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The growing tips of plants grow sterile; therefore, disease-free plants can be generated from them. How plants safeguard growing apices from pathogen infection is still a mystery. The shoot apical meristem (SAM) is one of the three stem cells niches that give rise to the above ground plant organs. This is very well explored; however, how signaling networks orchestrate immune responses against pathogen infections in the SAM remains unclear. To reconstruct a transcriptional framework of the differentially expressed genes (DEGs) pertaining to various SAM cellular populations, we acquired large-scale transcriptome datasets from the public repository Gene Expression Omnibus (GEO). We identify here distinct sets of genes for various SAM cellular populations that are enriched in immune functions, such as immune defense, pathogen infection, biotic stress, and response to salicylic acid and jasmonic acid and their biosynthetic pathways in the SAM. We further linked those immune genes to their respective proteins and identify interactions among them by mapping a transcriptome-guided SAM-interactome. Furthermore, we compared stem-cells regulated transcriptome with innate immune responses in plants showing transcriptional separation among their DEGs in Arabidopsis. Besides unleashing a repertoire of immune-related genes in the SAM, our analysis provides a SAM-interactome that will help the community in designing functional experiments to study the specific defense dynamics of the SAM-cellular populations. Moreover, our study promotes the essence of large-scale omics data re-analysis, allowing a fresh look at the SAM-cellular transcriptome repurposing data-sets for new questions.
Comprehensive bioinformatics identifies key microRNA players in ATG7-deficient lung fibroblasts
(2020)
Background: Deficient autophagy has been recently implicated as a driver of pulmonary fibrosis, yet bioinformatics approaches to study this cellular process are lacking. Autophagy-related 5 and 7 (ATG5/ATG7) are critical elements of macro-autophagy. However, an alternative ATG5/ATG7-independent macro-autophagy pathway was recently discovered, its regulation being unknown. Using a bioinformatics proteome profiling analysis of ATG7-deficient human fibroblasts, we aimed to identify key microRNA (miR) regulators in autophagy. Method: We have generated ATG7-knockout MRC-5 fibroblasts and performed mass spectrometry to generate a large-scale proteomics dataset. We further quantified the interactions between various proteins combining bioinformatics molecular network reconstruction and functional enrichment analysis. The predicted key regulatory miRs were validated via quantitative polymerase chain reaction. Results: The functional enrichment analysis of the 26 deregulated proteins showed decreased cellular trafficking, increased mitophagy and senescence as the major overarching processes in ATG7-deficient lung fibroblasts. The 26 proteins reconstitute a protein interactome of 46 nodes and miR-regulated interactome of 834 nodes. The miR network shows three functional cluster modules around miR-16-5p, miR-17-5p and let-7a-5p related to multiple deregulated proteins. Confirming these results in a biological setting, serially passaged wild-type and autophagy-deficient fibroblasts displayed senescence-dependent expression profiles of miR-16-5p and miR-17-5p. Conclusions: We have developed a bioinformatics proteome profiling approach that successfully identifies biologically relevant miR regulators from a proteomics dataset of the ATG-7-deficient milieu in lung fibroblasts, and thus may be used to elucidate key molecular players in complex fibrotic pathological processes. The approach is not limited to a specific cell-type and disease, thus highlighting its high relevance in proteome and non-coding RNA research.
CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss.
The process of viral integration into the host genome is an essential step of the HIV-1 life cycle. The viral integrase (IN) enzyme catalyzes integration. IN is an ideal therapeutic enzyme targeted by several drugs; raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), and bictegravir (BIC) having been approved by the USA Food and Drug Administration (FDA). Due to high HIV-1 diversity, it is not well understood how specific naturally occurring polymorphisms (NOPs) in IN may affect the structure/function and binding affinity of integrase strand transfer inhibitors (INSTIs). We applied computational methods of molecular modelling and docking to analyze the effect of NOPs on the full-length IN structure and INSTI binding. We identified 13 NOPs within the Cameroonian-derived CRF02_AG IN sequences and further identified 17 NOPs within HIV-1C South African sequences. The NOPs in the IN structures did not show any differences in INSTI binding affinity. However, linear regression analysis revealed a positive correlation between the Ki and EC50 values for DTG and BIC as strong inhibitors of HIV-1 IN subtypes. All INSTIs are clinically effective against diverse HIV-1 strains from INSTI treatment-naïve populations. This study supports the use of second-generation INSTIs such as DTG and BIC as part of first-line combination antiretroviral therapy (cART) regimens, due to a stronger genetic barrier to the emergence of drug resistance.
Insects have evolved an extraordinary range of nutritional adaptations to exploit other animals, plants, bacteria, fungi and soils as resources in terrestrial and aquatic environments. This special issue provides some new insights into the mechanisms underlying these adaptations. Contributions comprise lab and field studies investigating the chemical, physiological, cognitive and behavioral mechanisms that enable resource exploitation and nutrient intake regulation in insects. The collection of papers highlights the need for more studies on the comparative sensory ecology, underlying nutritional quality assessment, cue perception and decision making to fully understand how insects adjust resource selection and exploitation in response to environmental heterogeneity and variability.
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.
West African savannas are severely threatened with intensified land use and increasing degradation. Bees are important for terrestrial biodiversity as they provide native plant species with pollination services. However, little information is available regarding their mutualistic interactions with woody plant species. In the first network study from sub-Saharan West Africa, we investigated the effects of land-use intensity and climatic seasonality on plant–bee communities and their interaction networks. In total, we recorded 5686 interactions between 53 flowering woody plant species and 100 bee species. Bee-species richness and the number of interactions were higher in the low compared to medium and high land-use intensity sites. Bee- and plant-species richness and the number of interactions were higher in the dry compared to the rainy season. Plant–bee visitation networks were not strongly affected by land-use intensity; however, climatic seasonality had a strong effect on network architecture. Null-model corrected connectance and nestedness were higher in the dry compared to the rainy season. In addition, network specialization and null-model corrected modularity were lower in the dry compared to the rainy season. Our results suggest that in our study region, seasonal effects on mutualistic network architecture are more pronounced compared to land-use change effects. Nonetheless, the decrease in bee-species richness and the number of plant–bee interactions with an increase in land-use intensity highlights the importance of savanna conservation for maintaining bee diversity and the concomitant provision of ecosystem services.
Traps baited with attractive lures are increasingly used at entry-points and surrounding natural areas to intercept exotic wood-boring beetles accidentally introduced via international trade. Several trapping variables can affect the efficacy of this activity, including trap color. In this study, we tested whether species richness and abundance of jewel beetles (Buprestidae), bark and ambrosia beetles (Scolytinae), and their common predators (i.e., checkered beetles, Cleridae) can be modified using trap colors different to those currently used for surveillance of jewel beetles and bark and ambrosia beetles (i.e., green or black). We show that green and black traps are generally efficient, but also that many flower-visiting or dark-metallic colored jewel beetles and certain bark beetles are more attracted by other colors. In addition, we show that checkered beetles have color preferences similar to those of their Scolytinae preys, which limits using trap color to minimize their inadvertent removal. Overall, this study confirmed that understanding the color perception mechanisms in wood-boring beetles can lead to important improvements in trapping techniques and thereby increase the efficacy of surveillance programs.
