in the tumor. lntercellular junctions are lacking in MM cells from fish and humans. The FE technique showed considerable advantages in demonstrating membrane-surface peculiarities such as nuclear pores or pinocytotic vesicles. The FE replicas of fish melanomas are like those of humans. These findings may support the hypothesis that melanoma in fish and humans reflect the same biological phenomenon.
Behavioural adaptations have made the desert isopod Hemilepistus reaumuri the most successful herbivore and detritivore of the macrofauna of many arid areas in North Africa and Asia Minor. For survival and reproduction Hemilepistus is dependent on burrows. New burrows can only be dug during spring. With the time-consuming digging of a burrow, Hemilepistus has only made the first step towards solving its ecological problems. The burrows are vital and have to be continuously defended against competitors. This requirement is met by co-operation of individuals within the framework of a highly developed social behaviour. In spring adults form monogamous pairs in which partners recognize each other individually and later form, with their progeny, strictly closed family communities. Hemilepistus is compared with a Porcellio' sp. which has developed, convergently, a social behaviour which resembles that of Hemilepistus in many respects, but differs essentially in some aspects, partly reflecting differences in ecological requirements. This and a few other Porcellio species demonstrate some possible steps in the evolution of the social behaviour of Hemilepistus. The female Hemilepistus is-in contrast to Porcellio sp. - semelparous and the selective advantages of monogamy in its environment are not difficult to recognize. This chapter discusses how this mating system could have evolved and especially why monogamous behaviour is also the best method for the Hemilepistus male to maximize its reproductive success. The cohesion of pairs and of family communities in Hemilepistus is based on a highly developed chemical communication system. Individual- and family-specific badges owe their specificity to genetically determined discriminating substances. The nature of the badges raises a series of questions: e.g. since alien badges release aggression, how do parents avoid cannibalizing their young? Similar problems arise from the fact that family badges are mixtures of chemical compounds of very low volatility with the consequence that they can only be transferred by direct contact and that during moulting all substances are lost which an individual does not produce itself. It is shown that in solving these problems inhibiting properties (presumably substances) and learning play a dominant role.
Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.
Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.
The human gut is home for thousands of microbes that are important for human life. As most of these cannot be cultivated, metagenomics is an important means to understand this important community. To perform comparative metagenomic analysis of the human gut microbiome, I have developed SMASH (Simple metagenomic analysis shell), a computational pipeline. SMASH can also be used to assemble and analyze single genomes, and has been successfully applied to the bacterium Mycoplasma pneumoniae and the fungus Chaetomium thermophilum. In the context of the MetaHIT (Metagenomics of the human intestinal tract) consortium our group is participating in, I used SMASH to validate the assembly and to estimate the assembly error rate of 576.7 Gb metagenome sequence obtained using Illumina Solexa technology from fecal DNA of 124 European individuals. I also estimated the completeness of the gene catalogue containing 3.3 million open reading frames obtained from these metagenomes. Finally, I used SMASH to analyze human gut metagenomes of 39 individuals from 6 countries encompassing a wide range of host properties such as age, body mass index and disease states. We find that the variation in the gut microbiome is not continuous but stratified into enterotypes. Enterotypes are complex host-microbial symbiotic states that are not explained by host properties, nutritional habits or possible technical biases. The concept of enterotypes might have far reaching implications, for example, to explain different responses to diet or drug intake. We also find several functional markers in the human gut microbiome that correlate with a number of host properties such as body mass index, highlighting the need for functional analysis and raising hopes for the application of microbial markers as diagnostic or even prognostic tools for microbiota-associated human disorders.
Some members of the physiological human microbiome occasionally cause life-threatening disease even in immunocompetent individuals. A prime example of such a commensal pathogen is Neisseria meningitidis, which normally resides in the human nasopharynx but is also a leading cause of sepsis and epidemic meningitis. Using N. meningitidis as model organism, we tested the hypothesis that virulence of commensal pathogens is a consequence of within host evolution and selection of invasive variants due to mutations at contingency genes, a mechanism called phase variation. In line with the hypothesis that phase variation evolved as an adaptation to colonize diverse hosts, computational comparisons of all 27 to date completely sequenced and annotated meningococcal genomes retrieved from public databases showed that contingency genes are indeed enriched for genes involved in host interactions. To assess within-host genetic changes in meningococci, we further used ultra-deep whole-genome sequencing of throat-blood strain pairs isolated from four patients suffering from invasive meningococcal disease. We detected up to three mutations per strain pair, affecting predominantly contingency genes involved in type IV pilus biogenesis. However, there was not a single (set) of mutation(s) that could invariably be found in all four pairs of strains. Phenotypic assays further showed that these genetic changes were generally not associated with increased serum resistance, higher fitness in human blood ex vivo or differences in the interaction with human epithelial and endothelial cells in vitro. In conclusion, we hypothesize that virulence of meningococci results from accidental emergence of invasive variants during carriage and without within host evolution of invasive phenotypes during disease progression in vivo.
Commuting to work: Nucleolar long non-coding RNA control ribosome biogenesis from near and far
(2021)
Gene expression is an essential process for cellular growth, proliferation, and differentiation. The transcription of protein-coding genes and non-coding loci depends on RNA polymerases. Interestingly, numerous loci encode long non-coding (lnc)RNA transcripts that are transcribed by RNA polymerase II (RNAPII) and fine-tune the RNA metabolism. The nucleolus is a prime example of how different lncRNA species concomitantly regulate gene expression by facilitating the production and processing of ribosomal (r)RNA for ribosome biogenesis. Here, we summarise the current findings on how RNAPII influences nucleolar structure and function. We describe how RNAPII-dependent lncRNA can both promote nucleolar integrity and inhibit ribosomal (r)RNA synthesis by modulating the availability of rRNA synthesis factors in trans. Surprisingly, some lncRNA transcripts can directly originate from nucleolar loci and function in cis. The nucleolar intergenic spacer (IGS), for example, encodes nucleolar transcripts that counteract spurious rRNA synthesis in unperturbed cells. In response to DNA damage, RNAPII-dependent lncRNA originates directly at broken ribosomal (r)DNA loci and is processed into small ncRNA, possibly to modulate DNA repair. Thus, lncRNA-mediated regulation of nucleolar biology occurs by several modes of action and is more direct than anticipated, pointing to an intimate crosstalk of RNA metabolic events.
Insects exhibit complex systems of communication with chemical signalling being the most important mode. Although there are many studies on chemical communication in insects, the evolution of chemical signals is not well understood. Due to the conflict of interests between individuals, different selective pressures might act on sender and receiver. In this thesis I investigate different types of communication where either the sender, the receiver or both parties yield benefits. These studies were conducted with one digger wasp species, honeybees, one chrysidid wasp, and three ant species. Senders might benefit by exploiting existing preferences of receivers. Such sensory exploitation might influence the evolution of male signals that are designed to attract females. The sex pheromone of male European beewolves Philanthus triangulum (Hymenoptera, Crabronidae) might have evolved according to the sensory exploitation hypothesis. A three-step scenario is supported by our studies. First, a major component of the honeybee alarm pheromone, (Z)-11-eicosen-1-ol, is also found on the cuticles and in the air surrounding foraging honeybees. Second, it could be shown, that (Z)-11- eicosen-1-ol plays a crucial role as kairomone for prey identification of honeybees by beewolf females. Third, a reanalysis of the beewolf male sex pheromone shows a remarkable similarity of compounds between the pheromone and the honeybee cuticle, besides the co-occurrence of (Z)-11-eisosen-ol. The majority of the cuticular hydrocarbons of honeybees occur also in the headspace of foraging workers. These results strongly support the hypothesis that beewolf males evolved a pheromone that exploits the females’ pre-existing sensory sensitivity. In addition, the male sex pheromone shows a significantly higher similarity among brothers than among non-related individuals, which might enable beewolf females to discriminate against brothers and avoid detrimental effects of breeding. Together with the studies on the possible sensory exploitation this result shows that both, male and female beewolves probably gain more benefits than costs from the pheromone communication and, thus, the communication system as a whole can be regarded as cooperative. To maintain the reproductive division of labour in eusocial colonies, queens have to signal their presence and fecundity. In the ant Camponotus floridanus (Hymenoptera, Formicidae) queens mark their own eggs with a distinctive pattern of cuticular hydrocarbons. Two different hypotheses have been developed. One suggests a form of worker manipulation by the queen. The alternative hypothesis assumes a cooperative signal that provides information on the condition of the queen. The results of our investigation clearly favour the latter hypothesis. Chemical mimicry is a form of non-cooperative communication that benefits predominantly the sender. We provided conclusive evidence that the cockoo wasp, Hedychrum rutilans (Hymenoptera, Chrysididae), the primary brood parasitoid of Philanthus triangulum, evades recognition by beewolf females most probably by chemical mimicry of the odour of its host. Furthermore, the adaptation of the chemical signature in the social ant parasite Protomognathus americanus (Hymenoptera, Formicidae) to its Leptothorax (Hymenoptera, Formicidae) hosts was investigated. Although this parasite is principally adapted to its hosts’ cuticular hydrocarbon profile, there are still pronounced differences between the profiles of parasites and hosts. This might be explained by the trade-off, which the parasites faces when confronted locally with two host species with different cuticular hydrocarbon profiles. Non-cooperative communication in the sense that only receivers benefit was discovered in the exploitation of honeybees volatile cuticular hydrocarbons by beewolf females. By using emitted (Z)-11-eicosen-1-ol as a kairomone, the receiver, the beewolf female, yields the benefits and the sender, the honeybee prey, bears all the costs. The results of these studies contribute to the understanding of the evolution of cooperative and non-cooperative communication with chemical signals taking into account differential benefits for sender and/or receiver.
The instructive component of waggle dance communication has been shown to increase resource uptake of Apis mellifera colonies in highly heterogeneous resource environments, but an assessment of its relevance in temperate landscapes with different levels of resource heterogeneity is currently lacking. We hypothesized that the advertisement of resource locations via dance communication would be most relevant in highly heterogeneous landscapes with large spatial variation of floral resources. To test our hypothesis, we placed 24 Apis mellifera colonies with either disrupted or unimpaired instructive component of dance communication in eight Central European agricultural landscapes that differed in heterogeneity and resource availability. We monitored colony weight change and pollen harvest as measure of foraging success. Dance disruption did not significantly alter colony weight change, but decreased pollen harvest compared to the communicating colonies by 40%. There was no general effect of resource availability on nectar or pollen foraging success, but the effect of landscape heterogeneity on nectar uptake was stronger when resource availability was high. In contrast to our hypothesis, the effects of disrupted bee communication on nectar and pollen foraging success were not stronger in landscapes with heterogeneous compared to homogenous resource environments. Our results indicate that in temperate regions intra-colonial communication of resource locations benefits pollen foraging more than nectar foraging, irrespective of landscape heterogeneity. We conclude that the so far largely unexplored role of dance communication in pollen foraging requires further consideration as pollen is a crucial resource for colony development and health.
Climatic extreme events can cause the shift or disruption of plant-insect interactions due to altered plant quality, e.g. leaf carbon to nitrogen ratios, and phenology. However, the response of plant-herbivore interactions to extreme events and climatic gradients has been rarely studied, although climatic extremes will increase in frequency and intensity in the future and insect herbivores represent a highly diverse and functionally important group. We set up a replicated climate change experiment along elevational gradients in the German Alps to study the responses of three plant guilds and their herbivory by insects to extreme events (extreme drought, advanced and delayed snowmelt) versus control plots under different climatic conditions on 15 grassland sites. Our results indicate that elevational shifts in CN (carbon to nitrogen) ratios and herbivory depend on plant guild and season. CN ratios increased with altitude for grasses, but decreased for legumes and other forbs. In contrast to our hypotheses, extreme climatic events did not significantly affect CN ratios and herbivory. Thus, our study indicates that nutritional quality of plants and antagonistic interactions with insect herbivores are robust against seasonal climatic extremes. Across the three functional plant guilds, herbivory increased with nitrogen concentrations. Further, increased CN ratios indicate a reduction in nutritional plant quality with advancing season. Although our results revealed no direct effects of extreme climatic events, the opposing responses of plant guilds along elevation imply that competitive interactions within plant communities might change under future climates, with unknown consequences for plant-herbivore interactions and plant community composition.
Combined effects of climate change and extreme events on plants, arthropods and their interactions
(2013)
I. Global climate change directly and indirectly influences biotic and abiotic components of ecosystems. Changes in abiotic ecosystem components caused by climate change comprise temperature increases, precipitation changes and more frequently occurring extreme events. Mediated by these abiotic changes, biotic ecosystem components including all living organisms will also change. Expected changes of plants and animals are advanced phenologies and range shifts towards higher latitudes and altitudes which presumably induce changes in species interactions and composition. Altitudinal gradients provide an optimal opportunity for climate change studies, because they serve as natural experiments due to fast changing climatic conditions within short distances. In this dissertation two different approaches were conducted to reveal species and community responses to climate change. First, species richness and community trait analyses along an altitudinal gradient in the Bavarian Alps (chapters II, III) and second, climate change manipulation experiments under different climatic contexts (chapters IV, V, IV). II. We performed biodiversity surveys of butterfly and diurnal moth species on 34 grassland sites along an altitudinal gradient in the National Park Berchtesgaden. Additionally, we analysed the dominance structure of life-history traits in butterfly assemblages along altitude. Species richness of butterflies and diurnal moths decreased with increasing altitude. The dominance of certain life-history-traits changed along the altitudinal gradient with a higher proportion of larger-winged species and species with higher egg numbers towards higher altitudes. However, the mean egg maturation time, population density and geographic distribution within butterfly assemblages decreased with increasing altitude. Our results indicate that butterfly assemblages were mainly shaped by environmental filtering. We conclude that butterfly assemblages at higher altitudes will presumably lack adaptive capacity to future climatic conditions, because of specific trait combinations. III. In addition to butterfly and diurnal moth species richness we also studied plant species richness in combination with pollination type analyses along the altitudinal gradient. The management type of the alpine grasslands was also integrated in the analyses to detect combined effects of climate and management on plant diversity and pollination type. Plant species richness was highest at intermediate altitudes, whereby the management type influenced the plant diversity with more plant species at grazed compared to mown or non-managed grasslands. The pollination type was affected by both the changing climate along the gradient and the management type. These results suggest that extensive grazing can maintain high plant diversity along the whole altitudinal gradient. With ongoing climate change the diversity peak of plants may shift upwards, which can cause a decrease in biodiversity due to reduced grassland area but also changes in species composition and adaptive potential of pollination types. IV. We set up manipulation experiments on 15 grassland sites along the altitudinal gradient in order to determine the combined effects of extreme climatic events (extreme drought, advanced and delayed snowmelt) and elevation on the nutritional quality and herbivory rates of alpine plants. The leaf CN (carbon to nitrogen) ratio and the plant damage through herbivores were not significantly affected by the simulated extreme events. However, elevation influenced the CN ratios and herbivory rates of alpine plants with contrasting responses between plant guilds. Furthermore, we found differences in nitrogen concentrations and herbivory rates between grasses, legumes and forbs, whereas legumes had the highest nitrogen concentrations and were damaged most. Additionally, CN ratios and herbivory rates increased during the growing season, indicating a decrease of food plant quality during the growing season. Contrasting altitudinal responses of grasses, legumes and forbs presumably can change the dominance structure among these plant guilds with ongoing climate change. V. In this study we analysed the phenological responses of grassland species to an extreme drought event, advanced and delayed snowmelt along the altitudinal gradient. Advanced snowmelt caused an advanced beginning of flowering, whereas this effect was more pronounced at higher than at lower altitudes. Extreme drought and delayed snowmelt had rather low effects on the flower phenology and the responses did not differ between higher and lower sites. The strongest effect influencing flower phenology was altitude, with a declining effect through the season. The length of flowering duration was not significantly influenced by treatments. Our data suggest that plant species at higher altitudes may be more affected by changes in snowmelt timing in contrast to lowland species, as at higher altitudes more severe changes are expected. However, the risk of extreme drought events on flowering phenology seems to be low. VI. We established soil-emergence traps on the advanced snowmelt and control treatment plots in order to detect possible changes in abundances and emergence phenologies of five arthropod orders due to elevation and treatment. Additionally, we analysed the responses of Coleoptera species richness to elevation and treatment. We found that the abundance and species richness of Coleoptera increased with elevation as well as the abundance of Diptera. However, the abundance of Hemiptera decreased with elevation and the abundances of Araneae and Hymenoptera showed no elevational patterns. The advanced snowmelt treatment increased the abundances of Araneae and Hymenoptera. The emergence of soil-hibernating arthropods was delayed up to seven weeks at higher elevations, whereas advanced snowmelt did not influence the emergence phenology of arthropods immediately after snowmelt. With climate change earlier snowmelt will occur more often, which especially will affect soil-hibernating arthropods in alpine regions and may cause desynchronisations between species interactions. VII. In conclusion, we showed that alpine ecosystems are sensitive towards changing climate conditions and extreme events and that many alpine species in the Bavarian Alps are endangered. Many alpine species could exist under warmer climatic conditions, however they are expected to be outcompeted by more competitive lowland species. Furthermore, host-parasite or predator-prey interactions can be disrupted due to different responses of certain guilds to climate change. Understanding and predicting the complex dynamics and potential risks of future climate change remains a great challenge and therefore further studies analysing species and community responses to climate change are needed.
Cancer is one of the leading causes of death worldwide, with currently assessed chances to develop at least one cancer in a lifetime for about 20%. High cases rates and mortality require the development of new anticancer therapies and treatment strategies. Another important concern is toxicity normally associated with conventional therapy methods, such as chemo- and radiotherapy. Among many proposed antitumoral agents, oncolytic viruses are still one of the promising and fast-developing fields of research with almost a hundred studies published data on over 3000 patients since the beginning of the new millennia.
Among all oncolytic viruses, the Vaccinia virus is arguably one of the safest, with an extremely long and prominent history of use, since it was the one and only vaccine used in the Smallpox Eradication Program in the 1970s. Interestingly enough, it was the first oncolytic virus proven to have tumor tropism in vitro and in vivo in laboratory settings, and this year we can celebrate an unofficial 100th anniversary since the publication of the fact. While being highly immunogenic, Vaccinia virus DNA replication takes place in the cytoplasm of the infected cell, and virus genes never integrate into the host genome. Another advantage of using Vaccinia as an oncolytic agent is its high genome capacity, which allows inserting up to 25 kbps of exogenous genes, thus allowing to additionally arm the virus against the tumor.
Oncolytic virus action consists of two major parts: direct oncolysis and immune activation against the tumor, with the latter being the key to successful treatment. To this moment, preclinical research data are mostly generated in immunocompromised xenograft models, which have hurdles to be properly translated for clinical use. In the first part of the current study, fourteen different recombinant Vaccinia virus strains were tested in two different murine tumor cell lines and corresponding immunocompetent animal models. We found, that Copenhagen backbone Vaccinia viruses while being extremely effective in cell culture, do not show significant oncolytic efficacy in animals. In contrast, several of the LIVP backbone viruses tested (specifically, IL-2 expressing ones) have little replication ability when compared to the Copenhagen strain, but are able to significantly delay tumor growth and prolong survival of the treated animals. We have also noted cytokine related toxicity of the animals to be mouse strain specific.
We have also tested the virus with the highest therapeutic benefit in combination with romidepsin and cyclophosphamide. While the combination with histone deacetylase inhibitor romidepsin did not result in therapeutic benefit in our settings, the addition of cyclophosphamide significantly improved the efficacy of the treatment, at the same time reducing cytokine-associated toxicity of the IL-2 expressing virus.