1. Honeybees, which are among the most important pollinators globally, do not only collect pollen and nectar during foraging but may also disperse diverse microbes. Some of these can be deleterious to agricultural crops and forest trees, such as the bacterium Pantoea ananatis, an emerging pathogen in some systems. P. ananatis infections can lead to leaf blotches, die-back, bulb rot, and fruit rot. 2. We isolated P. ananatis bacteria from flowers with the aim of determining whether honeybees can sense these bacteria and if the bacteria affect behavioral responses of the bees to sugar solutions. 3. Honeybees decreased their responsiveness to different sugar solutions when these contained high concentrations of P. ananatis but were not deterred by solutions from which bacteria had been removed. This suggests that their reduced responsiveness was due to the taste of bacteria and not to the depletion of sugar in the solution or bacteria metabolites. Intriguingly, the bees appeared not to taste ecologically relevant low concentrations of bacteria. 4. Synthesis and applications. Our data suggest that honeybees may introduce P.ananatis bacteria into nectar in field-realistic densities during foraging trips and may thus affect nectar quality and plant fitness.
Trait variation in moths mirrors small-scaled ecological gradients in a tropical forest landscape
(2020)
Along environmental gradients, communities are expected to be filtered from the regional species pool by physical constraints, resource availability, and biotic interactions. This should be reflected in species trait composition. Using data on species-rich moth assemblages sampled by light traps in a lowland rainforest landscape in Costa Rica, we show that moths in two unrelated clades (Erebidae-Arctiinae; Geometridae) are much smaller-sized in oil palm plantations than in nearby old-growth forest, with intermediate values at disturbed forest sites. In old-growth forest, Arctiinae predominantly show aposematic coloration as a means of anti-predator defense, whereas this trait is much reduced in the prevalence in plantations. Similarly, participation in Müllerian mimicry rings with Hymenoptera and Lycidae beetles, respectively, is rare in plantations. Across three topographic types of old-growth forests, community-weighted means of moth traits showed little variation, but in creek forest, both types of mimicry were surprisingly rare. Our results emphasize that despite their mobility, moth assemblages are strongly shaped by local environmental conditions through the interplay of bottom–up and top–down processes. Assemblages in oil palm plantations are highly degraded not only in their biodiversity, but also in terms of trait expression.
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.
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.
Adding amino acids to a sucrose diet is not sufficient to support longevity of adult bumble bees
(2020)
Dietary macro-nutrients (i.e., carbohydrates, protein, and fat) are important for bee larval development and, thus, colony health and fitness. To which extent different diets (varying in macro-nutrient composition) affect adult bees and whether they can thrive on nectar as the sole amino acid source has, however, been little investigated. We investigated how diets varying in protein concentration and overall nutrient composition affected consumption, longevity, and breeding behavior of the buff-tailed bumble bee, Bombus terrestris (Hymenoptera: Apidae). Queenless micro-colonies were fed either natural nutrient sources (pollen), nearly pure protein (i.e., the milk protein casein), or sucrose solutions with low and with high essential amino acid content in concentrations as can be found in nectar. We observed micro-colonies for 110 days. We found that longevity was highest for pure pollen and lowest for pure sucrose solution and sucrose solution supplemented with amino acids in concentrations as found in the nectar of several plant species. Adding higher concentrations of amino acids to sucrose solution did only slightly increase longevity compared to sucrose alone. Consequently, sucrose solution with the applied concentrations and proportions of amino acids or other protein sources (e.g., casein) alone did not meet the nutritional needs of healthy adult bumble bees. In fact, longevity was highest and reproduction only successful in micro-colonies fed pollen. These results indicate that, in addition to carbohydrates and protein, adult bumble bees, like larvae, need further nutrients (e.g., lipids and micro-nutrients) for their well-being. An appropriate nutritional composition seemed to be best provided by floral pollen, suggesting that pollen is an essential dietary component not only for larvae but also for adult bees.
Under physiological conditions, protein synthesis controls cell growth and survival and is strictly regulated. Deregulation of protein synthesis is a frequent event in cancer. The majority of mutations found in colorectal cancer (CRC), including alterations in the WNT pathway as well as activation of RAS/MAPK and PI3K/AKT and, subsequently, mTOR signaling, lead to deregulation of the translational machinery. Besides mutations in upstream signaling pathways, deregulation of global protein synthesis occurs through additional mechanisms including altered expression or activity of initiation and elongation factors (e.g., eIF4F, eIF2α/eIF2B, eEF2) as well as upregulation of components involved in ribosome biogenesis and factors that control the adaptation of translation in response to stress (e.g., GCN2). Therefore, influencing mechanisms that control mRNA translation may open a therapeutic window for CRC. Over the last decade, several potential therapeutic strategies targeting these alterations have been investigated and have shown promising results in cell lines, intestinal organoids, and mouse models. Despite these encouraging in vitro results, patients have not clinically benefited from those advances so far. In this review, we outline the mechanisms that lead to deregulated mRNA translation in CRC and highlight recent progress that has been made in developing therapeutic strategies that target these mechanisms for tumor therapy.
Mesenchymal stem cells (MSCs) have considerable therapeutic abilities in various disorders, including hepatic fibrosis. They may be affected with different culture conditions. This study investigated, on molecular basics, the effect of pretreatment with eugenol on the characteristics of adipose tissue-derived MSCs (ASCs) in vitro and the implication of eugenol preconditioning on the in vivo therapeutic abilities of ASCs against CCl\(_4\)-induced hepatic fibrosis in rats. The effect of eugenol on ASCs was assessed using viability, scratch migration and sphere formation assays. Expressions of genes and proteins were estimated by immunofluorescence or qRT-PCR. For the in vivo investigations, rats were divided into four groups: the normal control group, fibrotic (CCl\(_4\)) group, CCl\(_4\)+ASCs group and CCl\(_4\) + eugenol-preconditioned ASCs (CCl\(_4\)+E-ASCs) group. Eugenol affected the viability of ASCs in a concentration- and time-dependent manner. Eugenol improved their self-renewal, proliferation and migration abilities and significantly increased their expression of c-Met, reduced expression 1 (Rex1), octamer-binding transcription factor 4 (Oct4) and nanog genes. Furthermore, E-ASCs showed more of a homing ability than ASCs and improved the serum levels of ALT, AST, albumin, total bilirubin and hyaluronic acid more efficient than ASCs in treating CCl\(_4\)-induced hepatic fibrosis, which was confirmed with histopathology. More interestingly, compared to the CCl\(_4\)+ASCs group, CCl\(_4\)+E-ASCs group showed a lower expression of inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), cluster of differentiation 163 (CD163) and tumor necrosis factor-α (TNF-α) genes and higher expression of matrix metalloproteinase (MMP)-9 and MMP-13 genes. This study, for the first time, revealed that eugenol significantly improved the self-renewal, migration and proliferation characteristics of ASCs, in vitro. In addition, we demonstrated that eugenol-preconditioning significantly enhanced the therapeutic abilities of the injected ASCs against CCl\(_4\)-induced hepatic fibrosis.
Nectar is crucial to maintain plant-pollinator mutualism. Nectar quality (nutritional composition) can vary strongly between individuals of the same plant species. The factors driving such inter-individual variation have however not been investigated closer. We investigated nectar quality of field scabious, Knautia arvensis in different grassland plant communities varying in species composition and richness to assess whether nectar quality can be affected by the surrounding plant community. We analyzed (with high performance liquid chromatography) the content of carbohydrates, overall amino acids, and essential amino acids. Amino acid and carbohydrate concentrations and proportions varied among plant individuals and with the surrounding plant community but were not related to the surrounding plant species richness. Total and individual carbohydrate concentrations were lowest, while proportions of the essential amino acids, valine, isoleucine, leucine (all phagostimulatory), and lysine were highest in plant species communities of the highest diversity. Our results show that K. arvensis nectar chemistry varies with the composition of the surrounding plant community, which may alter the taste and nutritional value and thus affect the plant’s visitor spectrum and visitation rate. However, the strong inter-individual variation in nectar quality requires additional studies (e.g., in semi-field studies) to disentangle different biotic and abiotic factors contributing to inter-individual nectar chemistry in a plant-community context.
Primeval forests in the temperate zone exist only as a few remnants, but theses serve as important reference areas for conservation. As key habitats, tree-related microhabitats (TreMs) are of intense interest to forest ecologists, but little is known about their natural composition and dynamics in different tree species. Beech forms a major part of the temperate forests that extend from Europe, home to European beech Fagus sylvatica L. (Fs), eastward to Iran, where Oriental beech Fagus orientalis Lipsky (Fo) is the dominant species. In this study, we compared TreMs in primeval forests of both species, using data from Fo growing in 25 inventory plots throughout the Hyrcanian forest belt in Iran and from Fs growing in a 9 ha permanent plot in the Uholka Forest of Ukraine. TreMs based on 47 types and 11 subgroups were recorded. Beech trees in the Hyrcanian forest had a higher mean diameter at breast height (dbh) than beech trees in Uholka and contained twice as many TreMs per hectare. Although the mean richness of TreMs per TreM bearing tree was similar in the two species, on the basis of the comparison single trees in two groups (n = 405 vs. 2251), the composition of the TreMs clearly differed, as the proportions of rot holes, root-buttress concavities, and crown deadwood were higher in the Hyrcanian Forest, and those of bark losses, exposed heartwood, and burrs and cankers higher in Uholka Forest. Estimates of TreMs dynamics based on dbh and using Weibull models showed a significantly faster cumulative increase of TreMs in Fo, in which saturation occurred already in trees with a dbh of 70–80 cm. By contrast, the increase in TreMs in Fs was continuous. In both species, the probability density was highest at a dbh of about 30 cm, but was twice as high in Fo. Because of limitations of our study design, the reason behind observed differences of TreM formation and composition between regions remains unclear, as it could be either result of the tree species or the environment, or their interaction. However, the observed differences were more likely the result of differences in the environment than in the two tree species. Nevertheless, our findings demonstrate that the Hyrcanian Forest, recently designated as a natural heritage site in Iran, is unique, not only as a tertiary relict or due to its endemic trees, herbs and arthropods, but also because of its TreMs, which form a distinct and rich habitat for associated taxa, including endemic saproxylic species.
Insect brood parasites have evolved a variety of strategies to avoid being detected by their hosts. Few previous studies on cuckoo wasps (Hymenoptera: Chrysididae), which are natural enemies of solitary wasps and bees, have shown that chemical mimicry, i.e., the biosynthesis of cuticular hydrocarbons (CHC) that match the host profile, evolved in several species. However, mimicry was not detected in all investigated host-parasite pairs. The effect of host range as a second factor that may play a role in evolution of mimicry has been neglected, since all previous studies were carried out on host specialists and at nesting sites where only one host species occurred. Here we studied the cuckoo wasp Parnopes grandior, which attacks many digger wasp species of the genus Bembix (Hymenoptera: Crabronidae). Given its weak host specialization, P. grandior may either locally adapt by increasing mimicry precision to only one of the sympatric hosts or it may evolve chemical insignificance by reducing the CHC profile complexity and/or CHCs amounts. At a study site harbouring three host species, we found evidence for a weak but appreciable chemical deception strategy in P. grandior. Indeed, the CHC profile of P. grandior was more similar to all sympatric Bembix species than to a non-host wasp species belonging to the same tribe as Bembix. Furthermore, P. grandior CHC profile was equally distant to all the hosts' CHC profiles, thus not pointing towards local adaptation of the CHC profile to one of the hosts' profile. We conducted behavioural assays suggesting that such weak mimicry is sufficient to reduce host aggression, even in absence of an insignificance strategy, which was not detected. Hence, we finally concluded that host range may indeed play a role in shaping the level of chemical mimicry in cuckoo wasps.
Symbiotic microbes help a myriad of insects acquire nutrients. Recent work suggests that insects also frequently associate with actinobacterial symbionts that produce molecules to help defend against parasites and predators. Here we explore a potential association between Actinobacteria and two species of fungus-farming ambrosia beetles, Xyleborinus saxesenii and Xyleborus affinis. We isolated and identified actinobacterial and fungal symbionts from laboratory reared nests, and characterized small molecules produced by the putative actinobacterial symbionts. One 16S rRNA phylotype of Streptomyces (XylebKG-1) was abundantly and consistently isolated from the galleries and adults of X. saxesenii and X. affinis nests. In addition to Raffaelea sulphurea, the symbiont that X. saxesenii cultivates, we also repeatedly isolated a strain of Nectria sp. that is an antagonist of this mutualism. Inhibition bioassays between Streptomyces griseus XylebKG-1 and the fungal symbionts from X. saxesenii revealed strong inhibitory activity of the actinobacterium toward the fungal antagonist Nectria sp. but not the fungal mutualist R. sulphurea. Bioassay guided HPLC fractionation of S. griseus XylebKG-1 culture extracts, followed by NMR and mass spectrometry, identified cycloheximide as the compound responsible for the observed growth inhibition. A biosynthetic gene cluster putatively encoding cycloheximide was also identified in S. griseus XylebKG-1. The consistent isolation of a single 16S phylotype of Streptomyces from two species of ambrosia beetles, and our finding that a representative isolate of this phylotype produces cycloheximide, which inhibits a parasite of the system but not the cultivated fungus, suggests that these actinobacteria may play defensive roles within these systems.
Background
The impact of hospital volume after rectal cancer surgery is seldom investigated. This study aimed to analyse the impact of annual rectal cancer surgery cases per hospital on postoperative mortality and failure to rescue.
Methods
All patients diagnosed with rectal cancer and who had a rectal resection procedure code from 2012 to 2015 were identified from nationwide administrative hospital data. Hospitals were grouped into five quintiles according to caseload. The absolute number of patients, postoperative deaths and failure to rescue (defined as in‐hospital mortality after a documented postoperative complication) for severe postoperative complications were determined.
Results
Some 64 349 patients were identified. The overall in‐house mortality rate was 3·9 per cent. The crude in‐hospital mortality rate ranged from 5·3 per cent in very low‐volume hospitals to 2·6 per cent in very high‐volume centres, with a distinct trend between volume categories (P < 0·001). In multivariable logistic regression analysis using hospital volume as random effect, very high‐volume hospitals (53 interventions/year) had a risk‐adjusted odds ratio of 0·58 (95 per cent c.i. 0·47 to 0·73), compared with the baseline in‐house mortality rate in very low‐volume hospitals (6 interventions per year) (P < 0·001). The overall postoperative complication rate was comparable between different volume quintiles, but failure to rescue decreased significantly with increasing caseload (15·6 per cent after pulmonary embolism in the highest volume quintile versus 38 per cent in the lowest quintile; P = 0·010).
Conclusion
Patients who had rectal cancer surgery in high‐volume hospitals showed better outcomes and reduced failure to rescue rates for severe complications than those treated in low‐volume hospitals.
Solitary bees are subject to a variety of pressures that cause severe population declines. Currently, habitat loss, temperature shifts, agrochemical exposure, and new parasites are identified as major threats. However, knowledge about detrimental bacteria is scarce, although they may disturb natural microbiomes, disturb nest environments, or harm the larvae directly. To address this gap, we investigated 12 Osmia bicornis nests with deceased larvae and 31 nests with healthy larvae from the same localities in a 16S ribosomal RNA (rRNA) gene metabarcoding study. We sampled larvae, pollen provisions, and nest material and then contrasted bacterial community composition and diversity in healthy and deceased nests. Microbiomes of pollen provisions and larvae showed similarities for healthy larvae, whilst this was not the case for deceased individuals. We identified three bacterial taxa assigned to Paenibacillus sp. (closely related to P. pabuli/amylolyticus/xylanexedens), Sporosarcina sp., and Bacillus sp. as indicative for bacterial communities of deceased larvae, as well as Lactobacillus for corresponding pollen provisions. Furthermore, we performed a provisioning experiment, where we fed larvae with untreated and sterilized pollens, as well as sterilized pollens inoculated with a Bacillus sp. isolate from a deceased larva. Untreated larval microbiomes were consistent with that of the pollen provided. Sterilized pollen alone did not lead to acute mortality, while no microbiome was recoverable from the larvae. In the inoculation treatment, we observed that larval microbiomes were dominated by the seeded bacterium, which resulted in enhanced mortality. These results support that larval microbiomes are strongly determined by the pollen provisions. Further, they underline the need for further investigation of the impact of detrimental bacterial acquired via pollens and potential buffering by a diverse pollen provision microbiome in solitary bees.