In the second part of the work, we analyzed the ability of adipose-derived mesenchymal stem cells to serve as a carrier for the oncolytic Vaccinia virus. We showed for the first time that the cells can be infected with the virus and can generate virus progeny. They are also able to survive for a substantially long time and, when injected into the bloodstream of tumor-bearing animals, produce the virus that is colonizing the tumor. Analysis of the systemic distribution of the cells after injection revealed that infected and uninfected cells are not distributed in the same manner, possibly suggesting that infected cells are getting recognized and cleared by an impaired immune system of athymic mice faster than non-infected cells. Despite this, injection of virus-loaded adipose-derived mesenchymal stem cells to human A549 tumor-bearing xenograft mice resulted in rapid tumor regression and reduced virus-related side effects of the treatment when compared to injection of the naked virus.
In conclusion, we have tested two different approaches to augmenting oncolytic Vaccinia virus therapy. First, the combination of recombinant Vaccinia virus expressing IL-2 and cyclophosphamide showed promising results in a syngeneic mouse model, despite the low permissivity of murine cells to the virus. Second, we loaded the oncolytic Vaccinia virus into mesenchymal stem cells and have proven that they can potentially serve as a vehicle for the virus.
Background
Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs.
Methods
Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni- and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC).
Results
RCC with and without miR-21 up- and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease.
Conclusions
A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis.
Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing
(2016)
Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect associations that are reproducible and significant after correction for multiple testing.
Background
The mechanisms by which vaccinia virus (VACV) interacts with the innate immune components are complex and involve different mechanisms. iNOS-mediated NO production by myeloid cells is one of the central antiviral mechanisms and this study aims to investigate specifically whether iNOS-mediated NO production by myeloid cells, is involved in tumor eradication following the virus treatment.
Methods
Human colon adenocarcinoma (HCT-116) xenograft tumors were infected by VACV. Infiltration of iNOS\(^{+}\) myeloid cell population into the tumor, and virus titer was monitored following the treatment. Single-cell suspensions were stained for qualitative and quantitative flow analysis. The effect of different myeloid cell subsets on tumor growth and colonization were investigated by depletion studies. Finally, in vitro culture experiments were carried out to study NO production and tumor cell killing. Student’s t test was used for comparison between groups in all of the experiments.
Results
Infection of human colon adenocarcinoma (HCT-116) xenograft tumors by VACV has led to recruitment of many CD11b\(^{+}\) ly6G\(^{+}\) myeloid-derived suppressor cells (MDSCs), with enhanced iNOS expression in the tumors, and to an increased intratumoral virus titer between days 7 and 10 post-VACV therapy. In parallel, both single and multiple rounds of iNOS-producing cell depletions caused very rapid tumor growth within the same period after virus injection, indicating that VACV-induced iNOS\(^{+}\) MDSCs could be an important antitumor effector component. A continuous blockade of iNOS by its specific inhibitor, L-NIL, showed similar tumor growth enhancement 7–10 days post-infection. Finally, spleen-derived iNOS+ MDSCs isolated from virus-injected tumor bearing mice produced higher amounts of NO and effectively killed HCT-116 cells in in vitro transwell experiments.
Conclusions
We initially hypothesized that NO could be one of the factors that limits active spreading of the virus in the cancerous tissue. In contrast to our initial hypothesis, we observed that PMN-MDSCs were the main producer of NO through iNOS and NO provided a beneficial antitumor effect, The results strongly support an important novel role for VACV infection in the tumor microenvironment. VACV convert tumor-promoting MDSCs into tumor-killing cells by inducing higher NO production.
Cofilin
(1999)
This study has identified cofilin, an actin binding protein, as a control element in the reorganization of the actin cytoskeleton which is highly relevant for T lymphocyte activation. Cofilin is regulated in its activity by reversible phosphorylation which is inducible by stimulation through accessory receptors such as CD2 and CD28. First it could be demonstrated that accessory receptor triggering induces the transient association of cofilin with the actin cytoskeleton and that only the dephosphorylated form of cofilin possesses the capacity to bind cytoskeletal actin in vivo. PI3-kinase inhibitors block both the dephosphorylation of cofilin and its association with the actin cytoskeleton. Importantly, cofilin, actin, PI3-kinase and one of its substrates, namely phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) which can bind to cofilin, co-localize within CD2-receptor caps. The cofilin/F-actin interaction has been identified as a crucial regulatory element for receptor cap formation and the strength of signal transduction. To this end, appropriately designed cell permeable non-toxic peptides that are homologous to actin binding motifs of the human cofilin sequence were introduced into untransformed human peripheral blood T lymphocytes. These peptides competitively and dose dependently inhibit the activation induced interaction of cofilin with the actin cytoskeleton in vivo. By this approach it was possible to study, for the first time, the functional consequences of this interaction in immunocompetent T cells. The present data demonstrate that inhibition of the actin/cofilin interaction in human T lymphocytes by means of these cofilin derived peptides abolishes receptor cap formation and strongly modulates functional T cell responses such as T cell proliferation, interleukin-2 production, cell surface expression of CD69, gIFN production, and CD95L expression. Importantly, receptor independent activation by PMA and calcium ionophore circumvents these peptide produced inhibitory effects on lymphocyte stimulation and places the cofilin/actin interaction to a proximal step in the cascade of signaling events following T cell activation via surface signals. The present results are novel since as yet no information existed regarding the molecular elements which link cell surface receptor stimulation directly to the resulting reorganization of the actin cytoskeleton.
Tropical rain forests and coral reefs are usually regarded as the epitome of complexity and diversity. The mechanisms, however, that allow so many species to coexist continuously, still need to be unraveled. Earlier equilibrium models explain community organization with a strict niche separation and specialization of the single species, achieved mainly by interspecific competition and consecutive resource partitioning. Recent non-equilibrium or stochastic models see stochastic factors ("intermediate disturbances") as more important. Such systems are characterized by broad niche overlaps and an unpredictable species composition. Mechanisms of coexistence are most interesting where species interactions are strongest and species packing is highest. This is the case within a functional group or guild where species use similar resources. In this project a community of seven closely related leaf beetle species (Chrysomelidae: Cassidinae) was investigated which coexist on a common host plant system (fam. Convovulaceae) in a tropical moist savanna (Ivory Coast, Comoé-Nationalpark). A broad overlap in the seasonal phenology of the leaf beetle species stood in contrast to a distinct spatial niche differentiation. The beetle community could be separated in a savanna-group (host plant: Ipomoea) and in a river side group (host plant: Merremia). According to a correspondence analysis the five species at the river side, using a common host plant, Merremia hederacea, proved to be predictable in their species composition. They showed a small scale niche differentiation along the light gradient (microhabitats). Laboratory studies confirmed differences in the tolerance towards high temperatures (up to 50°C in the field). Physiological trade-offs between phenology, microclimate and food quality seem best to describe patterns of resource use of the beetle species. Further a phylogeny based on mt-DNA sequencing of the beetle community was compared to its ecological resource use and the evolution of host plant use was reconstructed
A significant relatedness is of fundamental importance for the evolution and maintenance of social life (kin selection theory, Hamilton 1964a,b). Not only kin selection itself, but also more complex evolutionary theories make predictions on the occurrence of conflict and co-operation in animal societies. They all depend on the genetic relationships among individuals. Therefore, the study of unrelated, co-operating individuals provides a unique opportunity to critically test predictions based on these evolutionary theories. Using allozyme electrophoresis, the study species Pachycondyla villosa was found to represent three different species. Young queens in one of these species, provisionally called Pachycondyla cf. inversa, may co-operate during colony founding (pleometrosis). Approximately 50 per cent of all founding colonies collected near Itabuna, Brazil, consisted of two to five founding queens. Queens of P. cf. inversa have to forage for food (semi-claustral founding), and in founding associations only one queen specialised for this risky task. A microsatellite study showed that nestmate queens were typically not related. How can a division of labour be achieved, where one individual performs risky tasks to the favour of another individual to which it is not related? In contrast to the predictions made by group selectionists, this study provided clear evidence that the division of labour among co-foundresses of P. cf. inversa results from social competition: Co-foundresses displayed aggressive interactions and formed dominance hierarchies which predominantly served to force subordinates to forage. The frequency of queen antagonism increased with the duration since food was last added to the foraging arena. The social status was not, or only weakly associated with the reproductive status: As predicted by the reproductive skew theory, all foundresses laid eggs at similar rates, though the subordinate may be harassed during egg laying and occasionally, some of her eggs may be eaten by the dominant. The differential oophagy presumably was also reflected in a microsatellite study of foundress associations, which was conducted shortly after the first workers emerged: Here, the co-foundresses occasionally contributed unequally to the colony’s workers. Conflicts among workers or between workers and queens, e.g. over the division of labour or sex ratio, strongly depend on the genetic relationships among members of a colony. The number of two to five co-founding queens in polygynous colonies of P. cf. inversa, and the lack of relatedness among them, should lead to a decrease in the relatedness of workers. However, nestmate workers were closely related. Furthermore, worker relatedness may decrease as several queens were found to be multiply inseminated. Inbreeding coefficients were significantly different from zero in both queens and workers. No evidence for a geographical substructuring of the population was found. The deviation from random mating presumably was probably due to small, localised nuptial flights. Virgin queens do not mate near their natal nest and disperse before founding colonies. The analysis of cuticular hydrocarbons obtained from live queens revealed consistent differences between the patterns of cuticular hydrocarbons of queens with high vs. low rank: only high-ranking queens showed considerable amounts of cuticular pentadecane (n-C15) and heptadecene (n-C17:1). The presence of the two substances apparently was not associated with reproductive status. It is not yet known, if the two substances indeed serve to communicate high social status in P. cf. inversa. In experimentally assembled associations of two founding queens, queens engaged in aggressive interactions which already within one to twenty minutes resulted in stable dominance hierarchies. The queens attacking first usually won the contest and became dominant. Nest ownership at least for a couple of days did not influence the outcome of dominance interactions in the laboratory experiments, whereas queen body size apparently played an important role: In all eight trials, the larger queen became dominant. However, dominant queens from natural foundress associations were on average not larger than subordinates, suggesting that in the field, resident asymmetries might override size asymmetries only after a more prolonged period of nest ownership. Sequencing of the COI/COII region of mitochondrial DNA displayed sufficient variability for the study of the sociogenetic structure of the secondarily polygynous ant Pachycondyla obscuricornis: Six different haplotypes could be distinguished among six workers of different colonies from one study population in Costa Rica. The variability of other methods which were established (RFLPs, microsatellites, allozymes, and multilocus DNA fingerprinting) was too low for a further study on the genetic structure in P. obscuricornis.
A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the grain-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DHSa as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately SO times that of the combined E. coli catalases. The nucleutide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of tbe L. seeligeri catalase gene.
Chromosome translocations involving llpl3 have been associated with familial aniridia in two kindreds highlighting the chromosomal localization of the AN2 locus. This locus is also part of the WAGR complex (Wilros tumor, aniridia, genitourinary abnormalities, and mental retardation). In one kindred, the translocation is associated with a deletion, and probes for this region were used to identify and clone the breakpoints of the translocation in the second kindred. Comparison of phage restriction maps exclude the presence of any sizable deletion in this case. Sequences at the chromosome 11 breakpoint are conserved in multiple species, suggesting that the translocation falls within the AN2 gene.
Cloning and functional characterization of novel genes expressed preferentially in the human retina
(2005)
The human retina is a multi-layered neuronal tissue specialized for the reception and processing of visual information. The retina is composed of a great diversity of neuronal cell types including rod and cone photoreceptors, bipolar cells, ganglion cells, amacrine cells, horizontal cells and Müller glia. In response to light, a coordinated series of molecular events, the so-called phototransduction cascade, is triggered in photoreceptor cells and the signals from the photoreceptors are further processed by the bipolar and ganglion cells to the higher centers of the brain. The retina as highly complex system may be greatly susceptible to genetic defects which can lead to a wide range of disease phenotypes. Therefore, isolation and characterisation of the genes active in the human retina will facilitate our deeper understanding of retinal physiology and mechanisms underlying retinal degeneration and provide novel candidates for the retinal disease genes. To identify novel genes that are specifically or predominantly expressed in the human retina, a cDNA library enriched for retina specific transcripts was generated using suppression subtractive hybridization (SSH) technique. In total, 1113 clones were randomly isolated from the retina SSH cDNA library and partially sequenced. On the basis of BLASTN algorithm analysis these clones were classified into four categories including those with I) significant homology to known human genes (766/1113), II) significant homology to partial transcripts and hypothetical gene predictions (162/1113), III) no homology to known mRNAs (149/1113), and IV) vector sequences and clones derived from mitochondrial genes (36/1113). After correcting for redundancy, category I represented 234 known human genes and category II a total of 92unknown transcripts. Clones from category I, were selected for expression analysis by RT-PCR in a great number of human tissues. This resulted in the identification of 16 genes which were expressed exclusively in the retina, 13 which were highly expressed in the retina compared to other tissues, 12 genes which were specifically expressed in neuronal tissues and 48 ubiquitously expressed genes. Thus, our expression analysis resulted in the identification of 29 genes exclusively or abundantly transcribed in the human retina. Of those, retina specific genes L25,L33, L35, L37, L38 and L40 were selected for further analysis. To characterize the complete mRNA sequences of these transcripts a full-length human retina cDNA library was constructed. The analysis of the L25 gene revealed three splicing variants of the ABCC5 gene, consequently named ABCC5_SV1 (SV1), ABCC5_SV2 (SV2) and ABCC5_SV3 (SV3).These isoforms comprise the first five exons of ABCC5 and additional novel exons named 5a, 5b and 5c, generated by differential exon usage. The determined lengths of the three transcripts are 2039 bp, 1962 bp, and 1887 bp in size, respectively. RT-PCR, real-time PCR and Northern blot analysis of ABCC5 as well as the isoforms SV1, SV2 and SV3demonstrated high levels of expression for all transcripts in the retina compared to other tissues. Analysis of their nucleotide sequences revealed that inclusion of exon 5a in splicing variant SV1 produced a frame shift and premature termination codon (PTC). Our data show that this splice variant is the target of nonsense mediated mRNA decay (NMD). This was shown by inhibition of protein synthesis with antibiotics puromycin and anisomycin in human cell lines A-RPE 19 and Y79. Our analysis resulted in an increase of the PTC containing transcript and a decrease of the ABCC5 transcript. Conversely, the amount of both transcripts (SV1 and ABCC5) returned to pre-treatment levels after removal of the inhibitors. Together, our results suggest that alternative splicing of the ubiquitously expressed ABCC5 gene in addition to NMD is involved in retina-specific transcriptional regulation of the mRNA level of ABCC5. In contrast, additional experiments demonstrated that the levels of expression ofSV2 and SV3 isoforms do not appear to influence ABCC5 transcription. Several of the cloned genes were selected for additional genotyping of single nucleotide polymorphisms (SNPs) in order to construct their SNP maps which are going to be used for future association studies of complex disease AMD. Thus, identification of novel retinal genes and their functional characterization will further our elucidation of retinal physiology in general and in the diseased state in particular, by providing candidate retinal disease genes.
The gene for the FeS protein of the Rhodopseudomonas sphaeroides b/c1 complex was identified by means of crosshybridization with a segment of the gene encoding the corresponding FeS protein of Neurospora crassa. Plasmids (pRSF1-14) containing the cross-hybridizing region, covering in total 13.5 kb of chromosomal DNA, were expressed in vitro in a homologous system. One RSF plasmid directed the synthesis of all three main polypeptides of the R. sphaeroides blc1 complex: the FeS protein, cytochrome b and cytochrome c1• The FeS protein and cytochrome c1 were apparently synthesized as precursor fonns. None of the pRSF plasmids directed the synthesis of the 10-kd polypeptide found in b/c1 complex preparations. Partial sequencing of the cloned region was performed. Several sites of strong homology between R. sphaeroides and eukaryotic polypeptides of the b/c1 complex were identified. The genes encode the three b/c1 polypeptides in the order: (5') FeS protein, cytochrome b, cytochrome c1• The three genes are transcribed to give a polycistronic mRNA of 2.9 kb. This transcriptional unit has been designated the jbc operon; its coding capacity corresponds to the size of the polycistronic mRNA assuming that only the genes for the FeS protein (jbcF), cytochrome b (jbcß) and cytochrome c1 (jbcC) are present. This could indicate that these three subunits constitute the minimal catalytic unit of the b/c1 complex from photosynthetic membranes.
From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertionwas recloned as aPstl fragment into pJKK3-1, a shuttle vector which repücates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no extemal or intemal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at Ievels comparable to those ofthe B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin 0-producing strain of Streptococcus pyogenes or from üsteriolysin-producing strains of Usteria monoeytogenes, no positive hybridization signals were obtained. These data soggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.
Clonality analysis in B-Cell Chronic Lymphocytic Leukemia (B-CLL) associated with Richter's syndrome
(2006)
B-cell chronic lymphocytic leukemia (B-CLL) comprises 90% of chronic lymphoid leukemias in Western countries and patients with B-CLL have a heterogeneous clinical course. Approximately 3-5% of B-CLL patients encounter transformation to an aggressive lymphoma, mainly diffuse large B-cell lymphoma (DLBCL) or Hodgkin’s lymphoma (HL) which has been defined as Richter’s syndrome and is associated with a poor clinical outcome. The mutational status of the immunoglobulin heavy chain variable region (IgVH) gene not only implies the developmental stage at which the neoplastic transformation occurs in a given B-cell lymphoma, but also constitutes an important prognostic factor in B-CLL, since B-CLL patients with unmutated IgVH genes usually have a poor clinical outcome. Sparse molecular analyses performed in Richter’s syndrome so far suggest that it can occur in B-CLL patients carrying mutated or unmutated IgVH genes, and tumor cells in DLBCL or HL can be clonally identical to the B-CLL clone or arise as an independent, secondary lymphoma. To determine the clonal relationship between DLBCL or Hodgkin/Reed-Sternberg (HRS) cells and pre-existing B-CLL cells in a larger series, to identify the IgVH gene usage and the mutational status and to explore possible prognostic factors in B-CLL undergoing Richter’s transformation, we utilized a PCR-based GeneScan approach with subsequent sequencing of the IgVH genes. In cases with HRS/HRS-like cells laser capture microdissection (LCM) was employed to isolate these cells. In addition, a thorough morphological and immunohistochemical analysis was performed. In total, specimens from 48 patients were investigated including 40 cases of Richter’s syndrome and additional 8 cases of B-CLL cases with the presence of CD30-positive HRS-like cells. Among 40 cases of Richter’s syndrome, 34 B-CLL cases showed transformation to DLBCL and 6 cases transformed from B-CLL to HL. Sequencing was performed in 23 paired B-CLL and DLBCL cases. In 18 cases, B-CLL and DLBCL were clonally identical, whereas DLBCL developed as a clonally independent neoplasm in 5 patients. Among the clonally related pairs, 11 out of 15 cases carried unmutated IgVH genes in both the B-CLL and DLBCL component, whereas 5 of 6 B-CLL cases that showed transformation to HL carried mutated IgVH genes. HRS cells in two samples and HRS-like cells in one sample were clonally distinct from the B-CLL clone and infected by EBV, whereas one sample of HRS-like cells was related to the clone from the surrounding B-CLL cells and did not express latent membrane protein-1 (LMP1). The VH genes VH3-23, VH3-74, VH1-2 and VH3-9 were overused in B-CLL cases that transformed to DLBCL, whereas VH4-34 and VH3-48 were used in over half of the B-CLL cases with transformation to HL. Immunohistochemical staining of ZAP70 was significantly associated with unmutated IgVH genes in B-CLL cases undergoing Richter’s transformation. Clinical follow-up data could be obtained from 24 patients. The median survival times of B-CLL patients with transformation to DLBCL or HL were 7 and 21 months, respectively. No significantly different survival times were found between clonally related or unrelated cases, or between IgVH-mutated or -unmutated cases. We conclude that in Richter’s transformation, DLBCL can evolve by clonal transformation of the pre-existing B-CLL clone or occur as an independent, clonally unrelated neoplasm. In the majority of cases (78% in our series), B-CLL and DLBCL are clonally identical. In a subset of patients, however, DLBCL develops as an independent secondary neoplasm that is not clonally related to the B-CLL. Clonal transformation into DLBCL predominantly occurs in B-CLL patients with unmutated IgVH genes, whereas most B-CLL patients that show transformation to HL or CD30-positive HRS-like cells carry mutated IgVH genes. The tendency that IgVH-unmutated B-CLL transforms to DLBCL and IgVH-mutated B-CLL transforms to HL implies different transformation pathways in the two subtypes of Richter’s syndrome. In addition, important pathogenetic differences are likely to exist between DLBCL cases derived from a pre-existing B-CLL as compared to de novo DLBCL cases, since de novo DLBCL is usually characterized by mutated IgVH genes. The biased usage of IgVH genes in the two subtypes of Richter’s syndrome suggests a possible role for antigen involvement in tumorigenesis also in B-CLL cases that undergo Richter’s transformation. Finally, EBV-association in the HL variant of Richter’s syndrome occurs more frequently in clonally unrelated secondary malignancies.
Clinical evaluation of novel methods to determine dialysis parameters using conductivity cells
(2002)
Eine die letzten beiden Jahrzehnte anhaltende Diskussion über die erforderliche Dosis Dialyse, die ein Patient benötigt, ergab, daß der Harnstoff-basierte Kt/V-Wert signifikant mit der Morbidität von ‚end stage renal deasease' (ESRD)- Patienten korreliert. Wenn auch nicht vollständig akzeptiert, so scheint es doch zunehmende Übereinkunft zwischen Nephrologen, daß eine angemessene Dialysebehandlung von Patienten ohne Restdiurese im Rahmen eines 3x4 Stunden pro Woche- Schemas mindestens einen Kt/V-Wert von 1.2 bis 1.3 ergeben sollte, um auf lange Sicht die Lebensqualität zu sichern und die Morbidität und Mortalität niedrig zu halten. K ist die vom Dialysesystem erbrachte Clearance, t die Behandlungszeit und V das Harnstoff-Verteilungsvolumen, welches dem Gesamt-Körperwasser nahezu gleicht. Kt/V wird in der Dosiseinheit (ml Medikament pro ml Körperwasser) ausgedrückt und daher auch häufig als Dialyse-‚Dosis' bezeichnet, auch wenn darüber wegen der Zusammenfassung in nur einer Harnstoff-korrelierten Zahl konträr diskutiert wird. Diese Arbeit besitzt eine eher technische Prämisse und möchte sich nicht an dieser Diskussion beteiligen. Sie möchte dem interessierten Nephrologen lediglich eine patientenfreundliche, präzise, kostenneutrale und leicht zu handhabende technische Lösung an die Hand geben, um kontinuierlich die in Kt/V ausgedrückte Dialysedosis zu überwachen. Natürlich ist damit auch die Hoffnung verbunden, daß die Langzeit-Sterblichkeit verringert werden kann, wenn eine flächendeckende, zeitnahe Erfolgskontrolle der Dialyse ermöglicht wird. Die gewählte technische Lösung basiert auf der Äquivalenz der Diffusionskoeffizienten von gelöstem Harnstoff und Natriumchlorid. Es ist das zentrale Anliegen dieser Studie, festzustellen, ob das Diffusionsverhalten von NaCl und Harnstoff beim Durchtritt durch die Membran des Dialysefilters gleich ist. Der entscheidende Vorteil, der das Verfahren so leicht handhabbar macht, besteht darin, daß NaCl-Konzentrationen sehr genau durch die ohnehin in großer Zahl in Dialysegeräten verwendeten Leitfähigkeitsmeßzellen bestimmt werden können. Um die NaCl-Massenbilanz über den Dialysefilter zu bestimmen, benötigt man lediglich eine weitere Meßzelle, die stromab des Filters zu installieren ist. Die Messung von Harnstoff, die indirekt über die enzymatische Zerlegung in Ammonium-Ionen und das anschließende Erzeugen eines hoch zu verstärkenden elektrischen Potentials an einer Membran geschieht, ist komplizierter. Zudem ist eine geschlossene Kühlkette für das Enzym sicher zu stellen. Um eine leitfähigkeitsbasierte technische Lösung abzusichern, wurden zwei klinische Studien durchgeführt. In der ersten Studie wurde die Leitfähigkeit in Form von konstanten Stufenprofilen variiert. Sie wurde ausgehend von der Grundlinie für 7 min erhöht und anschließend für 7 min erniedrigt. Das Prinzip einer solchen Messung wurde erstmals 1982 in einer Patentschrift beschrieben. In einer Sequenz von 494 solchen Messungen in 206 automatisch aufgezeichneten Dialysesitzungen an 22 Patienten wurde gefunden, daß sich die Harnstoff- Clearance elektrolytisch mit einer Genauigkeit von -1.46+/-4.75% (Fehler+/-Standardabweichung) messen ließ. Die Messung von Kt/V gemäß dem Ein-Kompartment-Modell ergab eine ähnliche Genauigkeit von 2.88+/-4.15%. Obgleich diese Ergebnisse in Übereinstimmung mit anderen Studien stehen, wurde ein Effekt bemerkt, der nicht in Einklang mit der zunächst bestehenden Theorie zu bringen war. Dieser Effekt besteht darin, daß die Genauigkeit der elektrolytischen Clearancemessung vom beim Patienten vorhandenen Harnstoff-Verteilungsvolumen abhängig war. Weiter war es nicht bedeutungslos, welchen Teil des dreiteiligen Stufenprofils man zur Auswertung heranzog: Den Grundlinie-Hoch- Übergang, den von der Grundlinie zum Niedrig-Nieveau oder den Hoch-Niedrig- Übergang. Dies deutete auf einen Mangel an theoretischem Verständnis hin. Eine genaue weitere Untersuchung führte zu dem Ergebnis, daß unerwünschter NaCl-Transfer vom und zum Patienten die Ursache für die Abhängigkeit vom Verteilungsvolumen war. Es wurde daraufhin die Theorie dahingehend erweitert, daß dieser Effekt korrekt und plausibel beschrieben werden konnte. Aus dieser Erweiterung ergab sich die neue Forderung, den NaCl-Transfer bei der Messung weitestgehend zu minimieren. Die Benutzung von Stufenprofilen stellte hier jedoch an sich eine Limitierung dar, da in der zum Einnehmen eines stabilen Zustandes erforderlichen Zeit zuviel NaCl über die Membran transferiert wurde. Die Konsequenz war, von den Stufenprofilen auf kurze, dynamische Leitfähigkeitsboli überzugehen, die erlaubten, die NaCl-Gabe auf das Maß zu verringern, welches aufgrund der technischen Auflösung erforderlich war. Hierzu mußten jedoch die notwendigen mathematischen Algorithmen neu zugeschnitten werden. Nach diesem Schritt wurde eine weitere klinische Studie gestartet, die den Zweck verfolgte, das neue Verfahren am Patienten zu verifizieren. In dieser Studie mit 10 Patienten und 93 Dialysesitzungen, 264 Stufenprofil- und 173 Bolus-Dialysancemesssungen wurde gefunden, daß die Bolus-Messungen ihre zugehörigen blutseitigen Referenzmessungen mit außergewöhnlicher Genauigkeit von 0.06+/-4.76% trafen. Student's t-Test für gepaarte Daten ergab, daß sich die Datensätze nicht signifikant unterschieden (p=0.87). Die blutseitige Kt/V-Referenz auf der Basis des equilibrierten Einkompartment-Modells mit variablem Volumen wurde mit 5.32+/-3.9% getroffen, wobei eine Korrelation von 0.98 erzielt wurde. Die verbleibende Differenz von 5.32% wird der Vernachlässigung der Harnstoff-Erzeugung während der Messung zugeschrieben. Auch das Stufenprofil zeigte trotz seiner Abhängigkeit vom Verteilungsvolumen gegenüber dem gleichen Modell einen mittleren Fehler von 0.05+/-5% bei einer Korrelation von 0.96. Jedoch konnte es die Vernachlässigung der Harnstoff-Erzeugung nicht korrekt abbilden. Die kontinuierlich aufgenommenen Daten wurden auch nach dem 2-pool Modell untersucht, welches auch die Harnstoff-Erzeugung enthält sowie eine innere Kompartimentierung des Patienten annimmt und damit die tatsächlichen Verhältnisse besser beschreibt. Danach weicht das Bolus-Meßprinzip -3.04+/-14.3% von der Referenz ab (n.s.,p=0.13). Die relativ hohe Standardabweichung wird mit der Komplexität des Modells erklärt. Weiter ist aus der Theorie zum Na-Transfer eine vereinfachende Methode zur Messung des Na-Verteilungsvolumens abgeleitet worden. Diese Methode wurde in-vitro gegen ein Behältnis mit einer bekannten Menge an Dialysat geprüft. Es wurde ein mittlerer Fehler von -19.9+/-34% gefunden. Die Korrelation war 0.92 (n.s., p=0.916). Die gleiche Prüfung fand in-vivo gegen das Harnstoff-Verteilungsvolumen statt und ergab einen mittleren Fehler von -7.4+/-23.2% (r=0.71, n.s., p=0.39). Es hat den Anschein, als würden sich gemäß der Theorie der Dilution die Verteilungsvolumina für Natrium und für Harnstoff scheinbar nur wenig unterscheiden, obwohl sie sich absolut natürlich deutlich unterscheiden. In Anbetracht der erheblichen Vereinfachungen, die bei der Ableitung dieses Ansatzes gemacht wurden, scheint es ausgesprochen ermutigend, auf diesem Weg möglicherweise ein Verfahren entwickeln zu können, welches auf rein elektrolytischem Wege nun nicht mehr nur K, sondern auch V und damit alle zur Quantifizierung gesuchten Größen analytisch ermitteln kann. Weiter wurde im Rahmen dieser Arbeit anhand der analytisch ermittelten Volumina verglichen, ob man mit Hilfe anthropometrischer Formeln zur Ermittlung des Harnstoff-Verteilungsvolumens zu einer guten Abschätzung kommen kann. Es wurde gefunden, daß man das Ergebnis der Watson-Formel um ca. 13% vermindern kann und dann zu einem recht guten Wert für das tatsächliche Harnstoff-Verteilungsvolumen gelangt. Dies ist jedoch mit Vorsicht und Erfahrung zu tun, da sich damit Kt/V rechnerisch zum Nachteil des Patienten verändert. Auch ein elektrolytisches Verfahren mit empirischen Komponenten zur Ermittlung des Plasma-Natriums des Patienten wurde erprobt und konnte mit einer Genauigkeit von 4.3+/-1.2% den Laborwert vorhersagen. Zusammenfassend kann gesagt werden, daß sich im Rahmen dieser Arbeit die leitfähigkeitsbasierten Methoden zur Messung von einigen wichtigen Dialyseparametern als sehr nützlich für die klinische Praxis erwiesen haben und zudem mit keinem Zusatzaufwand für die Beteiligten verbunden sind. Das Ergebnis dieser Arbeit ist mittlerweile in größeren Stückzahlen in frei erhältliche Dialysegeräte implementiert worden. Die Erfahrung der ersten Zeit zeigt, daß das verwendete Prinzip von den Klinikern gut angenommen wird.
Aim: While elevational gradients in species richness constitute some of the best depicted patterns in ecology, there is a large uncertainty concerning the role of food resource availability for the establishment of diversity gradients in insects. Here, we
analysed the importance of climate, area, land use and food resources for determining diversity gradients of dung beetles along extensive elevation and land use gradients on Mt. Kilimanjaro, Tanzania.
Location: Mt. Kilimanjaro, Tanzania.
Taxon: Scarabaeidae (Coleoptera).
Methods: Dung beetles were recorded with baited pitfall traps at 66 study plots along a 3.6 km elevational gradient. In order to quantify food resources for the dung beetle community in form of mammal defecation rates, we assessed mammalian diversity and biomass with camera traps. Using a multi‐model inference framework and path analysis, we tested the direct and indirect links between climate, area, land use and mammal defecation rates on the species richness and abundance of dung beetles.
Results: We found that the species richness of dung beetles declined exponentially with increasing elevation. Human land use diminished the species richness of functional groups exhibiting complex behaviour but did not have a significant influence on total species richness. Path analysis suggested that climate, in particular temperature and to a lesser degree precipitation, were the most important predictors of dung beetle species richness while mammal defecation rate was not supported as a predictor variable.
Main conclusions: Along broad climatic gradients, dung beetle diversity is mainly limited by climatic factors rather than by food resources. Our study points to a predominant role of temperature‐driven processes for the maintenance and origination of species diversity of ectothermic organisms, which will consequently be subject to ongoing climatic changes.
Contemporary climate change leads to earlier spring phenological events in Europe. In forests, in which overstory strongly regulates the microclimate beneath, it is not clear if further change equally shifts the timing of leaf unfolding for the over- and understory of main deciduous forest species, such as Fagus sylvatica L. (European beech). Furthermore, it is not known yet how this vertical phenological (mis)match — the phenological difference between overstory and understory — affects the remotely sensed satellite signal. To investigate this, we disentangled the start of season (SOS) of overstory F.sylvatica foliage from understory F. sylvatica foliage in forests, within nine quadrants of 5.8 × 5.8 km, stratified over a temperature gradient of 2.5 °C in Bavaria, southeast Germany, in the spring seasons of 2019 and 2020 using time lapse cameras and visual ground observations. We explained SOS dates and vertical phenological (mis)match by canopy temperature and compared these to Sentinel-2 derived SOS in response to canopy temperature. We found that overstory SOS advanced with higher mean April canopy temperature (visual ground observations: −2.86 days per °C; cameras: −2.57 days per °C). However, understory SOS was not significantly affected by canopy temperature. This led to an increase of vertical phenological mismatch with increased canopy temperature (visual ground observations: +3.90 days per °C; cameras: +2.52 days per °C). These results matched Sentinel-2-derived SOS responses, as pixels of higher canopy height advanced more by increased canopy temperature than pixels of lower canopy height. The results may indicate that, with further climate change, spring phenology of F. sylvatica overstory will advance more than F. sylvatica understory, leading to increased vertical phenological mismatch in temperate deciduous forests. This may have major ecological effects, but also methodological consequences for the field of remote sensing, as what the signal senses highly depends on the pixel mean canopy height and the vertical (mis)match.
Climate change has created potential major threats to global biodiversity. The multiple components of climate change are projected to affect all pillars of biodiversity, from genes over species to biome level. Of particular concerns are "tipping points" where the exceedance of ecosystem thresholds will possibly lead to irreversible shifts of ecosystems and their functioning. As biodiversity underlies all goods and services provided by ecosystems that are crucial for human survival and wellbeing, this paper presents potential effects of climate change on biodiversity, its plausible impacts on human society as well as the setting in addressing a global crisis. Species affected by climate change may respond in three ways: change, move or die. Local species extinctions or a rapidly affected ecosystem as a whole respectively might move toward its particular "tipping point", thereby probably depriving its services to human society and ending up in a global crisis. Urgent and appropriate actions within various scenarios of climate change impacts on biodiversity, especially in tropical regions, are needed to be considered. Foremost a multisectoral approach on biodiversity issues with broader policies, stringent strategies and programs at international, national and local levels is essential to meet the challenges of climate change impacts on biodiversity.
Aim:
Temperature, food resources and top‐down regulation by antagonists are considered as major drivers of insect diversity, but their relative importance is poorly understood. Here, we used cavity‐nesting communities of bees, wasps and their antagonists to reveal the role of temperature, food resources, parasitism rate and land use as drivers of species richness at different trophic levels along a broad elevational gradient.
Location:
Mt. Kilimanjaro, Tanzania.
Taxon:
Cavity‐nesting Hymenoptera (Hymenoptera: Apidae, Colletidae, Megachilidae, Crabronidae, Sphecidae, Pompilidae, Vespidae).
Methods:
We established trap nests on 25 study sites that were distributed over similar large distances in terms of elevation along an elevational gradient from 866 to 1788 m a.s.l., including both natural and disturbed habitats. We quantified species richness and abundance of bees, wasps and antagonists, parasitism rates and flower or arthropod food resources. Data were analysed with generalized linear models within a multi‐model inference framework.
Results:
Elevational species richness patterns changed with trophic level from monotonically declining richness of bees to increasingly humped‐shaped patterns for caterpillar‐hunting wasps, spider‐hunting wasps and antagonists. Parasitism rates generally declined with elevation but were higher for wasps than for bees. Temperature was the most important predictor of both bee and wasp host richness patterns. Antagonist richness patterns were also well predicted by temperature, but in contrast to host richness patterns, additionally by resource abundance and diversity. The conversion of natural habitats through anthropogenic land use, which included biomass removal, agricultural inputs, vegetation structure and percentage of surrounding agricultural habitats, had no significant effects on bee and wasp communities.
Main conclusions:
Our study underpins the importance of temperature as a main driver of diversity gradients in ectothermic organisms and reveals the increasingly important role of food resources at higher trophic levels. Higher parasitism rates at higher trophic levels and at higher temperatures indicated that the relative importance of bottom‐up and top‐down drivers of species richness change across trophic levels and may respond differently to future climate change.
Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce 'click-AT-CLEM', a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.
Clerodendrumjistulosum Becc. is a true myrmecophyte as it offers nesting space for ants in hollow intemodes. In contrast to previous reports our investigations proved that these domatia open by themselves, thus providing cavities for a variety of different ant species. In Sarawak, Malaysia, we did not find an obligate relationship between C. jistulosum and a specific ant-partner. For comparison, studies on herbarium material of other Clerodendrum species were carried out a further species, C. deflexum from the Malay Peninsula and Sumatra presumably also is myrmecophytic.
The arrangement of transcriptional units in the loops of lampbrush chromosomes from oocyte nuclei of urodele amphibia and from primary nuclei of the green alga Acetabularia have been studied in the electron microscope using spread preparations. Loops with different patterns of arrangement of matrix units (i.e. to a first approximation, transcriptional units) can be distinguished: (i) loops consisting of one active transcriptional unit; (ii) loops containing one active transcriptional unit plus additional fibril-free, i.e. apparently untranscribed, intercepts that may include 'spacer' regions; (iii) loops containing two or more transcriptional units arranged in identical or changing polarities, with or without interspersed apparent spacer regions. Morphological details of the transcriptional complexes are described. The observations are not compatible with the concept that one loop reflects one and only one transcriptional unit but, rather, lead to a classification of loop types according to the arrangement of their transcriptional units. We propose that the lampbrush chromosome loop can represent a unit for the coordinate transcription of either one gene or a set of several (different) genes.
Cisplatin is a commonly used chemotherapeutic agent; however, its potential side effects, including gonadotoxicity and infertility, are a critical problem. Oxidative stress has been implicated in the pathogenesis of cisplatin-induced testicular dysfunction. We investigated whether kinetin use at different concentrations could alleviate gonadal injury associated with cisplatin treatment, with an exploration of the involvement of its antioxidant capacity. Kinetin was administered in different doses of 0.25, 0.5, and 1 mg/kg, alone or along with cisplatin for 10 days. Cisplatin toxicity was induced via a single IP dose of 7 mg/kg on day four. In a dose-dependent manner, concomitant administration of kinetin with cisplatin significantly restored testicular oxidative stress parameters, corrected the distorted sperm quality parameters and histopathological changes, enhanced levels of serum testosterone and testicular StAR protein expression, as well as reduced the up-regulation of testicular TNF-α, IL-1β, Il-6, and caspase-3, caused by cisplatin. It is worth noting that the testicular protective effect of the highest kinetin dose was comparable/more potent and significantly higher than the effects of vitamin C and the lowest kinetin dose, respectively. Overall, these data indicate that kinetin may offer a promising approach for alleviating cisplatin-induced reproductive toxicity and organ damage, via ameliorating oxidative stress and reducing inflammation and apoptosis.
The cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic factor that stabilises the c-Myc protein. CIP2A is overexpressed in several tumours, and expression levels are an independent marker for long-term outcome. To determine whether CIP2A expression is elevated in colon cancer and whether it might serve as a prognostic marker for survival, we analysed CIP2A mRNA expression by real-time PCR in 104 colon cancer samples. CIP2A mRNA was overexpressed in colon cancer samples and CIP2A expression levels correlated significantly with tumour stage. We found that CIP2A serves as an independent prognostic marker for disease-free and overall survival. Further, we investigated CIP2A-dependent effects on levels of c-Myc, Akt and on cell proliferation in three colon cancer cell lines by silencing CIP2A using small interfering (si) and short hairpin (sh) RNAs. Depletion of CIP2A substantially inhibited growth of colon cell lines and reduced c-Myc levels without affecting expression or function of the upstream regulatory kinase, Akt. Expression of CIP2A was found to be dependent on MAPK activity, linking elevated c-Myc expression to deregulated signal transduction in colon cancer.
Background and main text: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex and controversial clinical condition without having established causative factors. Increasing numbers of cases during past decade have created awareness among patients as well as healthcare professionals. Chronic viral infection as a cause of ME/CFS has long been debated. However, lack of large studies involving well-designed patient groups and validated experimental set ups have hindered our knowledge about this disease. Moreover, recent developments regarding molecular mechanism of pathogenesis of various infectious agents cast doubts over validity of several of the past studies.
Conclusions: This review aims to compile all the studies done so far to investigate various viral agents that could be associated with ME/CFS. Furthermore, we suggest strategies to better design future studies on the role of viral infections in ME/CFS.
1.Honeybees Apis mellifera and other pollinating insects suffer from pesticides in agricultural landscapes. Flupyradifurone is the active ingredient of a novel pesticide by the name of ‘Sivanto’, introduced by Bayer AG (Crop Science Division, Monheim am Rhein, Germany). It is recommended against sucking insects and marketed as ‘harmless’ to honeybees. Flupyradifurone binds to nicotinergic acetylcholine receptors like neonicotinoids, but it has a different mode of action. So far, little is known on how sublethal flupyradifurone doses affect honeybees.
2. We chronically applied a sublethal and field‐realistic concentration of flupyradifurone to test for long‐term effects on flight behaviour using radio‐frequency identification. We examined haematoxylin/eosin‐stained brains of flupyradifurone‐treated bees to investigate possible changes in brain morphology and brain damage.
3. A field‐realistic flupyradifurone dose of approximately 1.0 μg/bee/day significantly increased mortality. Pesticide‐treated bees initiated foraging earlier than control bees. No morphological damage in the brain was observed.
4. Synthesis and applications. The early onset of foraging induced by a chronical application of flupyradifurone could be disadvantageous for honeybee colonies, reducing the period of in‐hive tasks and life expectancy of individuals. Radio‐frequency identification technology is a valuable tool for studying pesticide effects on lifetime foraging behaviour of insects.
Polyploid genomes present a challenge for cytogenetic and genomic studies, due to the high number of similar size chromosomes and the simultaneous presence of hardly distinguishable paralogous elements. The karyotype of the Siberian sturgeon (Acipenser baerii) contains around 250 chromosomes and is remarkable for the presence of paralogs from two rounds of whole-genome duplications (WGD). In this study, we applied the sterlet-derived acipenserid satDNA-based whole chromosome-specific probes to analyze the Siberian sturgeon karyotype. We demonstrate that the last genome duplication event in the Siberian sturgeon was accompanied by the simultaneous expansion of several repetitive DNA families. Some of the repetitive probes serve as good cytogenetic markers distinguishing paralogous chromosomes and detecting ancestral syntenic regions, which underwent fusions and fissions. The tendency of minisatellite specificity for chromosome size groups previously observed in the sterlet genome is also visible in the Siberian sturgeon. We provide an initial physical chromosome map of the Siberian sturgeon genome supported by molecular markers. The application of these data will facilitate genomic studies in other recent polyploid sturgeon species.
Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are two different species of betaherpesviruses that integrate into sub-telomeric ends of human chromosomes, for which different prevalence rates of integration have been reported. It has been demonstrated that integrated viral genome is stable and is fully retained. However, study of chromosomally integrated viral genome in individuals carrying inherited HHV-6 (iciHHV-6) showed unexpected number of viral DR copies. Hence, we created an in vitro infection model and studied retention of full or partial viral genome over a period of time. We observed an exceptional event where cells retained viral direct repeats (DRs) alone in the absence of the full viral genome. Finally, we found evidence for non-telomeric integration of HHV-6A DR in both cultured cells and in an iciHHV-6 individual. Our results shed light on several novel features of HHV-6A chromosomal integration and provide valuable information for future screening techniques.
Biodiversity, a multidimensional property of natural systems, is difficult to quantify partly because of the multitude of indices proposed for this purpose. Indices aim to describe general properties of communities that allow us to compare different regions, taxa, and trophic levels. Therefore, they are of fundamental importance for environmental monitoring and conservation, although there is no consensus about which indices are more appropriate and informative. We tested several common diversity indices in a range of simple to complex statistical analyses in order to determine whether some were better suited for certain analyses than others. We used data collected around the focal plant Plantago lanceolata on 60 temperate grassland plots embedded in an agricultural landscape to explore relationships between the common diversity indices of species richness (S), Shannon's diversity (H'), Simpson's diversity (D-1), Simpson's dominance (D-2), Simpson's evenness (E), and Berger-Parker dominance (BP). We calculated each of these indices for herbaceous plants, arbuscular mycorrhizal fungi, aboveground arthropods, belowground insect larvae, and P.lanceolata molecular and chemical diversity. Including these trait-based measures of diversity allowed us to test whether or not they behaved similarly to the better studied species diversity. We used path analysis to determine whether compound indices detected more relationships between diversities of different organisms and traits than more basic indices. In the path models, more paths were significant when using H', even though all models except that with E were equally reliable. This demonstrates that while common diversity indices may appear interchangeable in simple analyses, when considering complex interactions, the choice of index can profoundly alter the interpretation of results. Data mining in order to identify the index producing the most significant results should be avoided, but simultaneously considering analyses using multiple indices can provide greater insight into the interactions in a system.
Chloroquine (CQ) treatment failure in Plasmodium falciparum parasites has been documented for decades, but the pharmacological explanation of this phenotype is not fully understood. Current concepts attribute CQ resistance to reduced accumulation of the drug at a given external CQ concentration ([CQ] ex) in resistant compared to sensitive parasites. The implication of this explanation is that the mechanisms of CQ-induced toxicity in resistant and sensitive strains are similar once lethal internal concentrations have been reached. To test this hypothesis, we investigated the mechanism of CQ-induced toxicity in CQ-sensitive (CQS) versus CQ-resistant (CQR) parasites by analyzing the time-course of cellular responses in these strains after exposure to varying [CQ] ex as determined in 72 h toxicity assays. Parasite killing was delayed in CQR parasites for up to 10 h compared to CQS parasites when exposed to equipotent [CQ] ex. In striking contrast, brief exposure (1 h) to lethal [CQ] ex in CQS but not CQR parasites caused the appearance of hitherto undescribed hemozoin (Hz)-containing compartments in the parasite cytosol. Hz-containing compartments were very rarely observed in CQR parasites even after CQ exposures sufficient to cause irreversible cell death. These findings challenge current concepts that CQ killing of malaria parasites is solely concentration-dependent, and instead suggest that CQS and CQR strains fundamentally differ in the consequences of CQ exposure.
Chlamydia trachomatis is an obligate intracellular pathogen that replicates inside a vacuole, the so-called inclusion. During replication by a biphasic life-cycle Chlamydia secrete via their type 3 secretion system various effector proteins into the inclusion lumen, the inclusion membrane or the host cell cytosol to form their favored replication niche. Chlamydia-infected cells are highly resistant against apoptosis since the replicative form of Chlamydia is non-infectious and premature cell death would cause complete loss of one Chlamydia generation. The bacteria block apoptosis by preventing mitochondrial outer membrane permeabilization. Various proteins with anti-apoptotic function are enriched in Chlamydia-infected cells such as Mcl-1, cIAP2, Survivin or HIF1α. The accumulation of these proteins is a result of increased gene expression and direct protein stabilization. However, the molecular mechanisms and involved bacterial effector proteins are mostly unknown.
With this work the molecular mechanisms of Mcl-1 stabilization and the participation of chlamydial factors were investigated. Mcl-1 is a member of the Bcl-2 protein family and has an extremely short half-life causing its permanent ubiquitination and subsequent degradation by the 26S proteasome under normal homeostasis whilst Mcl-1 accumulation results in apoptosis inhibition. It was shown that during C. trachomatis infection Mcl-1 ubiquitination is reduced causing its stabilization albeit no cellular ubiquitin-proteasome-system components are involved in this process. However, C. trachomatis express the two deubiquitinases ChlaDUB1 and ChlaDUB2 which are mostly uncharacterized. With this work the expression profile, subcellular localization, substrates and function of the deubiquitinases were investigated. It was shown that ChlaDUB1 is secreted to the surface of the inclusion where it interacts with Mcl-1 which is accumulated in the proximity of this compartment. By utilization of infection experiments, heterologous expression systems and in vitro experiments a direct interaction of ChlaDUB1 and Mcl-1 was demonstrated. Furthermore, it was shown that Mcl-1 is deubiquitinated by ChlaDUB1 causing its stabilization. During replicative phase of infection, ChlaDUB2 seems to be accumulated in the chlamydial particles. However, ChlaDUB2 substrates could not be identified which would give an indication for the physiological role of ChlaDUB2.
Since 2011, a protocol to transform C. trachomatis with artificial plasmid DNA is available. As part of this work the transformation of C. trachomatis with plasmid DNA suitable for the permanent or inducible protein overexpression on a routinely basis was established. In addition, the first targeted homologous recombination into the chlamydial genome to replace the ChlaDUB1 gene by a modified one was performed and validated. The targeted homologous recombination was also used to create a ChlaDUB1 knock-out mutant; however deletion of ChlaDUB1 seems to be lethal for C. trachomatis. Due to the fact that ChlaDUB1-lacking Chlamydia could not be obtained an inhibitor screen was performed and identified CYN312 as a potential ChlaDUB1 inhibitor. Application of CYN312 during infection interfered with chlamydial growth and reduced Mcl-1 quantity in infected cells. Furthermore, CYN312 treated Ctr-infected cells were significantly sensitized for apoptosis.
Taken together, C. trachomatis secretes the deubiquitinase ChlaDUB1 to the surface of the inclusion where it deubiquitinates Mcl-1 causing its accumulation in infected cells resulting in apoptosis resistance. Application of the ChlaDUB1 inhibitor CYN312 interferes with Mcl-1 stabilization sensitizing infected cells for apoptosis.
Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.
Human herpesvirus-6 (HHV-6) exists in latent form either as a nuclear episome or integrated into human chromosomes in more than 90% of healthy individuals without causing clinical symptoms. Immunosuppression and stress conditions can reactivate HHV-6 replication, associated with clinical complications and even death. We have previously shown that co-infection of Chlamydia trachomatis and HHV-6 promotes chlamydial persistence and increases viral uptake in an in vitro cell culture model. Here we investigated C. trachomatis-induced HHV-6 activation in cell lines and fresh blood samples from patients having Chromosomally integrated HHV-6 (CiHHV-6). We observed activation of latent HHV-6 DNA replication in CiHHV-6 cell lines and fresh blood cells without formation of viral particles. Interestingly, we detected HHV-6 DNA in blood as well as cervical swabs from C. trachomatis-infected women. Low virus titers correlated with high C. trachomatis load and vice versa, demonstrating a potentially significant interaction of these pathogens in blood cells and in the cervix of infected patients. Our data suggest a thus far underestimated interference of HHV-6 and C. trachomatis with a likely impact on the disease outcome as consequence of co-infection.
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.
In Brassicaceae, tissue damage triggers the mustard oil bomb i.e., activates the degradation of glucosinolates by myrosinases leading to a rapid accumulation of isothiocyanates at the site of damage. Isothiocyanates are reactive electrophilic species (RES) known to covalently bind to thiols in proteins and glutathione, a process that is not only toxic to herbivores and microbes but can also cause cell death of healthy plant tissues. Previously, it has been shown that subtoxic isothiocyanate concentrations can induce transcriptional reprogramming in intact plant cells. Glutathione depletion by RES leading to breakdown of the redox potential has been proposed as a central and common RES signal transduction mechanism. Using transcriptome analyses, we show that after exposure of Arabidopsis seedlings (grown in liquid culture) to subtoxic concentrations of sulforaphane hundreds of genes were regulated without depletion of the cellular glutathione pool. Heat shock genes were among the most highly up-regulated genes and this response was found to be dependent on the canonical heat shock factors A1 (HSFA1). HSFA1-deficient plants were more sensitive to isothiocyanates than wild type plants. Moreover, pretreatment of Arabidopsis seedlings with subtoxic concentrations of isothiocyanates increased resistance against exposure to toxic levels of isothiocyanates and, hence, may reduce the autotoxicity of the mustard oil bomb by inducing cell protection mechanisms.
A fundamental question in current biology concerns the translational mechanisms leading from genetic variability to phenotypes. Technologies have evolved to the extent that they can efficiently and economically determine an individual’s genomic composition, while at the same time big data on clinical profiles and diagnostics have substantially accumulated. Genome-wide association studies linking genomic loci to certain traits, however, remain limited in their capacity to explain the cellular mechanisms that underlie the given association. For most associations, gene expression has been blamed; yet given that transcript and protein abundance oftentimes do not correlate, that finding does not necessarily decrypt the underlying mechanism. Thus, the integration of further information is crucial to establish a model that could prove more accurate in predicting genotypic effects on the human organism.
In this work we describe the so-called proteotype as a feature of the cell that could provide a substantial link between genotype and phenotype. Rather than looking at the proteome as a set of independent molecules, we demonstrate a consistent modular architecture of the proteome that is driven by molecular cooperativity. Functional modules, especially protein complexes, can be further interrogated for differences between individuals and tackled as imprints of genetic and environmental variability. We also show that subtle stoichiometric changes of protein modules could have broader effects on the cellular system, such as the transport of specific molecular cargos.
The presented work also delineates to what extent temporal events and processes influence the stoichiometry of protein complexes and functional modules. The re-wiring of the glycolytic pathway for example is illustrated as a potential cause for an increased Warburg effect during the ageing of the human bone marrow. On top of analyzing protein abundances we also interrogate proteome dynamics in terms of stability and solubility transitions during the short temporal progression of the cell cycle. One of our main observations in the thesis encompass the delineation of protein complexes into respective sub-complexes according to distinct stability patterns during the cell cycle. This has never been demonstrated before, and is functionally relevant for our understanding of the dis- and assembly of large protein modules.
The insights presented in this work imply that the proteome is more than the sum of its parts, and primarily driven by variability in entire protein ensembles and their cooperative nature. Analyzing protein complexes and functional modules as molecular reflections of genetic and environmental variations could indeed prove to be a stepping stone in closing the gap between genotype and phenotype and customizing clinical treatments in the future.
The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes.
The Popeye domain containing (Popdc) gene family of membrane proteins is predominantly expressed in striated and smooth muscle tissues and has been shown to act as novel cAMP-binding proteins. In mice, loss of Popdc1 and Popdc2, respectively, affects sinus node function in the postnatal heart in an age and stress-dependent manner. In this thesis, I examined gene expression pattern and function of the Popdc gene family during zebrafish development with an emphasis on popdc2. Expression of the zebrafish popdc2 was exclusively present in cardiac and skeletal muscle during cardiac development, whereas popdc3 was expressed in striated muscle tissue and in distinct regions of the brain. In order to study the function of these genes, an antisense morpholino-based knockdown approach was used. Knockdown of popdc2 resulted in aberrant development of facial and tail musculature. In the heart, popdc2 morphants displayed irregular ventricular contractions with 2:1 and 3:1 ventricular pauses. Recordings of calcium transients using a transgenic indicator line Tg(cmlc2:gCaMP)s878 and selective plane illumination microscopy (SPIM) revealed the presence of an atrioventricular (AV) block in popdc2 morphants as well as a complete heart block. Interestingly, preliminary data revealed that popdc3 morphants developed a similar phenotype. In order to find a morphological correlate for the observed AV conduction defect, I studied the structure of the AV canal in popdc2 morphants using confocal analysis of hearts of the transgenic line Tg(cmlc2:eGFP-ras)s883, which outlines individual cardiac myocytes with the help of membrane-localized GFP. However, no evidence for morphological alterations was obtained. To ensure that the observed arrhythmia phenotype in the popdc2 morphant was based on a myocardial defect and not caused by defective valve development, live imaging was performed revealing properly formed valves. Thus, in agreement with the data obtained in knockout mice, popdc2 and popdc3 genes in zebrafish are involved in the regulation of cardiac electrical activity. However, both genes are not required for cardiac pacemaking, but they play essential roles in AV conduction. In order to elucidate the biological importance of cAMP-binding, wild type Popdc1 as well as mutants with a significant reduction in binding affinity for cAMP in vitro were overexpressed in zebrafish embryos. Expression of wild type Popdc1 led to a cardiac insufficiency phenotype characterized by pericardial edema and venous blood retention. Strikingly, the ability of the Popdc1 mutants to induce a cardiac phenotype correlated with the binding affinity for cAMP. These data suggest that cAMP-binding represents an important biological property of the Popdc protein family.
Flagellar motility and chemotaxis are essential virulence traits required for the ability of Helicobacter pylori to colonize the gastric mucosa. The flagellar regulatory network and the complex chemotaxis system of H. pylori are fundamentally different from other bacteria, despite many similarities. In H. pylori expression of the flagella is controlled by a complex regulatory cascade involving the two-component system FlgR-HP244, the sigma factors 54 and 28 and the anti-sigma 28 factor FlgM. Thus far, the input signal for histidine kinase HP244, which activates the transcriptional regulator FlgR, which triggers sigma factor 54-dependent transcription of the flagellar class 2 genes, is not known. Based on a yeast two-hybrid screen a highly significant protein-protein interaction between the H. pylori protein HP137 and both the histidine kinase HP244 and the flagellar hook protein HP908 (FlgE´) has been reported recently (Rain et al., 2001). So far, no function could be assigned to HP137. Interestingly, the interaction between HP137 and histidine kinase HP244 was observed in the characteristic block N sequence motif of the C-terminal ATP-binding kinase domain. In this work a potential role of HP137 in a feedback regulatory mechanism controlling the activity of histidine kinase HP244 in the flagellar regulation of H. pylori was investigated. Although the substitution of the gene encoding HP137 by a kanamycin cassette resulted in non-motile bacteria, the failure to restore motility by the reintroduction of hp137 in cis into the mutant strain, and the observation that HP137 has no significant effect on the activity of histidine kinase HP244 in vitro indicated that HP137 is not directly involved in flagellar regulation. Therefore, it was demonstrated that HP137 does not participate in the regulation of flagellar gene expression, neither in H. pylori nor in the closely related bacterium C. jejuni. Chemotactic signal transduction in H. pylori differs from the enterobacterial paradigm in several respects. In addition to a CheY response regulator protein (CheY1) H. pylori contains a CheY-like receiver domain (CheY2) which is C-terminally fused to the histidine kinase CheA. Furthermore, the genome of H. pylori encodes three CheV proteins consisting of an N-terminal CheW-like domain and a C-terminal receiver domain, while there are no orthologues of the chemotaxis genes cheB, cheR, and cheZ. To obtain insight into the mechanism controlling the chemotactic response of H. pylori the phosphotransfer reactions between the purified two-component signalling modules were investigated in vitro. Using in vitro phosphorylation assays it was shown that both H. pylori histidine kinases CheAY2 and CheA´ lacking the CheY-like domain (CheY2) act as ATP-dependent autokinases. Similar to other CheA proteins CheA´ shows a kinetic of phosphorylation represented by an exponential time course, while the kinetics of phosphorylation of CheAY2 is characterized by a short exponential time course followed by the hydrolysis of CheAY2~P. Therefore, it was demonstrated that the presence of the CheY2-like receiver domain influences the stability of the phosphorylated P1 domain of the CheA part of the bifunctional protein. Furthermore, it was proven that both CheY1 and CheY2 are phosphorylated by CheAY2 and CheA´~P and that the three CheV proteins mediate the dephosphorylation of CheA´~P, although with a clearly reduced efficiency as compared to CheY1 and CheY2. Moreover, CheA´ is capable of donating its phospho group to the CheY1 protein from C. jejuni and to CheY protein from E. coli. Retrophosphorylation experiments indicated that CheY1~P is able to transfer the phosphate group back to the HK CheAY2 and the receiver domain present in the bifunctional CheAY2 protein acts as a phosphate sink fine tuning the activity of the freely diffusible CheY1 protein, which is thought to interact with the flagellar motor. Hence, in this work evidence of a complex phosphorelay in the chemotaxis system was obtained which has similarities to other systems with multiple CheY proteins. The role of the CheV proteins remain unclear at the moment, but they might be engaged in a further fine regulation of the phosphate flow in this complex chemotaxis system and the independent function of the two domains CheA´ and CheY2 is not sufficient for normal chemotactic signalling in vivo.