The degree of spatial variation relative to temporal variation influences evolution of dispersal
(2020)
In the face of ongoing global climate and land use change, organisms have multiple possibilities to cope with the modification of their environment. The two main possibilities are to either adapt locally or disperse to a more suitable habitat. The evolution of both local adaptation and dispersal interacts and can be influenced by the spatial and temporal variation (of e.g. temperature or precipitation). In an individual based model (IBM), we explore evolution of phenotypes in landscapes with varying degree of spatial relative to global temporal variation in order to examine its influence on the evolution of dispersal, niche optimum and niche width. The relationship between temporal and spatial variation did neither influence the evolution of local adaptation in the niche optimum nor of niche widths. Dispersal probability is highly influenced by the spatio‐temporal relationship: with increasing spatial variation, dispersal probability decreases. Additionally, the shape of the distribution of the trait values over patch attributes switches from hump‐ to U‐shaped. At low spatial variance more individuals emigrate from average habitats, at high spatial variance more from extreme habitats. The comparatively high dispersal probability in extreme patches of landscapes with a high spatial variation can be explained by evolutionary succession of two kinds of adaptive response. Early in the simulations, extreme patches in landscapes with a high spatial variability act as sink habitats, where population persistence depends on highly dispersive individuals with a wide niche. With ongoing evolution, local adaptation of the remaining individuals takes over, but simultaneously a possible bet‐hedging strategy promotes higher dispersal probabilities in those habitats. Here, in generations that experience extreme shifts from the temporal mean of the patch attribute, the expected fitness becomes higher for dispersing individuals than for philopatric individuals. This means that under certain circumstances, both local adaptation and high dispersal probability can be selected for for coping with the projected environmental changes in the future.
Primary determinants of communities in deadwood vary among taxa but are regionally consistent
(2020)
The evolutionary split between gymnosperms and angiosperms has far‐reaching implications for the current communities colonizing trees. The inherent characteristics of dead wood include its role as a spatially scattered habitat of plant tissue, transient in time. Thus, local assemblages in deadwood forming a food web in a necrobiome should be affected not only by dispersal ability but also by host tree identity, the decay stage and local abiotic conditions. However, experiments simultaneously manipulating these potential community drivers in deadwood are lacking. To disentangle the importance of spatial distance and microclimate, as well as host identity and decay stage as drivers of local assemblages, we conducted two consecutive experiments, a 2‐tree species and 6‐tree species experiment with 80 and 72 tree logs, respectively, located in canopy openings and under closed canopies of a montane and a lowland forest. We sampled saproxylic beetles, spiders, fungi and bacterial assemblages from logs. Variation partitioning for community metrics based on a unified framework of Hill numbers showed consistent results for both studies: host identity was most important for sporocarp‐detected fungal assemblages, decay stage and host tree for DNA‐detected fungal assemblages, microclimate and decay stage for beetles and spiders and decay stage for bacteria. Spatial distance was of minor importance for most taxa but showed the strongest effects for arthropods. The contrasting patterns among the taxa highlight the need for multi‐taxon analyses in identifying the importance of abiotic and biotic drivers of community composition. Moreover, the consistent finding of microclimate as the primary driver for saproxylic beetles compared to host identity shows, for the first time that existing evolutionary host adaptions can be outcompeted by local climate conditions in deadwood.
Global sustainability agendas focus primarily on halting deforestation, yet the biodiversity crisis resulting from the degradation of remaining forests is going largely unnoticed. Forest degradation occurs through the loss of key ecological structures, such as dying trees and deadwood, even in the absence of deforestation. One of the main drivers of forest degradation is limited awareness by policy makers and the public on the importance of these structures for supporting forest biodiversity and ecosystem function. Here, we outline management strategies to protect forest health and biodiversity by maintaining and promoting deadwood, and propose environmental education initiatives to improve the general awareness of the importance of deadwood. Finally, we call for major reforms to forest management to maintain and restore deadwood; large, old trees; and other key ecological structures.
Increasing natural pest control in agricultural fields is an important aim of ecological intensification. Combined effects of landscape context and local placement of agri‐environmental schemes (AES) on natural pest control and within‐field distance functions of natural pest control agents have rarely been addressed but might affect the distribution of biocontrol providers. Importantly, it is currently unknown whether ecosystem services provided by adjacent AES are consistent for different crop types during crop rotation.
In this study, we assessed whether crop rotation from oilseed rape to cereals altered within‐field distance functions of ground‐dwelling predators from adjacent agri‐environmental fields along a gradient in landscape context. Additionally, we recorded crop pests, predation rates, parasitoids as well as crop yields on a total of 30 study sites.
Distance functions varied between trophic levels: Carabid richness decreased while densities of carabid beetles, staphylinid beetles as well as crop yields increased towards the field centres. Distance functions of parasitoids and pests were modulated by the amount of semi‐natural habitat in the surrounding landscape, while the effects of adjacent AES were limited.
Distance decay functions found for ground‐dwelling predators in oilseed rape in the previous year were not always present in cereals. Increasing distance to the field edge also increased effects of crop rotation on carabid beetle assemblages, indicating a source habitat function of field edges.
Synthesis and applications. Distance functions of natural pest control are not universal and the effects of agri‐environmental schemes (AES) in different adjacent crops during crop rotation vary and depend on ecological contrasts. A network of semi‐natural habitats and spatially optimized AES habitats can benefit pest control in agricultural landscapes, but constraints as a result of crop type need to be addressed by annually targeted, spatially shifting agri‐environment schemes for different crops.
Cataglyphis ants are known for their outstanding navigational abilities. They return to their inconspicuous nest after far‐reaching foraging trips using path integration, and whenever available, learn and memorize visual features of panoramic sceneries. To achieve this, the ants combine directional visual information from celestial cues and panoramic scenes with distance information from an intrinsic odometer. The largely vision‐based navigation in Cataglyphis requires sophisticated neuronal networks to process the broad repertoire of visual stimuli. Although Cataglyphis ants have been subjected to many neuroethological studies, little is known about the general neuronal organization of their central brain and the visual pathways beyond major circuits. Here, we provide a comprehensive, three‐dimensional neuronal map of synapse‐rich neuropils in the brain of Cataglyphis nodus including major connecting fiber systems. In addition, we examined neuronal tracts underlying the processing of visual information in more detail. This study revealed a total of 33 brain neuropils and 30 neuronal fiber tracts including six distinct tracts between the optic lobes and the cerebrum. We also discuss the importance of comparative studies on insect brain architecture for a profound understanding of neuronal networks and their function.
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.
Land‐use intensification leads to loss and degradation of habitats and is thus a major driver of biodiversity loss. Restoration strategies typically focus on promoting biodiversity but often neglect that land‐use intensification could have changed the underlying mechanisms of community assembly. Since assembly mechanisms determine the diversity and composition of communities, we propose that evaluation of restoration strategies should consider effects of restoration on biodiversity and community assembly. Using a multi‐taxon approach, we tested whether a strategy that promotes forest biodiversity by restoring deadwood habitats also affects assembly patterns.
We assessed saproxylic (i.e. deadwood‐dependent) beetles and fungi, as well as non‐saproxylic plants and birds in 68 beech forest plots in southern Germany, 8 years after the commencement of a restoration project. To assess changes in community assembly, we analysed the patterns of functional–phylogenetic diversity, community‐weighted mean (CWM) traits and their diversity. We hypothesized that restoration increases habitat amount and heterogeneity of deadwood and reduces canopy cover and thereby decreases the strength of environmental filters imposed by past silvicultural intensification, such as a low amount in deadwood.
With the restoration of deadwood habitats, saproxylic beetle communities became less functionally–phylogenetically similar, whereas the assembly patterns of saproxylic fungi and non‐saproxylic taxa remained unaffected by deadwood restoration. Among the traits analysed, deadwood diameter niche position of species was most strongly affected indicating that the enrichment of large deadwood objects led to lower functional–phylogenetical similarity of saproxylic beetles. Community assembly and traits of plants were mainly influenced by microclimate associated with changes in canopy cover.
Synthesis and applications. Our results indicate that the positive effects of deadwood restoration on saproxylic beetle richness are associated with an increase in deadwood amount. This might be linked to an increase in deadwood heterogeneity, and therefore decreasing management‐induced environmental filters. Deadwood enrichment can thus be considered an effective restoration strategy which reduces the negative effects of intense forest management on saproxylic taxa by not only promoting biodiversity but also by decreasing the environmental filters shaping saproxylic beetle communities, thus allowing the possibly for more interactions between species and a higher functional diversity.
DNA metabarcoding was utilized for a large‐scale, multiyear assessment of biodiversity in Malaise trap collections from the Bavarian Forest National Park (Germany, Bavaria). Principal component analysis of read count‐based biodiversities revealed clustering in concordance with whether collection sites were located inside or outside of the National Park. Jaccard distance matrices of the presences of barcode index numbers (BINs) at collection sites in the two survey years (2016 and 2018) were significantly correlated. Overall similar patterns in the presence of total arthropod BINs, as well as BINs belonging to four major arthropod orders across the study area, were observed in both survey years, and are also comparable with results of a previous study based on DNA barcoding of Sanger‐sequenced specimens. A custom reference sequence library was assembled from publicly available data to screen for pest or invasive arthropods among the specimens or from the preservative ethanol. A single 98.6% match to the invasive bark beetle Ips duplicatus was detected in an ethanol sample. This species has not previously been detected in the National Park.
Salvage logging, or logging after natural disturbances such as wildfires, insect outbreaks, and windstorms, is carried out to recover some of a forest's natural and/or economic capital. However, trade‐offs between management objectives and a lack of consensus on the ecological consequences of salvage logging impair science‐based decision making on the management of forests after natural disturbances. We conducted a global meta‐analysis of the impacts of salvage logging on regulating ecosystem services and on fuel loads, as a frequent post‐disturbance objective is preventing subsequent wildfires that could be fueled by the accumulation of dead trunks and branches. Salvage logging affected ecosystem services in a moderately negative way, regardless of disturbance type and severity, time elapsed since salvage logging, intensity of salvage logging, and the group of regulating ecosystem services being considered. However, prolonging the time between natural disturbance and salvage logging mitigated negative effects on regulating ecosystem services. Salvage logging had no overall effect on surface fuels; rather, different fuel types responded differently depending on the time elapsed since salvage logging. Delaying salvage logging by ~2–4 years may reduce negative ecological impacts without affecting surface fuel loads.
Plants may use different strategies to attract pollinators in long distance (e.g. floral display) and in short distance (e.g. ratio between differentially colored flowers) scales. The Verbenaceae Lantana canescens Kunth is a wide spread species in open sites of the Brazilian Pantanal wetland. Individuals of this generalist species can produce a variable number of open inflorescences with yellow and white flowers that are organized in whorls. In this study we tested the hypothesis that increased floral display (long distance attraction) and the ratio between yellow and white flowers (short distance attraction) enhances the number of pollinator species and individuals. We observed flower visitors and calculated floral parameters in 38 plots of 1 m2 each, that contained a varying number of flowering L. canescens individuals. Non-metric multidimensional scaling and Bray-Curtis distances were used to account for flower visitor composition and the relative visitation rate, respectively. We used a structural equation model to test the power of each predictor variable on the visitation rate and a covariance analysis to disentangle the effect of each independent variable on the frequency of plant-pollinator interactions. We found that the number of flower visitors and the visitation rate increased with increasing number of inflorescences. Disentangling long and short distance attraction indicated that the number of inflorescences (per plot) and the number of yellow flowers (yellowing effect) contributed most to flower visitation at long and short distance, respectively.
The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H\(_2\)O\(_2\) or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.
Primates often consume either bark or cambium (inner bark) as a fallback food tocomplete their diet during periods of food scarcity. Wild chimpanzees exhibit greatbehavioral diversity across Africa, as studies of new populations frequently reveal.Since 2014, we have been using a combination of camera traps and indirect signs tostudy the ecology and behavior of wild chimpanzees (Pan troglodytes verus) in ComoéNational Park, Ivory Coast, to document and understand the behavioral adaptations thathelp them to survive in a savanna–forest mosaic landscape. We found that Comoéchimpanzees peel the bark of the buttresses of kapok tree (Ceiba pentandra) trees to eatthe cambium underneath. Individuals of all sex/age classes across at least six neigh-boring communities peeled the bark, but only during the late rainy season andbeginning of the dry season, when cambium may represent an important fallback food.Baboons (Papio anubis) also target the same trees but mainly eat the bark itself. Mostof the bark-peeling wounds onCeibatrees healed completely within 2 years, seeminglywithout any permanent damage. We recorded chimpanzees visiting trees in early stagesof wound recovery but leaving them unpeeled. Only 6% of peeled trees (N= 53) werereexploited after a year, suggesting that chimpanzees waited for the rest of the trees toregrow the bark fully before peeling them again, thus using them sustainably. Manyhuman groups of hunter-gatherers and herders exploited cambium sustainably in thepast. The observation that similar sustainable bark-peeling behavior evolved in bothchimpanzees and humans suggests that it has an important adaptive value in harshenvironments when other food sources become seasonally scarce, by avoiding thedepletion of the resource and keeping it available for periods of scarcity.
Despite decades of scientific effort, there is still no consensus on the determinants of broad-scale gradients of animal diver-sity. We argue that general drivers of diversity are unlikely to be found among the narrowly defined taxa which are typically analyzed in studies of broad-scale diversity gradients because ecological niches evolve largely conservatively. This causes constraints in the use of available niche space leading to systematic differences in diversity gradients among taxa. We instead advocate studies of phylogenetically diverse animal communities along broad environmental gradients. Such multi-taxa communities are less constrained in resource use and diversification and may be better targets for testing major classical hypotheses on diversity gradients. Besides increasing the spatial scale in analyses, expanding the phylogenetic coverage may be a second way to achieve higher levels of generality in studies of broad-scale diversity gradients
Honeybees are very sensitive to their breeding temperature. Even slightly lower temperatures during larval development can significantly affect adult behavior. Several devices which are employed for killing the honeybee ectoparasite Varroa destructor rely on short-term hyperthermia in the honeybee hive. The device used here applies 43.7 °C for 2 h, which is highly effective in killing the mites. We study how short-term hyperthermia affects worker brood and behavior of emerging adult bees. Sucrose responsiveness was strongly reduced after treatment of larvae early or late of larval development. Hyperthermia significantly enhanced life span, particularly in bees receiving treated early in larval development. To ask whether increased life span correlated with foraging performance, we used radio frequency identification (RFID). Onset and offset of foraging behavior as well as foraging trip duration and lifetime foraging effort were unaffected by hyperthermia treatment as prepupa.