The unicellular pathogen Trypanosoma brucei is the causative agent of African
trypanosomiasis, an endemic disease prevalent in sub-Saharan Africa. Trypanosoma brucei alternates between a mammalian host and the tsetse fly vector. The extracellular parasite survives in the mammalian bloodstream by periodically exchanging their ˈvariant surface glycoproteinˈ (VSG) coat to evade the host immune response. This antigenic variation is achieved through monoallelic expression of one VSG variant from subtelomeric ˈbloodstream
form expression sitesˈ (BES) at a given timepoint. During the differentiation from the bloodstream form (BSF) to the procyclic form (PCF) in the tsetse fly midgut, the stage specific surface protein is transcriptionally silenced and replaced by procyclins. Due to their subtelomeric localization on the chromosomes, VSG transcription and silencing is partly regulated by homologues of the mammalian telomere complex such as TbTRF, TbTIF2 and TbRAP1 as well as by ˈtelomere-associated proteinsˈ (TelAPs) like TelAP1. To gain more insights into transcription regulation of VSG genes, the identification and characterization of other TelAPs is critical and has not yet been achieved. In a previous study, two biochemical approaches were used to identify other novel TelAPs. By using ˈco-immunoprecipitationˈ (co-IP) to enrich possible interaction partners of TbTRF and by affinity chromatography using telomeric repeat oligonucleotides, a listing of TelAP candidates has been conducted. With this approach TelAP1 was identified as a novel component of the telomere complex, involved in the kinetics of transcriptional BES silencing during BSF to PCF differentiation. To gain further insights into the telomere complex composition, other previously enriched proteins were characterized through a screening process using RNA interference to deplete potential candidates. VSG expression profile changes and overall proteomic changes after depletion were analyzed by mass spectrometry. With this method, one can gain insights into the functions of the proteins and their involvement in VSG expression site regulation. To validate the interaction of proteins enriched by co-IP with TbTRF and TelAP1 and to identify novel interaction proteins, I performed reciprocal affinity purifications of the four most promising candidates (TelAP2, TelAP3, PPL2 and PolIE) and additionally confirmed colocalization of two candidates with TbTRF via immunofluorescence (TelAP2, TelAP3). TelAP3 colocalizes with TbTRF and potentially interacts with TbTRF, TbTIF2, TelAP1 and TelAP2, as well as with two translesion polymerases PPL2 and PolIE in BSF. PPL2 and PolIE seem to be in close contact to each other at the telomeric ends and fulfill different roles as only PolIE is involved in VSG regulation while PPL2 is not. TelAP2 was previously characterized to be associated with telomeres by partially colocalizing with TbTRF and cells show a VSG derepression phenotype when the protein was depleted. Here I show that TelAP2 interacts with the telomere-binding proteins TbTRF and TbTIF2 as well as with the telomere-associated protein TelAP1 in BSF and that TelAP2 depletion results in a loss of TelAP1 colocalization with TbTRF in BSF.
In conclusion, this study demonstrates that characterizing potential TelAPs is effective in gaining insights into the telomeric complex's composition and its role in VSG regulation in Trypanosoma brucei. Understanding these interactions could potentially lead to new therapeutic targets for combatting African trypanosomiasis.
African trypanosomiasis is a disease endemic to sub-Saharan Africa. It affects humans as well as wild and domestic animals. The human form of the disease is known as sleeping sickness and the animal form as nagana, which are usually fatal if left untreated. The cause of African trypanosomiasis is the unicellular parasite Trypanosoma brucei. During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host the parasite multiplies as bloodstream form (BSF) extracellularly in the bloodstream or the lymphatic system. Survival of BSF parasites relies on immune evasion by antigenic variation of surface proteins because its extracellular lifestyle leads to direct exposure to immune responses. At any given time each BSF cell expresses a single type of variant surface glycoprotein (VSG) on its surface from a large repertoire. The active VSG is transcribed from one of 15 specialized subtelomeric domains, termed bloodstream expression sites (BESs). The remaining 14 BESs are silenced. This monoallelic expression and periodic switching of the expressed VSG enables to escape the immune response and to establish a persistent infection in the mammalian host. During developmental differentiation from BSF to the insect vector-resident procyclic form (PCF), the active BES is transcriptionally silenced to stop VSG transcription. Thus, all 15 BESs are inactive in the PCF cells as surface protein expression is developmentally regulated.
Previous reports have shown that the telomere complex components TbTRF, TbRAP1 and TbTIF2 are involved in VSG transcriptional regulation. However, the precise nature of their contribution remains unclear. In addition, no information is available about the role of telomeres in the initiation and regulation of developmental BES silencing. To gain insights into the regulatory mechanisms of telomeres on VSG transcription and developmental repression it is therefore essential to identify the complete composition of the trypanosome telomere complex.
To this end, we used two complementary biochemical approaches and quantitative label-free interactomics to determine the composition of telomere protein complexes in T. brucei. Firstly, using a telomeric pull-down assay we found 17 potential telomere-binding proteins including the known telomere-binding proteins TbTRF and TbTIF2. Secondly, by performing a co-immunoprecipitation experiment to elucidate TbTRF interactions we co-purified five proteins. All of these five proteins were also enriched with telomeric DNA in the pull-down assay.
To validate these data, I characterized one of the proteins found in both experiments (TelBP1). In BSF cells, TelBP1 co-localizes with TbTRF and interacts with already described telomere-binding proteins such as TbTRF, TbTIF2 and TbRAP1 indicating that TelBP1 is a novel component of the telomere complex in trypanosomes. Interestingly, protein interaction studies in PCF cells suggested a different telomere complex composition compared to BSF cells. In contrast to known members of the telomere complex, TelBP1 is dispensable for cell viability indicating that its function might be uncoupled from the known telomere-binding proteins. Overexpression of TelBP1 had also no effect on cell viability, but led to the discovery of two additional shorter isoforms of TelBP1. However, their source and function remained elusive.
Although TelBP1 is not essential for cell viability, western blot analysis revealed a 4-fold upregulation of TelBP1 in the BSF stage compared to the PCF stage supporting the concept of a dynamic telomere complex composition. We observed that TelBP1 influences the kinetics of transcriptional BES silencing during developmental transition from BSF to PCF. Deletion of TelBP1 caused faster BES silencing compared to wild-type parasites.
Taken together, TelBP1 function illustrates that developmental BES silencing is a fine-tuned process, which involves stage-specific changes in telomere complex formation.
In this thesis we have used Drosophila melanogaster as a model organism to investigate proteins and their putative interacting partners that are directly or indirectly involved in the release of neurotransmitters at the synapse. We have used molecular techniques to investigate conserved synaptic proteins, synapsin and synapse associated protein of 47 kD (SAP47), and a putative interaction partner of SAP47, tubulin binding chaperone E-like (TBCEL). SAP47 and synapsins are highly conserved synaptic vesicle associated proteins in Drosophila melanogaster. To further investigate the role and function of Sap47 and Syn genes, we had earlier generated the null mutants by P-element mutagenesis (Funk et al., 2004; Godenschwege et al., 2004). Western blots and ELISA of brain homogenates from Sap47156 null mutants showed the presence of up-regulated phospho-synapsin in comparison to wild-type (CS) and the presence of up-regulated phospho-synapsin was partially abolished when a pan-neuronal rescue of SAP47 was performed by the Gal4- UAS technique. Thus, the results suggest a qualitative and quantitative modulation of synapsin by SAP47. At the transcript level, we did not observe any difference in content of Syn transcript in Sap47156 and wild-type CS flies. The question of a direct molecular interaction between SAP47 and synapsin was investigated by co-immunoprecipitation (Co-IP) experiments and we did not find any stable interactions under the several IP conditions we tested. The possibility of Sap47 as a modifier of Syn at the genetic level was investigated by generating and testing homozygous double null mutants of Sap47 and Syn. The Syn97, Sap47156 double mutants are viable but have a reduced life span and decreased locomotion when compared to CS. In 2D-PAGE analysis of synapsins we identified trains of spots corresponding to synapsins, suggesting that synapsin has several isoforms and each one of them is posttranslationally modified. In an analysis by Blue native-SDS-PAGE (BN-SDS-2D- PAGE) and Western blot we observed synapsin and SAP47 signals to be present at 700-900 kDa and 200-250 kDa, respectively, suggesting that they are part of large but different complexes. We also report the possibility of Drosophila synapsin forming homo- and heteromultimers, which has also been reported for synapsins of vertebrates. In parallel to the above experiments, phosphorylation of synapsins in Drosophila was studied by IP techniques followed by 1D-SDS gel electrophoresis and mass spectrometry (in collaboration with S. Heo and G. Lubec). We identified and verified 5 unique phosphorylation sites in Drosophila synapsin from our MS analysis. Apart from phosphorylation modifications we identified several other PTMs which have not been verified. The significance of these phosphorylations and other identified PTMs needs to be investigated further and their implications for synapsin function and Drosophila behavior has to be elucidated by further experiments. In a collaborative project with S. Kneitz and N. Nuwal, we investigated the effects of Sap156 and Syn97 mutations by performing a whole Drosophila transcriptome microarray analysis of the individual null mutants and the double mutants (V2 and V3). We obtained several candidates which were significantly altered in the mutants. These genes need to be investigated further to elucidate their interactions with Sap47 and Syn. In another project, we investigated the role and function of Drosophila tubulin- binding chaperone E-like (Tbcel, CG12214). The TBCEL protein has high homology to vertebrate TBCE-like (or E-like) which has high sequence similarity to tubulin-binding chaperone E (TBCE) (hence the name TBCE-Like). We generated an anti-TBCEL polyclonal antiserum (in collaboration with G. Krohne). According to flybase, the Tbcel gene has only one exon and codes for two different transcripts by alternative transcription start sites. The longer transcript RB is present only in males whereas the shorter transcript RA is present only in females. In order to study the gene function we performed P- element jump-out mutagenesis to generate deletion mutants. We used the NP4786 (NP) stock which has a P(GawB) insertion in the 5’ UTR of the Tbcel gene. NP4786 flies are homozygous lethal due to a second-site lethality as the flies are viable over a deficiency (Df) chromosome (a deletion of genomic region spanning the Tbcel gene and other upstream and downstream genes). We performed the P-element mutagenesis twice. In the first trial we obtained only revertants and the second experiment is still in progress. In the second attempt, jump-out was performed over the deficiency chromosome to prevent homologous chromosome mediated double stranded DNA repair. During the second mutagenesis an insertion stock G18151 became available. These flies had a P-element insertion in the open reading frame (ORF) of the Tbcel gene but was homozygous viable. Western blots of fresh tissue homogenates of NP/Df and G18151 flies probed with anti-TBCEL antiserum showed no TBCEL signal, suggesting that these flies are Tbcel null mutants. We used these flies for further immunohistochemical analyses and found that TBCEL is specifically expressed in the cytoplasm of cyst cells of the testes and is associated with the tubulin of spermatid tails in wild-type CS, whereas in NP/Df and G18151 flies the TBCEL staining in the cyst cells was absent and there was a disruption of actin investment cones. We also found enrichment of TBCEL staining around the actin investment cone. These results are also supported by the observation that the enhancer trap expression of the NP4786 line is localised to the cyst cells, similar to TBCEL expression. Also, male fertility of NP/Df and G18151 flies was tested and they were found to be sterile with few escapers. Thus, these results suggest that TBCEL is involved in Drosophila spermatogenesis with a possible role in the spermatid elongation and individualisation process.
Genes involved in sex determination and differentiation have been identified in mice, humans, chickens, reptiles, amphibians and teleost fishes. However, little is known of their functional conservation, and it is unclear whether there is a common set of genes shared by all vertebrates. Coelacanths, basal Sarcopterygians and unique "living fossils", could help establish an inventory of the ancestral genes involved in these important developmental processes and provide insights into their components. In this study 33 genes from the genome of Latimeria chalumnae and from the liver and testis transcriptomes of Latimeria menadoensis, implicated in sex determination and differentiation, were identified and characterized and their expression levels measured. Interesting findings were obtained for GSDF, previously identified only in teleosts and now characterized for the first time in the sarcopterygian lineage; FGF9, which is not found in teleosts; and DMRT1, whose expression in adult gonads has recently been related to maintenance of sexual identity. The gene repertoire and testis-specific gene expression documented in coelacanths demonstrate a greater similarity to modern fishes and point to unexpected changes in the gene regulatory network governing sexual development.
Multiple myeloma (MM) is a disease of terminally differentiated B-cells which accumulate in the bone marrow leading to bone lesions, hematopoietic insufficiency and hypercalcemia. Genetically, MM is characterized by a great heterogeneity. A recent next-generation sequencing approach resulted in the identification of a signaling network with an accumulation of mutations in receptor-tyrosine kinases (RTKs), adhesion molecules and downstream effectors. A deep-sequencing amplicon approach of the coding DNA sequence of the six RTKs EPHA2, EGFR, ERBB3, IGF1R, NTRK1 and NTRK2 was conducted in a patient cohort (75 MM samples and 68 corresponding normal samples) of the “Deutsche Studiengruppe Multiples Myelom (DSMM)” to further elucidate the role of RTKs in MM. As an initial approach the detected mutations were correlated with cytogenetic abnormalities and clinical data in the course of this thesis. RTK mutations were present in 13% of MM patients of the DSMM XI trial and accumulated in the ligand-binding and tyrosine-kinase domain. The newly identified mutations were associated with an adverse patient survival, but not with any cytogenetic abnormality common in MM. Especially rare patient-specific SNPs (single nucleotide polymorphism) had a negative impact on patient survival. For a more comprehensive understanding of the role of rare RTK SNPs in MM, a second amplicon sequencing approach was performed in a patient cohort of the DSMM XII trial that included 75 tumor and 184 normal samples. This approach identified a total of 23 different mutations in the six RTKs EPHA2, EGFR, ERBB3, IGF1R, NTRK1 and NTRK2 affecting 24 patients. These mutations could furthermore be divided into 20 rare SNPs and 3 SNVs (single nucleotide variant). In contrast to the first study, the rare SNPs were significantly associated with the adverse prognostic factor del17p.
IGF1R was among the most commonly mutated RTKs in the first amplicon sequencing approach and is known to play an important role in diverse cellular processes such as cell proliferation and survival. To study the role of IGF1R mutations in the hard-to-transfect MM cells, stable IGF1R-knockdown MM cell lines were established. One of the knockdown cell lines (L363-C/C9) as well as a IGF1R-WT MM cell line (AMO1) were subsequently used for the stable overexpression of WT IGF1R and mutant IGF1R (N1129S, D1146N). Overall, an impact on the MAPK and PI3K/AKT signaling pathways was observed upon the IGF1R knockdown as well as upon WT and mutant IGF1R overexpression. The resulting signaling pattern, however, differed between different MM cell lines used in this thesis as well as in a parallel performed master thesis which further demonstrates the great heterogeneity described in MM.
Taken together, the conducted sequencing and functional studies illustrate the importance of RTKs and especially of IGF1R and its mutants in the pathogenesis of MM. Moreover, the results support the potential role of IGF1R as a therapeutic target for a subset of MM patients with mutated IGF1R and/or IGF1R overexpression.
Glioblastoma multiforme (GBM) is one of the most frequent and malignant forms of brain cancer in adults. The prognosis is poor with a median survival time of 12-15 months. There is a broad range of alternative treatment options studied in preclinical and clinical trials for GBM. One alternative treatment option is oncolytic virotherapy, defined as the use of replication‐competent viruses that selectively infect and destroy cancer cells while leaving, non‐transformed cells unharmed. Vaccinia virus (VACV) is one favorable candidate. Although oncolytic viruses can kill tumor cells grown in vitro with high efficiency, they often exhibit reduced replication capacity in vivo suggesting that physiological aspects of the tumor microenvironment decrease the virus’ therapeutic potential. The percentage and composition of immune cells varies between cancer types and patients and is investigated as a biomarker in several studies. Making oncolytic virotherapy successful for GBM, it is necessary to understand the individual tumor biology, the interaction with the microenvironment and immune system.
It was demonstrated that the attenuated VACV wild-type (wt) isolate LIVP 1.1.1 replicate and lyse the murine GL261 glioma cell line in vitro. In the following, the replication efficacy was characterized in a comparative approach in vivo. Immunocompetent C57BL/6 (wt) mice and immunodeficient mouse strains of different genetic background C57BL/6 athymic and Balb/c athymic mice were used. In addition, subcutaneous and intracranial locations were compared. The results revealed viral replication exclusively in Balb/c athymic mice with subcutaneous tumors but in none of the other models.
In the following, the tumor microenvironment of the subcutaneous tumor models at the time of infection was performed. The study showed that implantation of the same tumor cells in different mouse strains resulted in a different tumor microenvironment with a distinct composition of immune cells. Highest differences were detected between immunodeficient and immunocompetent mice. The study showed major differences in the expression of MHCII with strongest expression in C57BL/6 wt and weakest in Balb/c athymic tumors. In the following, the influence of the phenotypic change associated with the upregulation of MHCII on GL261 tumor cells on viral replication was analyzed. Comparison of C57BL/6 wt and C57BL/6 IFN-γ knockout mice revealed endogenous IFN-γ levels to upregulate MHCII on GL261 tumor cells and to reduce viral replication in C57BL/6 wt mice. Analysis of single cell suspensions of tumor homogenates of C57BL/6 and Balb/c athymic mice showed that the IFN-γ-mediated anti-tumor effect was a reversible effect. Furthermore, reasons for inhibition of virus replication in orthotopic glioma models were elucidated. By immunohistochemical analysis it was shown that intratumoral amounts of Iba1+ microglia and GFAP+ astrocytes in Gl261 gliomas was independent from intratumoral VACV injection. Based on these findings virus infection in glioma, microglia and astrocytes was compared and analyzed in cell culture. In contrast to the GL261 glioma cells, replication was barely detectable in BV-2 microglia and IMA2.1 astrocytic cells. Co-culture experiments revealed that microglia compete for virus uptake in cell culture. It was further shown that BV-2 cells showed apoptotic characteristics after VACV infection while GL261 cells showed signs of necrotic cell death. Additionally, in BV-2 cells with M1-phenotype a further reduction of viral replication and inhibition of cell lysis was detected. Infection of IMA 2.1 cells was independent of the M1/M2-phenotype. Application of BV-2 microglia with M1-phenotype onto organotypic slice cultures with implanted GL261 tumors resulted in reduced infection of BV-2 cells with LIVP 1.1.1, whereas GL261 cells were significantly infected.