The fruit fly Drosophila is a prime model in circadian research, but still little is known about its circadian regulation of metabolism. Daily rhythmicity in levels of several metabolites has been found, but knowledge about hydrophobic metabolites is limited. We here compared metabolite levels including lipids between period\(^{01}\) (per\(^{01}\)) clock mutants and Canton-S wildtype (WT\(_{CS}\)) flies in an isogenic and non-isogenic background using LC–MS. In the non-isogenic background, metabo-lites with differing levels comprised essential amino acids, kynurenines, pterinates, glycero(phospho)lipids, and fatty acid esters. Notably, detectable diacylglycerols (DAG) and acylcarnitines (AC), involved in lipid metabolism, showed lower levels in per\(^{01}\) mutants. Most of these differences disappeared in the isogenic background, yet the level differences for AC as well as DAG were consistent for fly bodies. AC levels were dependent on the time of day in WTCS in phase with food consumption under LD conditions, while DAGs showed weak daily oscillations. Two short-chain ACs continued to cycle even in constant darkness. per\(^{01}\) mutants in LD showed no or very weak diel AC oscillations out of phase with feeding activity. The low levels of DAGs and ACs in per\(^{01}\) did not correlate with lower total food consumption, body mass or weight. Clock mutant flies showed higher sensitivity to starvation independent of their background-dependent activity level. Our results suggest that neither feeding, energy storage nor mobilisation is significantly affected in per\(^{01}\) mutants, but point towards impaired mitochondrial activity, supported by upregulation of the mitochondrial stress marker 4EBP in the clock mutants
Biological systems are dynamic and three-dimensional but many techniques allow only static and two-dimensional observation of cells. We used three-dimensional (3D) lattice light-sheet single-molecule localization microscopy (dSTORM) to investigate the complex interactions and distribution of single molecules in the plasma membrane of whole cells. Different receptor densities of the adhesion receptor CD56 at different parts of the cell highlight the importance and need of three-dimensional observation and analysis techniques.
Background
The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), has escalated rapidly to a global pandemic stretching healthcare systems worldwide to their limits. Surgeonshave had to immediately react to this unprecedented clinical challenge by systematically repurposing surgical wards.
Purpose
To provide a detailed set of guidelines developed in a surgical ward at University Hospital Wuerzburg to safelyaccommodate the exponentially rising cases of SARS-CoV-2 infected patients without compromising the care of emergencysurgery and oncological patients or jeopardizing the well-being of hospital staff.
Conclusions
The dynamic prioritization of SARS-CoV-2 infected and surgical patient groups is key to preserving life whilemaintaining high surgical standards. Strictly segregating patient groups in emergency rooms, non-intensive care wards andoperating areas prevents viral spread while adequately training and carefully selecting hospital staff allow them to confidentlyand successfully undertake their respective clinical duties.
Aim: Despite increasing interest in β-diversity, that is the spatial and temporal turnover of species, the mechanisms underlying species turnover at different spatial scales are not fully understood, although they likely differ among different functional groups. We investigated the relative importance of dispersal limitations and the environmental filtering caused by vegetation for local, multi-taxa forest communities differing in their dispersal ability, trophic position and body size.
Location: Temperate forests in five regions across Germany.
Methods: In the inter-region analysis, the independent and shared effects of the regional spatial structure (regional species pool), landscape spatial structure (dispersal limitation) and environmental factors on species turnover were quantified with a 1-ha grain across 11 functional groups in up to 495 plots by variation partitioning. In the intra-region analysis, the relative importance of three environmental factors related to vegetation (herb and tree layer composition and forest physiognomy) and spatial structure for species turnover was determined.
Results: In the inter-region analysis, over half of the explained variation in community composition (23% of the total explained 35%) was explained by the shared effects of several factors, indicative of spatially structured environmental filtering. Among the independent effects, environmental factors were the strongest on average over 11 groups, but the importance of landscape spatial structure increased for less dispersive functional groups. In the intra-region analysis, the independent effect of plant species composition had a stronger influence on species turnover than forest physiognomy, but the relative importance of the latter increased with increasing trophic position and body size.
Main conclusions: Our study revealed that the mechanisms structuring assemblage composition are associated with the traits of functional groups. Hence, conservation frameworks targeting biodiversity of multiple groups should cover both environmental and biogeographical gradients. Within regions, forest management can enhance β-diversity particularly by diversifying tree species composition and forest physiognomy.
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.
Head and neck squamous cell carcinoma (HNSCC) is known to overexpress a variety of receptor tyrosine kinases, such as the HGF receptor Met. Like other malignancies, HNSCC involves a mutual interaction between the tumor cells and surrounding tissues and cells. We hypothesized that activation of HGF/Met signaling in HNSCC influences glucose metabolism and therefore substantially changes the tumor microenvironment. To determine the effect of HGF, we submitted three established HNSCC cell lines to mRNA sequencing. Dynamic changes in glucose metabolism were measured in real time by an extracellular flux analyzer. As expected, the cell lines exhibited different levels of Met and responded differently to HGF stimulation. As confirmed by mRNA sequencing, the level of Met expression was associated with the number of upregulated HGF-dependent genes. Overall, Met stimulation by HGF leads to increased glycolysis, presumably mediated by higher expression of three key enzymes of glycolysis. These effects appear to be stronger in Met\(^{high}\)-expressing HNSCC cells. Collectively, our data support the hypothesized role of HGF/Met signaling in metabolic reprogramming of HNSCC.
Specialization of plant-pollinator interactions increases with temperature at Mt. Kilimanjaro
(2020)
Aim: Species differ in their degree of specialization when interacting with other species, with significant consequences for the function and robustness of ecosystems. In order to better estimate such consequences, we need to improve our understanding of the spatial patterns and drivers of specialization in interaction networks.
Methods: Here, we used the extensive environmental gradient of Mt. Kilimanjaro (Tanzania, East Africa) to study patterns and drivers of specialization, and robustness of plant–pollinator interactions against simulated species extinction with standardized sampling methods. We studied specialization, network robustness and other network indices of 67 quantitative plant–pollinator networks consisting of 268 observational hours and 4,380 plant–pollinator interactions along a 3.4 km elevational gradient. Using path analysis, we tested whether resource availability, pollinator richness, visitation rates, temperature, and/or area explain average specialization in pollinator communities. We further linked pollinator specialization to different pollinator taxa, and species traits, that is, proboscis length, body size, and species elevational ranges.
Results: We found that specialization decreased with increasing elevation at different levels of biological organization. Among all variables, mean annual temperature was the best predictor of average specialization in pollinator communities. Specialization differed between pollinator taxa, but was not related to pollinator traits. Network robustness against simulated species extinctions of both plants and pollinators was lowest in the most specialized interaction networks, that is, in the lowlands.
Conclusions: Our study uncovers patterns in plant–pollinator specialization along elevational gradients. Mean annual temperature was closely linked to pollinator specialization. Energetic constraints, caused by short activity timeframes in cold highlands, may force ectothermic species to broaden their dietary spectrum. Alternatively or in addition, accelerated evolutionary rates might facilitate the establishment of specialization under warm climates. Despite the mechanisms behind the patterns have yet to be fully resolved, our data suggest that temperature shifts in the course of climate change may destabilize pollination networks by affecting network architecture.
Neutral sphingomyelinase-2 (NSM2) is a member of a superfamily of enzymes responsible for conversion of sphingomyelin into phosphocholine and ceramide at the cytosolic leaflet of the plasma membrane. Upon specific ablation of NSM2, T cells proved to be hyper-responsive to CD3/CD28 co-stimulation, indicating that the enzyme acts to dampen early overshooting activation of these cells. It remained unclear whether hyper-reactivity of NSM2-deficient T cells is supported by a deregulated metabolic activity in these cells. Here, we demonstrate that ablation of NSM2 activity affects metabolism of the quiescent CD4\(^+\) T cells which accumulate ATP in mitochondria and increase basal glycolytic activity. This supports enhanced production of total ATP and metabolic switch early after TCR/CD28 stimulation. Most interestingly, increased metabolic activity in resting NSM2-deficient T cells does not support sustained response upon stimulation. While elevated under steady-state conditions in NSM2-deficient CD4\(^+\) T cells, the mTORC1 pathway regulating mitochondria size, oxidative phosphorylation, and ATP production is impaired after 24 h of stimulation. Taken together, the absence of NSM2 promotes a hyperactive metabolic state in unstimulated CD4\(^+\) T cells yet fails to support sustained T cell responses upon antigenic stimulation.
Folliculin Controls the Intracellular Survival and Trans-Epithelial Passage of Neisseria gonorrhoeae
(2020)
Neisseria gonorrhoeae, a Gram-negative obligate human pathogenic bacterium, infects human epithelial cells and causes sexually transmitted diseases. Emerging multi-antibiotic resistant gonococci and increasing numbers of infections complicate the treatment of infected patients. Here, we used an shRNA library screen and next-generation sequencing to identify factors involved in epithelial cell infection. Folliculin (FLCN), a 64 kDa protein with a tumor repressor function was identified as a novel host factor important for N. gonorrhoeae survival after uptake. We further determined that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for its survival in the cells by modulating autophagy. In addition, FLCN was also required to maintain cell to cell contacts in the epithelial layer. In an infection model with polarized cells, FLCN inhibited the polarized localization of E-cadherin and the transcytosis of gonococci across polarized epithelial cells. In conclusion, we demonstrate here the connection between FLCN and bacterial infection and in particular the role of FLCN in the intracellular survival and transcytosis of gonococci across polarized epithelial cell layers.
Comparison of the central human and mouse platelet signaling cascade by systems biological analysis
(2020)
Background
Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC).
Results
The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12.
Conclusions
Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.
Objective
The biological interpretation of gene expression measurements is a challenging task. While ordination methods are routinely used to identify clusters of samples or co-expressed genes, these methods do not take sample or gene annotations into account. We aim to provide a tool that allows users of all backgrounds to assess and visualize the intrinsic correlation structure of complex annotated gene expression data and discover the covariates that jointly affect expression patterns.
Results
The Bioconductor package covRNA provides a convenient and fast interface for testing and visualizing complex relationships between sample and gene covariates mediated by gene expression data in an entirely unsupervised setting. The relationships between sample and gene covariates are tested by statistical permutation tests and visualized by ordination. The methods are inspired by the fourthcorner and RLQ analyses used in ecological research for the analysis of species abundance data, that we modified to make them suitable for the distributional characteristics of both, RNA-Seq read counts and microarray intensities, and to provide a high-performance parallelized implementation for the analysis of large-scale gene expression data on multi-core computational systems. CovRNA provides additional modules for unsupervised gene filtering and plotting functions to ensure a smooth and coherent analysis workflow.
The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca\(^{2+}\) increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca\(^{2+}\) concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca\(^{2+}\) rise led to an increase in mitochondrial Ca\(^{2+}\) concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca\(^{2+}\) homeostasis and induces cytoplasmic Ca\(^{2+}\) overload, which results in both apoptotic and necrotic cell death in parallel or succession.
IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca\(^{2+}\) overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca\(^{2+}\) homeostasis."
Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions.
MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.
Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4x to 10x expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10-20nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 +/- 7.7nm. Imaging of lipid bilayers using light microscopy is challenging. Here the authors label cells using a short chain click-compatible ceramide to visualize mammalian and bacterial membranes with expansion microscopy.
The amyotrophic lateral sclerosis (ALS) neurodegenerative disorder has been associated with multiple genetic lesions, including mutations in the gene for fused in sarcoma (FUS), a nuclear-localized RNA/DNA-binding protein. Neuronal expression of the pathological form of FUS proteins in Caenorhabditis elegans results in mislocalization and aggregation of FUS in the cytoplasm, and leads to impairment of motility. However, the mechanisms by which the mutant FUS disrupts neuronal health and function remain unclear. Here we investigated the impact of ALS-associated FUS on motor neuron health using correlative light and electron microscopy, electron tomography, and electrophysiology. We show that ectopic expression of wild-type or ALS-associated human FUS impairs synaptic vesicle docking at neuromuscular junctions. ALS-associated FUS led to the emergence of a population of large, electron-dense, and filament-filled endosomes. Electrophysiological recording revealed reduced transmission from motor neurons to muscles. Together, these results suggest a pathological effect of ALS-causing FUS at synaptic structure and function organization.
The enrichment of deadwood is essential for the conservation of saproxylic biodiversity in managed forests. However, existing strategies focus on a cost‐intensive increase of deadwood amount, while largely neglecting increasing deadwood diversity.
Deadwood objects, that is logs and branches, from six tree species were experimentally sun exposed, canopy shaded and artificially shaded for 4 years, after which the alpha‐, beta‐ and gamma‐diversity of saproxylic beetles, wood‐inhabiting fungi and spiders were analysed. Analyses of beta‐diversity included the spatial distance between exposed deadwood objects. A random‐drawing procedure was used to identify the combination of tree species and sun exposure that yielded the highest gamma‐diversity at a minimum of exposed deadwood amount.
In sun‐exposed plots, species numbers in logs were higher than in shaded plots for all taxa, while in branches we observed the opposite for saproxylic beetles. Tree species affected the species numbers only of saproxylic beetles and wood‐inhabiting fungi. The beta‐diversity of saproxylic beetles and wood‐inhabiting fungi among logs was influenced by sun exposure and tree species, but beta‐diversity of spiders by sun exposure only. For all saproxylic taxa recorded in logs, differences between communities increased with increasing spatial distance.
A combination of canopy‐shaded Carpinus logs and sun‐exposed Populus logs resulted in the highest species numbers of all investigated saproxylic taxa among all possible combinations of tree species and sun‐exposure treatments.
Synthesis and applications. We recommend incorporating the enrichment of different tree species and particularly the variation in sun exposure into existing strategies of deadwood enrichment. Based on the results of our study, we suggest to combine the logs of softwood broadleaf tree species (e.g. Carpinus, Populus), hardwood broadleaf tree species (e.g. Quercus) and coniferous tree species (e.g. Pinus) under different conditions of sun exposure and distribute them spatially in a landscape to maximize the beneficial effects on overall diversity.
Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to off-spring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.
Tropical mountain forests contribute disproportionately to terrestrial biodiversity but little is known about insect diversity in the canopy and how it is distributed between tree species. We sampled tree-specific arthropod communities from 28 trees by canopy fogging and analysed beetle communities which were first morphotyped and then identified by their DNA barcodes. Our results show that communities from forests at 1100 and 1700 m a.s.l. are almost completely distinct. Diversity was much lower in the upper forest while community structure changed from many rare, less abundant species to communities with a pronounced dominance structure. We also found significantly higher beta-diversity between trees at the lower than higher elevation forest where community similarity was high. Comparisons on tree species found at both elevations reinforced these results. There was little species overlap between sites indicating limited elevational ranges. Furthermore, we exploited the advantage of DNA barcodes to patterns of haplotype diversity in some of the commoner species. Our results support the advantage of fogging and DNA barcodes for community studies and underline the need for comprehensive research aimed at the preservation of these last remaining pristine forests.
Background
Valid indicators are required to measure surgical quality. These ideally should be sensitive and selective while being easy to understand and adjust. We propose here the MTL30 quality indicator which takes into account 30-day mortality, transfer within 30 days, and a length of stay of 30 days as composite markers of an uneventful operative/postoperative course.