Taken together, the analyzed GL261 tumors were imprinted by the immunologic and genetic background in which they grow. The experimental approach applied in this thesis can be used as suitable model which reflects the principles of personalized medicine
In an additional project, based on gene expression data and bioinformatic analyses, the biological role and function of the anti-apoptotic factor AVEN was analyzed with regard to oncolytic VACV therapy. Besides a comparison of the replication efficacy of GLV-1h68 and VACV-mediated cell killing of four human tumor cell lines, it was shown that AVEN was expressed in all analyzed cells. Further, shown for HT-29 and 1936-MEL, the knockdown of AVEN by siRNA in cell culture resulted in an increase of apoptotic characteristics and a decrease of VACV infection. These findings provide essential insights for future virus development.
Characterization of motility and erythrocyte adherence as virulence factors in African trypanosomes
(2018)
Pathogens causing African animal trypanosomiasis (AAT), the major livestock disease in sub-Saharan Africa, belong to the salivarian group of the African trypanosomes, which are transmitted by the bite of the tsetse fly (Glossina spec.). T. vivax, T. congolense and T. brucei brucei are major pathogens of cattle in particular, causing nagana, with dramatic socio-economic consequences for the affected regions. The parasites additionally have a huge reservoir of other livestock and wild animal hosts. T. brucei, the species which also includes the subspecies pathogenic to humans causing sleeping sickness, has been extensively studied as the cultivatable model trypanosome. But less is known about the other salivarian species, which are not routinely held in culture, if at all possible. A hallmark of trypanosomal lifestyle is the protozoan flagellates incessant motility, which enables them to populate an enormous range of habitats in very diverse hosts. We were now able to characterize, for the first time with high spatiotemporal resolution microscopy, the swimming behaviour and mechanism of the most relevant salivarian species isolated directly from blood. We show the influence of viscosity on the motility of bloodstream form (BSF) cells and simulate their movement between erythrocytes, giving a clear picture of how all analyzed species move under varying environmental conditions. We show that although the basic mechanism of flagellar motility applies to all analyzed species, there are clear morphological differences that produce different reactions to the physical environment. We could define specific conditions for highly increased swimming persistence and speed for compared to the behaviour in standard culture. These results have important implications for the parasites survival strategies in the host, e.g. regarding the capacity for antibody clearance. Although we show all species to effectively remove antibodies from the cell surface, T. congolense differed markedly in its motility behaviour, which gives rise to interesting questions about this species behaviour in the bloodstream. Most of the T. congolense parasites (and to a lesser extent T. vivax) adhere to sheep erythrocytes. Further in vitro studies showed that T. congolense and T. vivax adhered to rabbit, goat, pig and cattle erythrocytes- but binding behaviour was absent in murine blood. Notably, both T. brucei and T. evansi lacked adherence to all studied host erythrocytes. Generally, attachment to blood cells caused reduction of swimming velocities. Judging from its cell architecture, as well as the motility studies in higher media viscosity and in micropillar arrays, T. congolense is not adapted to swim at high speeds in the mammalian bloodstream. Low swimming speeds could allow these purely intravascular parasites to remain bound to the host erythrocytes.
Characterization of memories and ignorant (S6KII) mutants in operant conditioning in the heat-box
(2002)
Learning and memory processes of operant conditioning in the heat-box were analysed. Age, sex, and larval desity were not critical parameters influencing memory, while low or high activity levels of flies were negatively correlated with their performance. In a search for conditioning parameters leading to high retention scores, intermittent training was shown to give better results than continuous training. As the memory test is the immediate continuation of the conditioning phase just omitting reinforcement, we obtain a memory which consists of two components: a spatial preference for one side of the chamber and a stay-where-you-are effect in which the side preference is contaminated by the persistence of heat avoidance. Intermittent training strengthens the latter. In the next part, memory retention was investigated. Flies were trained in one chamber and tested in a second one after a brief reminder training. With this direct transfer, memory scores reflect an associative learning process in the first chamber. To investigate memory retention after extended time periods, indirect transfer experiments were performed. The fly was transferred to a different environment between training and test phases. With this procedure an after-effect of the training was still observed two hours later. Surprisingly, exposure to the chamber without conditioning also lead to a memory effect in the indirect transfer experiment. This exposure effect revealed a dispositional change that facilitates operant learning during the reminder training. The various memory effects are independent of the mushroom bodies. The transfer experiments and yoked controls proved that the heat-box records an associative memory. Even two hours after the operant conditioning procedure, the fly remembers that its position in the chamber controls temperature. The cAMP signaling cascade is involved in heat-box learning. Thus, amnesiac, rutabaga, and dunce mutants have an impaired learning / memory. Searching for, yet unknown, genes and signaling cascades involved in operant conditioning, a Drosophila melanogaster mutant screen with 1221 viable X-chromosome P-element lines was performed. 29 lines with consistently reduced heat avoidance/ learning or memory scores were isolated. Among those, three lines have the p[lacW] located in the amnesiac ORF, confirming that with the chosen candidate criteria the heat-box is a useful tool to screen for learning and /or memory mutants. The mutant line ignP1 (8522), which is defective in the gene encoding p90 ribosomal S6 kinase (S6KII), was investigated. The P-insertion of line ignP1 is the first Drosophila mutation in the ignorant (S6KII) gene. It has the transposon inserted in the first exon. Mutant males are characterized by low training performance, while females perform well in the standard experiment. Several deletion mutants of the ignorant gene have been generated. In precise jumpouts the phenotype was reverted. Imprecise jumpouts with a partial loss of the coding region were defective in operant conditioning. Surprisingly, null mutants showed wild-type behavior. This might indicate an indirect effect of the mutated ignorant gene on learning processes. In classical odor avoidance conditioning, ignorant null mutants showed a defect in the 3-min, 30-min, and 3-hr memory, while the precise jumpout of the transposon resulted in a reversion of the behavioral phenotype. Deviating results from operant and classical conditioning indicate different roles for S6KII in the two types of learning.
Characterization of a novel putative factor involved in host adaptation in Trypanosoma brucei
(2016)
Trypanosomes are masters of adaptation to different host environments
during their complex life cycle. Large-scale proteomic approaches provide information on changes at
the cellular level in a systematic way. However, a detailed work on single components is necessary
to understand the adaptation mechanisms on a molecular level. Here we have performed a detailed
characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation
factor (Tb927.11.2400) identified previously in a SILAC-based comparative proteome study.
Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form
(PCF) stage specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin like
(TbFlabarinL) and demonstrate that it is a result of a gene duplication event, which occurred in
African trypanosomes. TbFlabarinL is not essential for growth of the parasites under cell culture
conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We
generated a TbFlabarinL-specific antibody and showed that it localizes in the flagellum. The
co-immunoprecipitation experiment together with a biochemical cell fractionation indicated a dual
association of TbFlabarinL with the flagellar
membrane and the components of the paraflagellar rod.
Monolayer or suspension cell cultures are of only limited value as experimental models for human cancer. Therefore, more sophisticated, three-dimensional culture systems like spheroid cultures or histocultures are used, which more closely mimic the tumor in individual patients compared to monolayer or suspension cultures. As tissue culture or tissue engineering requires more sophisticated culture, specialized in vitro techniques may also improve experimental tumor models. In the present work, a new miniaturized hollow-fiber bioreactor system for mammalian cell culture in small volumes (up to 3 ml) is characterized with regard to transport characteristics and growth of leukemic cell lines (chapter 2). Cell and medium compartment are separated by dialysis membranes and oxygenation is accomplished using oxygenation membranes. Due to a transparent housing, cells can be observed by microscopy during culture. The leukemic cell lines CCRF-CEM, HL-60 and REH were cultivated up to densities of 3.5 x 107/ml without medium change or manipulation of the cells. Growth and viability of the cells in the bioreactor were the same or better, and the viable cell count was always higher compared to culture in Transwellâ plates. As shown using CCRF-CEM cells, growth in the bioreactor was strongly influenced and could be controlled by the medium flow rate. As a consequence, consumption of glucose and generation of lactate varied with the flow rate. Influx of low molecular weight substances in the cell compartment could be regulated by variation of the concentration in the medium compartment. Thus, time dependent concentration profiles (e.g. pharmacokinetic profiles of drugs) can be realized as illustrated using glucose as a model compound. Depending on the molecular size cut-off of the membranes used, added growth factors like GM-CSF and IL-3 as well as factors secreted from the cells are retained in the cell compartment for up to one week. Second, a method for monitoring cell proliferation the hollow-fiber bioreactor by use of the Alamar BlueTM dye was developed (chapter 3). Alamar BlueTM is a non-fluorescent compound which yields a fluorescent product after reduction e.g. by living cells. In contrast to the MTT-assay, the Alamar BlueTM-assay does not lead to cell death. However, when not removed from the cells, the Alamar BlueTM dye shows a reversible, time- and concentration-dependent growth inhibition as observed for leukemic cell lines. When applied in the medium compartment of a hollow-fiber bioreactor system, the dye is delivered to the cells across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensity can be measured in medium samples reflecting growth of the cells in the cell compartment. This procedure offers several advantages. First, exposure of the cells to the dye can be reduced compared to conventional culture in plates. Second, handling steps are minimized since no sample of the cells needs to be taken for readout. Moreover, for the exchange of medium, a centrifugation step can be avoided and the cells can be cultivated further. Third, the method allows to discriminate between cell densities of 105, 106 and 107 of proliferating HL-60 cells cultivated in the cell compartment of the bioreactor. Measurement of fluorescence in the medium compartment is more sensitive compared to glucose or lactate measurement for cell counts below 106 cells/ml, in particular. In conclusion, the Alamar BlueTM-assay combined with the hollow-fiber bioreactor offers distinct advantages for the non-invasive monitoring of cell viability and proliferation in a closed system. In chapter 4 the use of the hollow-fiber bioreactor as a tool for toxicity testing was investigated, as current models for toxicity as well as efficacy testing of drugs in vitro allow only limited conclusions with regard to the in vivo situation. Examples of the drawbacks of current test systems are the lack of realistic in vitro tumor models and difficulties to model drug pharmacokinetics. The bioreactor proved to be pyrogen free and is steam-sterilizable. Leukemic cell lines like HL-60 and primary cells such as PHA-stimulated lymphocytes can be grown up to high densities of 1-3 x 107 and analyzed during growth in the bioreactor by light-microscopy. The cytostatic drug Ara-C shows a dose-dependent growth inhibition of HL-60 cells and a dose-response curve similar to controls in culture plates. The bioreactor system is highly flexible since several systems can be run in parallel, soluble drugs can be delivered continuously via a perfusion membrane and gaseous compounds via an oxygenation membrane which also allows to control pO2 and pH (via pCO2) during culture in the cell compartment. The modular concept of the bioreactor system allows realization of a variety of different design properties, which may lead to an improved in vitro system for toxicity testing by more closely resembling the in vivo situation. Whereas several distinct advantages of the new system have been demonstrated, more work has to be done to promote in vitro systems in toxicity testing and drug development further and to reduce the need for animal tests.
(1) Background: about 10% of Wilms Tumor (WT) patients have a malformation or cancer predisposition syndrome (CPS) with causative germline genetic or epigenetic variants. Knowledge on CPS is essential for genetic counselling. (2) Methods: this retrospective analysis focused on 2927 consecutive patients with WTs registered between 1989 and 2017 in the SIOP/GPOH studies. (3) Results: Genitourinary malformations (GU, N = 66, 2.3%), Beckwith-Wiedemann spectrum (BWS, N = 32, 1.1%), isolated hemihypertrophy (IHH, N = 29, 1.0%), Denys-Drash syndrome (DDS, N = 24, 0.8%) and WAGR syndrome (N = 20, 0.7%) were reported most frequently. Compared to others, these patients were younger at WT diagnosis (median age 24.5 months vs. 39.0 months), had smaller tumors (349.4 mL vs. 487.5 mL), less often metastasis (8.2% vs. 18%), but more often nephroblastomatosis (12.9% vs. 1.9%). WT with IHH was associated with blastemal WT and DDS with stromal subtype. Bilateral WTs were common in WAGR (30%), DDS (29%) and BWS (31%). Chemotherapy induced reduction in tumor volume was poor in DDS (0.4% increase) and favorable in BWS (86.9% reduction). The event-free survival (EFS) of patients with BWS was significantly (p = 0.002) worse than in others. (4) Conclusions: CPS should be considered in WTs with specific clinical features resulting in referral to a geneticist. Their outcome was not always favorable.
In a variety of established tumour cell lines, but also in primary mammary epithelial cells metalloprotease-dependent transactivation of the EGFR, and EGFR characteristic downstream signalling events were observed in response to stimulation with physiological concentrations of GPCR agonists such as the mitogens LPA and S1P as well as therapeutically relevant concentrations of cannabinoids. Moreover, this study reveals ADAM17 and HB-EGF as the main effectors of this mechanism in most of the cancer cell lines investigated. However, depending on the cellular context and GPCR agonist, various different members of the ADAM family are selectively recruited for specific ectodomain shedding of proAR and/or proHB-EGF and subsequent EGFR activation. Furthermore, biological responses induced by LPA or S1P such as migration in breast cancer and HNSCC cells, depend on ADAM17 and proHB-EGF/proAR function, respectively, suggesting that highly abundant GPCR ligands may play a role in tumour development and progression. Moreover, EGFR signal transactivation could be identified as the mechanistic link between cannabinoid receptors and the activation of mitogen activated protein kinases (MAPK) ERK1/2 as well as pro-survival Akt/PKB signalling. Depending on the cellular context, cannabinoid-induced signal cross-communication was mediated by shedding of proAmphiregulin and/or proHB-EGF by ADAM17. Most importantly, our data show that concentrations of THC comparable to those detected in the serum of patients after THC administration accelerate proliferation of cancer cells instead of apoptosis and thereby may contribute to cancer progression in patients.
Characterisation of Metalloprotease-mediated EGFR Signal Transactivation after GPCR Stimulation
(2011)
In the context of metalloprotease-mediated transactivation of the epidermal growth factor receptor, different monoclonal antibodies against ADAM17 / TACE were characterized for their ability to block the sheddase. Activity of some of them was observed at doses between 2µg/mL and 10µg/mL. Kinetic analyses showed their activity starting at around 30 minutes. In cellular assays performed with the antibodies, especially upon treatment of cells with sphingosine-1-phosphate a reduction in proliferation was observed with some candidates. Moreover this study provides potential new roles for ß-Arrestins. Their involvement in the triple membrane-passing signal pathway of EGFR transactivation was shown. Furthermore, in overexpressing cellular model systems, an interaction between ADAM17 and ß-Arrestin1 could be observed. Detailed analysis discovered that phosphorylation of ß-Arrestin1 is crucial for this interaction. Additionally, the novel mechanism of UV-induced EGFR transactivation was extended to squamous cell carcinoma. The mechanism happens in a dose dependent manner and requires a metalloprotease to shed the proligand Amphiregulin. The involvement of both ADAM9 and ADAM17, being the metalloproteases responsible for this cleavage, was shown for SCC9 cells.
Fish of the genus Xiphophorus belong to the oldest animal models in cancer research. The oncogene responsible for the generation of spontaneous aggressive melanoma encodes for a mutated epidermal growth factor receptor (Egfr) and is called xmrk for Xiphophorus melanoma receptor kinase. Xmrk constitutive activation mechanisms and subsequent signaling pathways have already been investigated and charaterized but it is still unknown if Egfr ligands may also play a role in Xmrk-driven melanoma formation. To investigate the potential role of Egfr ligands in Xmrk-driven melanoma, I firstly analyzed the evolution of teleost and tetrapod Egfr/Egfr ligand systems. I especially focused on the analysis on the medaka fish, a closely related species to Xiphophorus, for which the whole genome has been sequenced. I could identify all seven Egfr ligands in medaka and could show that the two teleost-specific Egfr copies of medaka display dissimilar expression patterns in adult tissues together with differential expression of Egfr ligand subsets, arguing for subfunctionalization of receptor functions in this fish. Our phylogenetic and synteny analyses supported the hypothesis that only one gene in the chordate ancestor gave rise to the diversity of Egfr ligands found in vertebrate genomes today. I also could show that the Egfr extracellular subdomains implicated in ligand binding are not evolutionary conserved between tetrapods and teleosts, making the use of heterologous ligands in experiments with fish cells debatable. Despite its well understood and straight-forward process, Xmrk-driven melanomagenesis in Xiphophorus is problematic to further investigate in vivo. Our laboratory recently established a new melanoma animal model by generating transgenic mitf::xmrk medaka fishes, a Xiphophorus closely related species offering many more advantages. These fishes express xmrk under the control of the pigment-cell specific Mitf promoter. During my PhD thesis, I participated in the molecular analysis of the stably transgenic medaka and could show that the Xmrk-induced signaling pathways are similar when comparing Xiphophorus with transgenic mitf::xmrk medaka. These data together with additional RNA expression, protein, and histology analyses showed that Xmrk expression under the control of a pigment cell-specific promoter is sufficient to induce melanoma in the transgenic medaka, which develop very stereotyped tumors, including uveal and extracutaneous melanoma, with early onset during larval stages. To further investigate the potential role of Egfr ligands in Xmrk-driven melanoma, I made use of two model systems. One of them was the above mentioned mitf::xmrk medaka, the other was an in-vitro cell culture system, where the EGF-inducible Xmrk chimera HERmrk is stably expressed in murine melanocytes. Here I could show that HERmrk activation strongly induced expression of amphiregulin (Areg) and heparin-binding EGF-like growth factor (Hbegf) in melanocytes. This regulation was dependent on the MAPK and SRC signaling pathways. Moreover, upregulation of Adam10 and Adam17, the two major sheddases of Egfr ligands, was observed. I also could demonstrate the functionality of the growth factors by invitro analyses. Using the mitf::xmrk medaka model I could also show the upregulation of a subset of ligand genes, namely egf, areg, betacellulin (btc) and epigen (epgn) as well as upregulation of medaka egfrb in tumors from fish with metastatic melanoma. All these results converge to support an Xmrk-induced autocrine Egfr ligand loop. Interestingly, my in-vitro experiments with conditioned supernatant from medaka Egf- and Hbegf-producing cells revealed that not only Xiphophorus Egfrb, but also the pre-activated Xmrk could be further stimulated by the ligands. Altogether, I could show with in-vitro and in-vivo experiments that Xmrk is capable of inducing a functional autocrine Egfr ligand loop. These data confirm the importance of autocrine loops in receptor tyrosine kinase (RTK)-dependent cancer development and show the possibility for a constitutively active RTK to strengthen its oncogenic signaling by ligand binding.
Corynebacterium glutamicum is together with C. callunae and C. efficiens a member of the diverse group of mycolic-acid containing actinomycetes, the mycolata. These bacteria are potent producer of glutamate, lysine and other amino acids on industrial scale. The cell walls of most actinomycetes contain besides an arabinogalactan-peptidoglycan complex large amounts of mycolic acids. This three-layer envelope is called MAP (mycolyl-arabinogalactan-peptidoglycan) complex and it represents a second permeability barrier beside the cytoplasmic membrane similar to the outer membrane of Gram-negative bacteria. In analogy to the situation in the outer membrane of Gram-negative bacteria, channels are present in the mycolic acid layer of the mycobacterial cell wall for the passage of hydrophilic solutes. Molecular studies have provided far-reaching findings on the amino acid flux and its balance in C. glutamicum in general, but the L-glutamate export still remains unknown. The properties of the outer layers, typical of mycolata, seem to be of major importance in this process, and diffusion seems to play a key role for this part of the cell wall. The major aim of this thesis was to identify and study novel channel-forming proteins of the amino acid producers C. glutamicum, C. callunae and C. efficiens. Cell wall extracts of the organisms were investigated and a novel pore-forming protein, named PorH, that is homologue in all three organisms, was detected and characterized. PorHC.glut was isolated from C. glutamicum cells cultivated in minimal medium. The protein was identified in lipid bilayer experiments and purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The purified protein forms cation-selective channels with a diameter of about 2.2 nm and an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. Organic solvent extracts were used to study the permeability properties of the cell wall of C. callunae and C.efficiens. The cell extracts contained channel-forming activity, the corresponding proteins were purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column and named PorHC.call and PorHC.eff. Channels formed by PorHC.call are cation-selective with a diameter of about 2.2 nm and an average single-channel conductance of 3 nS, whereas PorHC.eff forms slightly anion selective channels with an average single-channel conductance of 2.3 nS in 1 M KCl in the lipid bilayer assay. The PorH proteins were partially sequenced and the corresponding genes, which were designated as porH, were identified in the published genome sequence of C. glutamicum and C. efficiens. The chromosome of C. callunae is not sequenced, but PorHC.call shows a high homology to PorHC.eff and PorHC.glut. The proteins have no N-terminal extension, only the inducer methionine, which suggests that secretion of the proteins could be very similar to that of PorAC.glut of C. glutamicum. PorHC.glut is coded in the bacterial chromosome by a gene that is localized in the vincinity of the porAC.glut gene, within a putative operon formed by 13 genes that are encoded by the minus strand. Both porins are cotranscribed and coexist in the cell wall, which was demonstrated in RT-PCR and immunological detection experiments. The arrangement of porHC.glut and porAC.glut on the chromosome is similar to that of porBC.glut and porCC.glut and it was found that PorAC.glut, PorHC.glut, PorBC.glut and PorCC.glut coexist in the cell wall of C. glutamicum. The molecular mass of about 6 kDa of the PorH channel forming proteins is rather small and suggests that the cell wall channels are formed by oligomers. A possibly hexameric form was demonstrated for PorHC.glut in Western blot analysis with anti- PorHC.glut antibodies. Secondary structure predictions for PorHC.glut, PorHC.call and PorHC.eff predict that a stretch of about 42 amino acids of PorHC.glut and 28 amino acids of PorHC.call and PorHC.eff forms amphipathic -helices with a total length of 6.3 nm and 4.2 nm respectively. This should be sufficient to cross the mycolic acid layer. Another objective of this work was to establish an heterologous expression system for corynebacterial channel-forming proteins, to investigate the channel-forming properties of the up to now only hypothetical porins PorA, PorB, PorC from C. efficiens and PorC from C. glutamicum. We could demonstrate with recombinant expression experiments in E. coli that porBC.eff and porCC.eff encode for channel-forming proteins. They are, like PorBC.glut, anion-selective with a similar single-channel conductance of 1 nS in 1 M KCl.
The ability to produce toxins is spread among a huge variety of bacterial strains. A very prominent class of bacterial protein toxins is the family of binary AB toxins sharing a common mode of intoxication. A pore forming component B binds and translocates an enzymatic component A into the cytosol of target cells exhibiting a fatal mode of action. These components are supposed to be not toxic themselves but both required for cell toxicity. Anthrax toxin produced by the Gram-positive bacteria Bacillus anthracis is the best studied binary toxin especially since its use as a biological weapon in the context of the attacks of 9/11 in 2001. In contrast to other binary toxins, Anthrax toxin possesses two different enzymatic components, edema factor (EF), a calcium- and calmodulin-dependent adenylat-cyclase and lethal factor (LF), a zinc-dependent metalloprotease. Protective antigen (PA) is the pore-forming component responsible for binding and translocation. Clostridium botulinum possesses in addition to the well known botulinum toxin (Botox) a variety of other toxins, such as the binary C2 toxin. C2 toxin is composed of the binding and translocation moiety C2II and the enzymatic moiety C2I acting as an actin-ADP-ribosyltransferase. In this study, the mode of translocation and the binding kinetics to the enzymatic component were studied in a biophysical experimental setup. In chapter 2, the binding of the N-terminal fractions EFN and LFN to the PA channel are analyzed in artificial bilayer membranes revealing lower binding affinity compared to full-length EF and LF. Other biophysical properties like voltage-dependency and ionic-strength dependency are not influenced. The results suggest that additional forces are involved in the binding process, than those concerning the N-terminus exclusively, as it was supposed previously. As the treatment of an Anthrax infection with antibiotics is often medicated very late due to the lack of early symptoms, tools to prevent intoxication are required. 4-aminoquinolones like chloroquine are known to block the PA channel, thereby inhibiting intoxication but they also lead to severe side-effects. In chapter 3 new promising agents are described that bind to PA in artificial bilayer systems, elucidating common motives and features which are necessary for binding to PA in general. The possible interaction of Anthrax and C2 toxin is investigated by measuring the binding of one enzymatic component to the respective other toxin’s pore (chapter 4). Interestingly, in vitro experiments using the black lipid bilayer assay show that PA is able to bind to C2I resulting in half saturation constants in the nanomolar range. Furthermore, in vivo this combination of toxin components exhibits cell toxicity in human cell lines. This is first-time evidence that a heterologous toxin combination is functional in in vitro and in vivo systems. In contrast, C2II is able to bind to EF as well as to LF in vitro, whereas in in vivo studies almost no toxic effect is detected. In the case of PA, an N-terminal His6-tag attached to the enzymatic subunit increased the binding affinity (chapter 5). A His6-tag attached to not related proteins also led to high binding affinities, providing the possibility to establish PA as a general cargo protein. In chapter 6 a set of different molecules and proteins is summarized, which are either related or not related to binary toxins, PA is able to bind. In first line, the presence of positive charges is found to be responsible for binding to PA which is in accordance to the fact that PA is highly cation selective. Furthermore, we present evidence that different cationic electrolytes serve as a binding partner to the PA channel. In the last decade another toxin has aroused public attention as it was found to be responsible for a rising number of nosocomial infections: Clostridium difficile CDT toxin. The mode of action of the enzymatic subunit CDTa is similar to C2I of C2 toxin, acting as an ADP-ribosylating toxin. The channel forming and binding properties of CDT toxin are studied in artificial bilayer membranes (chapter 7). We found that two different types of channels are formed by the B component CDTb. The first channel is similar to that of iota toxin’s Ib of Clostridium perfringens with comparable single channel conductance, selectivity and binding properties to the enzymatic subunit CDTa. The formation of this type of channel is cholesterol-dependent, whereas in the absence of cholesterol another kind of channel is observed. This channel has a single channel conductance which is rather high compared to all other binary toxin channels known so far, it is anion selective and does not show any binding affinity to the enzymatic component CDTa. The results reveal completely new insights in channel formation properties and the flexibility of a pore-forming component. Additionally, these findings suggest further possibilities of toxicity of the pore forming component itself which is not known for any other binary toxin yet. Therefore, the pathogenic role of this feature has to be studied in detail.
The morphology of nucleolar and non-nucleolar (Iampbrush chromosome loops) chromatin was studied in the electron microscope during states of reduced transcriptional activity in amphibian oocytes (Xenopus laevis, Triturus alpestris, T. cristatus). Reduced transcriptional activity was observed in maturing stages of oocyte development and after treatment with an inhibitor, actinomycin D. Strands of nucleolar chromatin appear smooth and thin, and contain only few, if any, nucleosomal particles in the transcribed units. This is true whether they are densely or only sparsely covered with lateral ribonucleoprotein fibrils. This smooth and non-nucleosomal character is also predominant in the interspersed, apparently nontranscribed rDNA spacer regions. During inactivation, however, nucleolar chromatin frequently and progressively assumes a beaded appearance in extended fibril-free-that is, apparently nontranscribed - regions. I n either fUll-grown 00- cytes or late after drug treatment, most of the nucleolar chromatin is no longer smooth and thin, but rather shows a beaded configuration indistinguishable from inactive non - nucleolar chromatin. In many chromatin strands, transitions of fibril-associated regions of smooth character into beaded regions wihout lateral fibrils are seen. Similarly, in the non-nucleolar chromatin of the retracting lampbrush chromosome loops, reduced transcriptional activity is correlated with a change from smooth to beaded morphology. Here, however, beaded regions are also commonly found interspersed between the more or less distant bases of the lateral fibrils, the putative transcriptional complexes. I n both sorts of chromatin, detergents (in particular Sarkosyl) that remove most of the chromatin proteins including histones from the DNA axis but leave the RNA polymerases of the transcriptional complexes attached were used to discriminate between polymerases and nucleosomal particles. The results suggest that nucleosomes are absent in heavily transcribed chromatin regions but are reformed after inactivation. In contrast to the findings with inactivated nucleolar genes, in lampbrush chromosome loops the beaded nucleosomal configuration appears to be assumed also in regions within transcriptional units that, perhaps temporarily, are not involved in transcription.
Background: The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector.
Results: To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using quantitative confocal microscopy.
Conclusions: The obtained transcriptome data demonstrate the regulations of gene expression immediately following malaria parasite transmission to the mosquito. Our findings support the identification of proteins important for sexual reproduction and further development of the mosquito midgut stages and provide insights into the genetic basis of the rapid adaption of Plasmodium to the insect vector.
Various types of cancer involve aberrant cell cycle regulation. Among the pathways responsible for tumor growth, the YAP oncogene, a key downstream effector of the Hippo pathway, is responsible for oncogenic processes including cell proliferation, and metastasis by controlling the expression of cell cycle genes. In turn, the MMB multiprotein complex (which is formed when B-MYB binds to the MuvB core) is a master regulator of mitotic gene expression, which has also been associated with cancer. Previously, our laboratory identified a novel crosstalk between the MMB-complex and YAP. By binding to enhancers of MMB target genes and promoting B-MYB binding to promoters, YAP and MMB co-regulate a set of mitotic and cytokinetic target genes which promote cell proliferation. This doctoral thesis addresses the mechanisms of YAP and MMB mediated transcription, and it characterizes the role of YAP regulated enhancers in transcription of cell cycle genes.
The results reported in this thesis indicate that expression of constitutively active, oncogenic YAP5SA leads to widespread changes in chromatin accessibility in untransformed human MCF10A cells. ATAC-seq identified that newly accessible and active regions include YAP-bound enhancers, while the MMB-bound promoters were found to be already accessible and remain open during YAP induction. By means of CRISPR-interference (CRISPRi) and chromatin immuniprecipitation (ChIP), we identified a role of YAP-bound enhancers in recruitment of CDK7 to MMB-regulated promoters and in RNA Pol II driven transcriptional initiation and elongation of G2/M genes. Moreover, by interfering with the YAP-B-MYB protein interaction, we can show that binding of YAP to B-MYB is also critical for the initiation of transcription at MMB-regulated genes. Unexpectedly, overexpression of YAP5SA also leads to less accessible chromatin regions or chromatin closing. Motif analysis revealed that the newly closed regions contain binding motifs for the p53 family of transcription factors. Interestingly, chromatin closing by YAP is linked to the reduced expression and loss of chromatin-binding of the p53 family member Np63. Furthermore, I demonstrate that downregulation of Np63 following expression of YAP is a key step in driving cellular migration.
Together, the findings of this thesis provide insights into the role of YAP in the chromatin changes that contribute to the oncogenic activities of YAP. The overexpression of YAP5SA not only leads to the opening of chromatin at YAP-bound enhancers which together with the MMB complex stimulate the expression of G2/M genes, but also promotes the closing of chromatin at ∆Np63 -bound regions in order to lead to cell migration.
Background: Chagas disease (CD) is a major burden in Latin America, expanding also to non-endemic countries. A gold standard to detect the CD causing pathogen Trypanosoma cruzi is currently not available. Existing real time polymerase chain reactions (RT-PCRs) lack sensitivity and/or specificity. We present a new, highly specific RT-PCR for the diagnosis and monitoring of CD. Material and Methods: We analyzed 352 serum samples from Indigenous people living in high endemic CD areas of Colombia using three leading RT-PCRs (k-DNA-, TCZ-, 18S rRNA-PCR), the newly developed one (NDO-PCR), a Rapid Test/enzyme-linked immuno sorbent assay (ELISA), and immunofluorescence. Eighty-seven PCR-products were verified by sequence analysis after plasmid vector preparation. Results: The NDO-PCR showed the highest sensitivity (92.3%), specificity (100%), and accuracy (94.3%) for T. cruzi detection in the 87 sequenced samples. Sensitivities and specificities of the kDNA-PCR were 89.2%/22.7%, 20.5%/100% for TCZ-PCR, and 1.5%/100% for the 18S rRNA-PCR. The kDNA-PCR revealed a 77.3% false positive rate, mostly due to cross-reactions with T. rangeli (NDO-PCR 0%). TCZ- and 18S rRNA-PCR showed a false negative rate of 79.5% and 98.5% (NDO-PCR 7.7%), respectively. Conclusions: The NDO-PCR demonstrated the highest specificity, sensitivity, and accuracy compared to leading PCRs. Together with serologic tests, it can be considered as a reliable tool for CD detection and can improve CD management significantly.
The MuvB multiprotein complex, together with B-MYB and FOXM1 (MMB-FOXM1), plays an essential role in cell cycle progression by regulating the transcription of genes required for mitosis and cytokinesis. In many tumors, B-MYB and FOXM1 are overexpressed as part of the proliferation signature. However, the transcriptional targets that are important for oncogenesis have not been identified. Given that mitotic kinesins are highly expressed in cancer cells and that selected kinesins have been reported as target genes of MMB-FOXM1, we sought to determine which mitotic kinesins are directly regulated by MMB-FOXM1. We demonstrate that six mitotic kinesins and two microtubule-associated non-motor proteins (MAPs) CEP55 and PRC1 are direct transcriptional targets of MuvB, B-MYB and FOXM1 in breast cancer cells.
Suppression of KIF23 and PRC1 strongly suppressed proliferation of MDA-MB-231 cells. The set of MMB-FOXM1 regulated kinesins genes and 4 additional kinesins which we referred to as the mitotic kinesin signature (MKS) is linked to poor outcome in breast cancer patients. Thus, mitotic kinesins could be used as prognostic biomarker and could be potential therapeutic targets for the treatment of breast cancer.
Neisseria gonorrhoeae are Gram-negative bacteria with diplococcal shape. As an obligate human pathogen, it is the causative agent of gonorrhoea, a sexually transmitted disease. Gonococci colonize a variety of mucosal tissues, mainly the urogenital tract in men and women. Occasionally N. gonorrhoeae invades the bloodstream, leading to disseminated gonococcal infection. These bacteria possess a repertoire of virulence factors, which expression patterns can be adapted to the environmental conditions of the host. Through the accumulation of antibiotic resistances and in absence of vaccines, some neisserial strains have the potential to spread globally and represent a major public health threat. Therefore, it is necessary to understand the exact molecular mechanisms underlying the successful infection and progression of gonococci within their host. This deeper understanding of neisserial infection and survival mechanisms is needed for the development of new therapeutic agents.
In this work, the role of host-cell sphingolipids on the intracellular survival of N. gonorrhoeae was investigated. It was shown that different classes of sphingolipids strongly interact with invasive gonococci in epithelial cells. Therefore, novel and highly specific clickable sphingolipid analogues were applied to study these interactions with this pathogen. The formation of intra- and extracellular sphingosine vesicles, which were able to target gonococci, was observed. This direct interaction led to the uptake and incorporation of sphingosine into the neisserial membrane. Together with in vitro results, sphingosine was identified as a potential bactericidal reagent as part of the host cell defence. By using different classes of sphingolipids and their clickable analogues, essential structural features, which seem to trigger the bacterial uptake, were detected. Furthermore, effects of key enzymes of the sphingolipid signalling pathway were tested in a neutrophil infection model.
In conclusion, the combination of click chemistry and infection biology made it possible to shed some light on the dynamic interplay between cellular sphingosine and N. gonorrhoeae. Thereby, a possible “catch-and-kill” mechanism could have been observed.
Small bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in a very few cases has the role in metabolism been demonstrated. Here, we performed a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 (NgncR_162/163) and their impact on the metabolism of Neisseria gonorrhoeae. These sRNAs have been reported to control genes of the citric acid and methylcitric acid cycles by posttranscriptional negative regulation. By transcriptome analysis, we now expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially in the uptake of glycine, phenylalanine, and branched-chain amino acids. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and, hence, probably affect biosyntheses of nucleotides, vitamins, and other amino acids via the supply of one-carbon (C\(_1\)) units. Indeed, these roles were confirmed by metabolomics and metabolic flux analysis, which revealed a bipartite metabolic network with glucose degradation for the supply of anabolic pathways and the usage of amino acids via the citric acid cycle for energy metabolism. Thus, by combined deep RNA sequencing (RNA-seq) and metabolomics, we significantly extended the regulon of NgncR_162/163 and demonstrated the role of NgncR_162/163 in the regulation of central metabolic pathways of the gonococcus.
Animal circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. Here we dissect the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. We also show that the central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals.
Chlamydia are Gram-negative obligate intracellular bacteria responsible for a wide spectrum of relevant diseases. Due to their biphasic developmental cycle Chlamydia depend on an intact host cell for replication and establishment of an acute infection. Chlamydia have therefore evolved sophisticated strategies to inhibit programmed cell death (PCD) induced by a variety of stimuli and to subvert the host immune system. This work aimed at elucidating whether an infection with C. trachomatis can influence the cellular response to double-stranded RNA (dsRNA). The synthesis of dsRNA is a prominent feature of viral replication inside infected cells that can induce both PCD and the activation of a cellular innate immune response. In order to mimic chlamydial and viral co-infections, Chlamydia-infected cells were transfected with polyinosinic:polycytidylic acid (polyI:C), a synthetic dsRNA. In the first part of this work it was investigated whether C. trachomatis-infected host cells could resist apoptosis induced by polyI:C. A significant reduction in apoptosis, determined by PARP cleavage and DNA fragmentation, could be observed in infected cells. It could be shown that processing of the initiator caspase-8 was inhibited in infected host cells. This process was dependent on early bacterial protein synthesis and was specific for dsRNA because apoptosis induced by TNFalpha was not blocked at the level of caspase-8. Interestingly, the activation of cellular factors involved in apoptosis induction by dsRNA, most importantly PKR and RNase L, was not abrogated in infected cells. Instead, RNA interference experiments revealed the crucial role of cFlip, a cellular caspase-8 inhibitor, for chlamydial inhibition of dsRNA-induced apoptosis. First data acquired by co-immunoprecipitation experiments pointed to an infection-induced concentration of cFlip in the dsRNA-induced death complex of caspase-8 and FADD. In the second part of this work, the chlamydial influence on the first line of defense against viral infections, involving expression of interferons and interleukins, was examined. Activation of the interferon regulatory factor 3 (IRF-3) and the NF-kappaB transcription factor family member p65, both central regulators of the innate immune response to dsRNA, was altered in Chlamydia-infected epithelial cells. polyI:C-induced degradation of IkappaB-alpha, the inhibitor of NF-kappaB, was accelerated in infected cells which was accompanied by a change in nuclear translocation of the transcription factor. Translocation of IRF-3, in contrast, was significantly blocked upon infection. Together the data presented here demonstrate that infection with C. trachomatis can drastically alter the cellular response to dsRNA and imply an impact of chlamydial infections on the outcome of viral super-infections.
Members of the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family are important regulators of the actin cytoskeleton dynamics. VASP functions as well as its interactions with other proteins are regulated by phosphorylation at three sites - serine157 (S157), serine239 (S239), and threonine278 (T278) in humans. cAMP- and cGMP- dependent protein kinases phosphorylate S157 and S239, respectively. In contrast, the kinase responsible for T278 was as yet unknown and identified in the first part of this thesis. In a screen for T278 phosphorylating kinases using a phospho-specific antibody against phosphorylated T278 AMP-activated protein kinase (AMPK) was identified in endothelial cells. Mutants of AMPK with altered kinase-activity modulate T278-phosphorylation levels in cells. AMPK-driven T278-phosphorylation impaired stress fiber formation and changed cell morphology in living cells. AMPK is a fundamental sensor of cellular and whole body energy homeostasis. Zucker Diabetic Fatty (ZDF) rats, which are an animal model for type II diabetes mellitus, were used to analyze the impact of phosphorylated T278 in vivo. AMPK-activity and T278-phosphorylation were substantially reduced in arterial vessel walls of ZDF rats in comparison to control animals. These findings demonstrate that VASP is a new AMPK substrate, that VASP phosphorylation mediates the effects of metabolic regulation on actin cytoskeleton rearrangements, and that this signaling system becomes down-regulated in diabetic vessel disorders in rats. In the second part of this thesis, a functional analysis of differential VASP phosphorylations was performed. To systematically address VASP phosphorylation patterns, a set of VASP phosphomimetic mutants was cloned. These mutants enable the mimicking of defined phosphorylation patterns and the specific analysis of single kinase-mediated phosphorylations. VASP localization to the cell periphery was increased by S157- phosphorylation and modulated by phosphorylation at S239 and T278. Latter phosphorylations synergistically reduced actin polymerization. In contrast, S157- phosphorylation had no effect on actin-dynamics. Taken together, the results of the second part show that phosphorylation of VASP serves as a fine regulator of localization and actin polymerization activity. In summary, this study revealed the functions of VASP phosphorylations and established novel links between signaling pathways and actin cytoskeleton rearrangement.
In initial experiments, the well characterized VACV strain GLV-1h68 and three wild-type LIVP isolates were utilized to analyze gene expression in a pair of autologous human melanoma cell lines (888-MEL and 1936 MEL) after infection. Microarray analyses, followed by sequential statistical approaches, characterized human genes whose transcription is affected specifically by VACV infection. In accordance with the literature, those genes were involved in broad cellular functions, such as cell death, protein synthesis and folding, as well as DNA replication, recombination, and repair. In parallel to host gene expression, viral gene expression was evaluated with help of customized VACV array platforms to get better insight over the interplay between VACV and its host. Our main focus was to compare host and viral early events, since virus genome replication occurs early after infection. We observed that viral transcripts segregated in a characteristic time-specific pattern, consistent with the three temporal expression classes of VACV genes, including a group of genes which could be classified as early-stage genes. In this work, comparison of VACV early replication and respective early gene transcription led to the identification of seven viral genes whose expression correlated strictly with replication. We considered the early expression of those seven genes to be representative for VACV replication and we therefore referred to them as viral replication indicators (VRIs). To explore the relationship between host cell transcription and viral replication, we correlated viral (VRI) and human early gene expression. Correlation analysis revealed a subset of 114 human transcripts whose early expression tightly correlated with early VRI expression and thus early viral replication. These 114 human molecules represented an involvement in broad cellular functions. We found at least six out of 114 correlates to be involved in protein ubiquitination or proteasomal function. Another molecule of interest was the serine-threonine protein kinase WNK lysine-deficient protein kinase 1 (WNK1). We discovered that WNK1 features differences on several molecular biological levels associated with permissiveness to VACV infection. In addition to that, a set of human genes was identified with possible predictive value for viral replication in an independent dataset. A further objective of this work was to explore baseline molecular biological variances associated with permissiveness which could help identifying cellular components that contribute to the formation of a permissive phenotype. Therefore, in a subsequent approach, we screened a set of 15 melanoma cell lines (15-MEL) regarding their permissiveness to GLV-1h68, evaluated by GFP expression levels, and classified the top four and lowest four cell lines into high and low permissive group, respectively. Baseline gene transcriptional data, comparing low and highly permissive group, suggest that differences between the two groups are at least in part due to variances in global cellular functions, such as cell cycle, cell growth and proliferation, as well as cell death and survival. We also observed differences in the ubiquitination pathway, which is consistent with our previous results and underlines the importance of this pathway in VACV replication and permissiveness. Moreover, baseline microRNA (miRNA) expression between low and highly permissive group was considered to provide valuable information regarding virus-host co-existence. In our data set, we identified six miRNAs that featured varying baseline expression between low and highly permissive group. Finally, copy number variations (CNVs) between low and highly permissive group were evaluated. In this study, when investigating differences in the chromosomal aberration patterns between low and highly permissive group, we observed frequent segmental amplifications within the low permissive group, whereas the same regions were mostly unchanged in the high group. Taken together, our results highlight a probable correlation between viral replication, early gene expression, and the respective host response and thus a possible involvement of human host factors in viral early replication. Furthermore, we revealed the importance of cellular baseline composition for permissiveness to VACV infection on different molecular biological levels, including mRNA expression, miRNA expression, as well as copy number variations. The characterization of human target genes that influence viral replication could help answering the question of host cell response to oncolytic virotherapy and provide important information for the development of novel recombinant vaccinia viruses with improved features to enhance replication rate and hence trigger therapeutic outcome.
The honeybee Apis mellifera is a social insect well known for its complex behavior and the ability to learn tasks associated with central place foraging, such as visual navigation or to learn and remember odor-reward associations. Although its brain is smaller than 1mm² with only 8.2 x 105 neurons compared to ~ 20 x 109 in humans, bees still show amazing social, cognitive and learning skills. They express an age – related division of labor with nurse bees staying inside the hive and performing tasks like caring for the brood or cleaning, and foragers who collect food and water outside the hive. This challenges foragers with new responsibilities like sophisticated navigation skills to find and remember food sources, drastic changes in the sensory environment and to communicate new information to other bees. Associated with this plasticity of the behavior, the brain and especially the mushroom bodies (MBs) - sensory integration and association centers involved in learning and memory formation – undergo massive structural and functional neuronal alterations. Related to this background my thesis on one hand focuses on neuronal plasticity and underlying molecular mechanisms in the MBs that accompany the nurse – forager transition.
In the first part I investigated an endogenous and an internal factor that may contribute to the nurse - forager phenotype plasticity and the correlating changes in neuronal network in the MBs: sensory exposure (light) and juvenile hormone (JH). Young bees were precociously exposed to light and subsequently synaptic complexes (microglomeruli, MG) in the MBs or respectively hemolymph juvenile hormone (JH) levels were quantified. The results show that light input indeed triggered a significant decrease in MG density, and mass spectrometry JH detection revealed an increase in JH titer. Interestingly light stimulation in young bees (presumably nurse bees) triggered changes in MG density and JH levels comparable to natural foragers. This indicates that both sensory stimuli as well as the endocrine system may play a part in preparing bees for the behavioral transition to foraging.
Considering a connection between the JH levels and synaptic remodeling I used gene knockdown to disturb JH pathways and artificially increase the JH level. Even though the knockdown was successful, the results show that MG densities remained unchanged, showing no direct effect of JH on synaptic restructuring.
To find a potential mediator of structural synaptic plasticity I focused on the calcium-calmodulin-dependent protein kinase II (CaMKII) in the second part of my thesis. CaMKII is a protein known to be involved in neuronal and behavioral plasticity and also plays an important part in structural plasticity reorganizing synapses. Therefore it is an interesting candidate for molecular mechanisms underlying MG reorganization in the MBs in the honeybee. Corresponding to the high abundance of CaMKII in the learning center in vertebrates (hippocampus), CaMKII was shown to be enriched in the MBs of the honeybee. Here I first investigated the function of CaMKII in learning and memory formation as from vertebrate work CaMKII is known to be associated with the strengthening of synaptic connections inducing long term potentiation and memory formation. The experimental approach included manipulating CaMKII function using 2 different inhibitors and a specific siRNA to create a CaMKII knockdown phenotype. Afterwards bees were subjected to classical olfactory conditioning which is known to induce stable long-term memory. All bees showed normal learning curves and an intact memory acquisition, short-term and mid-term memory (1 hour retention). However, in all cases long-term memory formation was significantly disrupted (24 and 72 hour retention). These results suggests the necessity of functional CaMKII in the MBs for the induction of both early and late phases of long-term memory in honeybees. The neuronal and molecular bases underlying long-term memory and the resulting plasticity in behavior is key to understanding higher brain function and phenotype plasticity. In this context CaMKII may be an important mediator inducing structural synaptic and neuronal changes in the MB synaptic network.
Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models.
Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status
(2014)
Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.
Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.
Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH\(_2\), led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE\(_2\), which correlated with the upregulation of numerous NF\(_k\)B-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu.
Carrion plays an essential role in shaping the structure and functioning of ecosystems and has far‐reaching implications for biodiversity conservation. The change in availability and type of carcasses throughout ecosystems can involve negative effects for scavenging communities. To address this issue, there have been recent conservation management measures of carrion provision in natural systems. However, the optimal conditions under which exposing carcasses to optimize conservation outcomes are still limited. Here, we used camera traps throughout elevational and vegetational gradients to monitor the consumption of 48 deer carcasses over a study period of six years by evaluating 270,279 photographs resulting out of 15,373 trap nights. We detected 17 species visiting carcass deployments, including five endangered species. Our results show that large carcasses, the winter season, and a heterogeneous surrounding habitat enhanced the frequency of carcass visits and the species richness of scavenger assemblages. Contrary to our expectations, carcass species, condition (fresh/frozen), and provision schedule (continuous vs single exposure) did not influence scavenging frequency or diversity. The carcass visitation frequency increased with carcass mass and lower temperatures. The effect of large carcasses was especially pronounced for mesopredators and the Eurasian lynx (Lynx lynx). Lynx were not too influenced in its carrion acquisition by the season, but exclusively preferred remote habitats containing higher forest cover. Birds of prey, mesopredators, and top predators were also positively influenced by the visiting rate of ravens (Corvus corax), whereas no biotic or abiotic preferences were found for wild boars (Sus scrofa). This study provides evidence that any ungulate species of carrion, either in a fresh or in previously frozen condition, attracts a high diversity of scavengers especially during winter, thereby supporting earlier work that carcass provisions may support scavenger communities and endangered species.
Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13C6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C3-compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C3-substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens.
Defense against biotic or abiotic stresses is one of the benefits of living in symbiosis. Leaf-cutting ants, which live in an obligate mutualism with a fungus, attenuate thermal and desiccation stress of their partner through behavioral responses, by choosing suitable places for fungus-rearing across the soil profile. The underground environment also presents hypoxic (low oxygen) and hypercapnic (high carbon dioxide) conditions, which can negatively influence the symbiont. Here, we investigated whether workers of the leaf-cutting ant Acromyrmex lundii use the CO\(_{2}\) concentration as an orientation cue when selecting a place to locate their fungus garden, and whether they show preferences for specific CO\(_{2}\) concentrations. We also evaluated whether levels preferred by workers for fungus-rearing differ from those selected for themselves. In the laboratory, CO\(_{2}\) preferences were assessed in binary choices between chambers with different CO\(_{2}\) concentrations, by quantifying number of workers in each chamber and amount of relocated fungus. Leaf-cutting ants used the CO\(_{2}\) concentration as a spatial cue when selecting places for fungus-rearing. A. lundii preferred intermediate CO\(_{2}\) levels, between 1 and 3%, as they would encounter at soil depths where their nest chambers are located. In addition, workers avoided both atmospheric and high CO\(_{2}\) levels as they would occur outside the nest and at deeper soil layers, respectively. In order to prevent fungus desiccation, however, workers relocated fungus to high CO\(_{2}\) levels, which were otherwise avoided. Workers’ CO\(_{2}\) preferences for themselves showed no clear-cut pattern. We suggest that workers avoid both atmospheric and high CO\(_{2}\) concentrations not because they are detrimental for themselves, but because of their consequences for the symbiotic partner. Whether the preferred CO\(_{2}\) concentrations are beneficial for symbiont growth remains to be investigated, as well as whether the observed preferences for fungus-rearing influences the ants’ decisions where to excavate new chambers across the soil profile.
Several oncolytic viruses (OVs) including various human and canine adenoviruses, canine distemper virus, herpes-simplex virus, reovirus, and members of the poxvirus family, such as vaccinia virus and myxoma virus, have been successfully tested for canine cancer therapy in preclinical and clinical settings. The success of the cancer virotherapy is dependent on the ability of oncolytic viruses to overcome the attacks of the host immune system, to preferentially infect and lyse cancer cells, and to initiate tumor-specific immunity. To date, several different strategies have been developed to overcome the antiviral host defense barriers. In our study, we used canine adipose-derived mesenchymal stem cells (cAdMSCs) as a “Trojan horse” for the delivery of oncolytic vaccinia virus Copenhagen strain to achieve maximum oncolysis against canine soft tissue sarcoma (CSTS) tumors. A single systemic administration of vaccinia virus-loaded cAdMSCs was found to be safe and led to the significant reduction and substantial inhibition of tumor growth in a CSTS xenograft mouse model. This is the first example that vaccinia virus-loaded cAdMSCs could serve as a therapeutic agent against CSTS tumors.
Can Joint Carbon and Biodiversity Management in Tropical Agroforestry Landscapes Be Optimized?
(2012)
Managing ecosystems for carbon storage may also benefit biodiversity conservation, but such a potential 'win-win' scenario has not yet been assessed for tropical agroforestry landscapes. We measured above-and below-ground carbon stocks as well as the species richness of four groups of plants and eight of animals on 14 representative plots in Sulawesi, Indonesia, ranging from natural rainforest to cacao agroforests that have replaced former natural forest. The conversion of natural forests with carbon stocks of 227-362 Mg C ha\(^{-1}\) to agroforests with 82-211 Mg C ha\(^{-1}\) showed no relationships to overall biodiversity but led to a significant loss of forest-related species richness. We conclude that the conservation of the forest-related biodiversity, and to a lesser degree of carbon stocks, mainly depends on the preservation of natural forest habitats. In the three most carbon-rich agroforestry systems, carbon stocks were about 60% of those of natural forest, suggesting that 1.6 ha of optimally managed agroforest can contribute to the conservation of carbon stocks as much as 1 ha of natural forest. However, agroforestry systems had comparatively low biodiversity, and we found no evidence for a tight link between carbon storage and biodiversity. Yet, potential win-win agroforestry management solutions include combining high shade-tree quality which favours biodiversity with cacao-yield adapted shade levels.
Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding, and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system.
Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency.
Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mice display increased cell densities in the DR at embryonic stages E13.5, E17.5, and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5.
Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that Cdh13 deficiency affects the cell density of the developing DR and the posterior innervation of the prefrontal cortex (PFC), and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.
1. Pollination services of cacao are crucial for global chocolate production, yet remain critically understudied, particularly in regions of origin of the species. Notably, uncertainties remain concerning the identity of cacao pollinators, the influence of landscape (forest distance) and management (shade cover) on flower visitation and the role of pollen deposition in limiting fruit set.
2. Here, we aimed to improve understanding of cacao pollination by studying limiting factors of fruit set in Peru, part of the centre of origin of cacao. Flower visitors were sampled with sticky insect glue in 20 cacao agroforests in two biogeographically distinct regions of Peru, across gradients of shade cover and forest distance. Further, we assessed pollen quantities and compared fruit set between naturally and manually pollinated flowers.
3. The most abundant flower visitors were aphids, ants and thrips in the north and thrips, midges and parasitoid wasps in the south of Peru. We present some evidence of increasing visitation rates from medium to high shade (40%–95% canopy closure) in the dry north, and opposite patterns in the semi-humid south, during the wet season.
4. Natural pollination resulted in remarkably low fruit set rates (2%), and very low pollen deposition. After hand pollination, fruit set more than tripled (7%), but was still low.
5. The diversity and high relative abundances of herbivore flower visitors limit our ability to draw conclusions on the functional role of different flower visitors. The remarkably low fruit set of naturally and even hand pollinated flowers indicates that other unaddressed factors limit cacao fruit production. Such factors could be, amongst others, a lack of effective pollinators, genetic incompatibility or resource limitation. Revealing efficient pollinator species and other causes of low fruit set rates is therefore key to establish location-specific management strategies and develop high yielding native cacao agroforestry systems in regions of origin of cacao
C60 fullerene as an effective nanoplatform of alkaloid Berberine delivery into leukemic cells
(2019)
A herbal alkaloid Berberine (Ber), used for centuries in Ayurvedic, Chinese, Middle-Eastern, and native American folk medicines, is nowadays proved to function as a safe anticancer agent. Yet, its poor water solubility, stability, and bioavailability hinder clinical application. In this study, we have explored a nanosized carbon nanoparticle—C60 fullerene (C60)—for optimized Ber delivery into leukemic cells. Water dispersions of noncovalent C60-Ber nanocomplexes in the 1:2, 1:1, and 2:1 molar ratios were prepared. UV–Vis spectroscopy, dynamic light scattering (DLS), and atomic force microscopy (AFM) evidenced a complexation of the Ber cation with the negatively charged C60 molecule. The computer simulation showed that π-stacking dominates in Ber and C\(_{60}\) binding in an aqueous solution. Complexation with C\(_{60}\) was found to promote Ber intracellular uptake. By increasing C\(_{60}\) concentration, the C\(_{60}\)-Ber nanocomplexes exhibited higher antiproliferative potential towards CCRF-CEM cells, in accordance with the following order: free Ber < 1:2 < 1:1 < 2:1 (the most toxic). The activation of caspase 3/7 and accumulation in the sub-G1 phase of CCRF-CEM cells treated with C\(_{60}\)-Ber nanocomplexes evidenced apoptosis induction. Thus, this study indicates that the fast and easy noncovalent complexation of alkaloid Ber with C\(_{60}\) improved its in vitro efficiency against cancer cells.
C14ORF39/SIX6OS1 is a constituent of the synaptonemal complex and is essential for mouse fertility
(2016)
Meiotic recombination generates crossovers between homologous chromosomes that are essential for genome haploidization. The synaptonemal complex is a ‘zipper’-like protein assembly that synapses homologue pairs together and provides the structural framework for processing recombination sites into crossovers. Humans show individual differences in the number of crossovers generated across the genome. Recently, an anonymous gene variant in C14ORF39/SIX6OS1 was identified that influences the recombination rate in humans. Here we show that C14ORF39/SIX6OS1 encodes a component of the central element of the synaptonemal complex. Yeast two-hybrid analysis reveals that SIX6OS1 interacts with the well-established protein synaptonemal complex central element 1 (SYCE1). Mice lacking SIX6OS1 are defective in chromosome synapsis at meiotic prophase I, which provokes an arrest at the pachytene-like stage and results in infertility. In accordance with its role as a modifier of the human recombination rate, SIX6OS1 is essential for the appropriate processing of intermediate recombination nodules before crossover formation.
Protein-protein interaction (PPI) studies are gaining momentum these days due to the plethora of various high-throughput experimental methods available for detecting PPIs. Proteins create complexes and networks by functioning in harmony with other proteins and here in silico network biology hold the promise to reveal new functionality of genes as it is very difficult and laborious to carry out experimental high-throughput genetic screens in living organisms. We demonstrate this approach by computationally screening C. elegans conserved homologs of already reported human tumor suppressor and aging associated genes. We select by this nhr-6, vab-3 and gst-23 as predicted longevity genes for RNAi screen. The RNAi results demonstrated the pro-longevity effect of these genes. Nuclear hormone receptor nhr-6 RNAi inhibition resulted in a C. elegans phenotype of 23.46% lifespan reduction. Moreover, we show that nhr-6 regulates oxidative stress resistance in worms and does not affect the feeding behavior of worms. These findings imply the potential of nhr-6 as a common therapeutic target for aging and cancer ailments, stressing the power of in silico PPI network analysis coupled with RNAi screens to describe gene function.