Methods
Patients documented in the StuDoQ|Colon and StuDoQ|Rectal carcinoma register of the German Society for General and Visceral Surgery (DGAV) were analyzed with regard to the effects of patient and tumor-related risk factors as well as postoperative complications on the MTL30.
Results
In univariate analysis, the MTL30 correlated significantly with patient and tumor-related risk factors such as ASA score (p<0.001), age (p<0.001), or UICC stage (p<0.001). There was a high sensitivity for the postoperative occurrence of complications such as re-operations (p<0.001) or subsequent bleeding (p<0.001), as well as a significant correlation with the CDC classification (p<0.001). In multivariate analysis, patient-related risk factors and postoperative complications significantly increased the odds ratio for a positive MTL30. A negative MTL30 showed a high specify for an uneventful operative and postoperative course.
Conclusion
The MTL30 is a valid indicator of colorectal surgical quality.
Forests are increasingly affected by natural disturbances. Subsequent salvage logging, a widespread management practice conducted predominantly to recover economic capital, produces further disturbance and impacts biodiversity worldwide. Hence, naturally disturbed forests are among the most threatened habitats in the world, with consequences for their associated biodiversity. However, there are no evidence-based benchmarks for the proportion of area of naturally disturbed forests to be excluded from salvage logging to conserve biodiversity. We apply a mixed rarefaction/extrapolation approach to a global multi-taxa dataset from disturbed forests, including birds, plants, insects and fungi, to close this gap. We find that 757% (mean +/- SD) of a naturally disturbed area of a forest needs to be left unlogged to maintain 90% richness of its unique species, whereas retaining 50% of a naturally disturbed forest unlogged maintains 73 +/- 12% of its unique species richness. These values do not change with the time elapsed since disturbance but vary considerably among taxonomic groups. Salvage logging has become a common practice to gain economic returns from naturally disturbed forests, but it could have considerable negative effects on biodiversity. Here the authors use a recently developed statistical method to estimate that ca. 75% of the naturally disturbed forest should be left unlogged to maintain 90% of the species unique to the area.
Staphylococcus aureus is a common cause of bacteremia that can lead to severe complications once the bacteria exit the bloodstream and establish infection in secondary organs. Despite its clinical relevance, little is known about the bacterial factors facilitating the development of these metastatic infections. Here, we used an S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) to identify genes that are critical for efficient bacterial colonization of secondary organs in a murine model of metastatic bloodstream infection. Our transposon screen identified a LysR-type transcriptional regulator (LTTR), which was required for efficient colonization of secondary organs such as the kidneys in infected mice. The critical role of LTTR in secondary organ colonization was confirmed using an isogenic mutant deficient in the expression of LTTR. To identify the set of genes controlled by LTTR, we used an S. aureus strain carrying the LTTR gene in an inducible expression plasmid. Gene expression analysis upon induction of LTTR showed increased transcription of genes involved in branched-chain amino acid biosynthesis, a methionine sulfoxide reductase, and a copper transporter as well as decreased transcription of genes coding for urease and components of pyrimidine nucleotides. Furthermore, we show that transcription of LTTR is repressed by glucose, is induced under microaerobic conditions, and required trace amounts of copper ions. Our data thus pinpoints LTTR as an important element that enables a rapid adaptation of S. aureus to the changing host microenvironment.
IMPORTANCE Staphylococcus aureus is an important pathogen that can disseminate via the bloodstream and establish metastatic infections in distant organs. To achieve a better understanding of the bacterial factors facilitating the development of these metastatic infections, we used in this study a Staphylococcus aureus transposon mutant library in a murine model of intravenous infection, where bacteria first colonize the liver as the primary infection site and subsequently progress to secondary sites such as the kidney and bones. We identified a novel LysR-type transcriptional regulator (LTTR), which was specifically required by S. aureus for efficient colonization of secondary organs. We also determined the transcriptional activation as well as the regulon of LTTR, which suggests that this regulator is involved in the metabolic adaptation of S. aureus to the host microenvironment found in secondary infection sites.
Ants build complex nest structures by reacting to simple, local stimuli. While underground nests result from the space generated by digging, some leaf- and grass-cutting ants also construct conspicuous aboveground turrets around nest openings. We investigated whether the selection of specific building materials occurs during turret construction in Acromyrmex fracticornis grass-cutting ants, and asked whether single building decisions at the beginning can modify the final turret architecture. To quantify workers' material selection, the original nest turret was removed and a choice between two artificial building materials, thin and thick sticks, was offered for rebuilding. Workers preferred thick sticks at the very beginning of turret construction, showed varying preferences thereafter, and changed to prefer thin sticks for the upper, final part of the turret, indicating that they selected different building materials over time to create a stable structure. The impact of a single building choice on turret architecture was evaluated by placing artificial beams that divided a colony's nest entrance at the beginning of turret rebuilding. Splitting the nest entrance led to the self-organized construction of turrets with branched galleries ending in multiple openings, showing that the spatial location of a single building material can strongly influence turret morphology.
The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution. Here, using computational integration of multi-omics data, the authors provide a detailed transcriptome and translatome of herpes simplex virus 1 (HSV-1), including previously unidentified ORFs and N-terminal extensions. The study also provides a HSV-1 genome browser and should be a valuable resource for further research.
Lentic freshwater organisms are influenced by a multitude of factors, including geomorphology, hydrology, anthropogenic impacts and climate change. Organisms that depend on patchy resources such as water beetles may also be sensitive to anthropogenic habitat degradation, like pollution, eutrophication, water level or management alteration.
To assess composition and ecological trends in the water beetle communities of Central Europe, we sampled water beetles (Dytiscidae, Haliplidae, Noteridae) in 33 water bodies in Southern Germany from 1991 to 2018. We used manual, time‐standardised capture during three periods: between 1991 and 1995, 2007 and 2008, and 2017 and 2018.
During the 28‐year survey period, we captured a total of 81 species. We found annual declines in both species number (ca −1%) and abundance (ca −2%). Also, community composition showed significant changes over time. The significant impact of pH on the community composition suggests that the recorded changes through time partly reflect natural succession processes. However, a pronounced decline of beetle species belonging to the moor‐related beetle associations indicated that Central European water beetles are also threatened by non‐successional factors, including desiccation, increased nitrogen input and/or mineralisation, and the loss of specific habitats. This trend to physiographical homogenisation resulted in corresponding community composition shifts.
To effectively protect endangered species, conservation strategies need to be aimed at regularly creating new water bodies with mineralic bottom substratum, and maintenance of moor water bodies that represent late successional stages.
Salvage logging following natural disturbances may alter the natural successional trajectories of biological communities by affecting the occurrences of species, functional groups and evolutionary lineages. However, few studies have examined whether dissimilarities between bird communities of salvaged and unsalvaged forests are more pronounced for rare species, functional groups and evolutionary lineages than for their more common counterparts.
We compiled data on breeding bird assemblages from nine study areas in North America, Europe and Asia, covering a 17‐year period following wildfire or windstorm disturbances and subsequent salvage logging. We tested whether dissimilarities based on non‐shared species, functional groups and evolutionary lineages (a) decreased or increased over time and (b) the responses of rare, common and dominant species varied, by using a unified statistical framework based on Hill numbers and null models.
We found that dissimilarities between bird communities caused by salvage logging persisted over time for rare, common and dominant species, evolutionary lineages and for rare functional groups. Dissimilarities of common and dominant functional groups increased 14 years post disturbance.
Salvage logging led to significantly larger dissimilarities than expected by chance. Functional dissimilarities between salvaged and unsalvaged sites were lower compared to taxonomic and phylogenetic dissimilarities. In general, dissimilarities were highest for rare, followed by common and dominant species.
Synthesis and applications. Our research demonstrates that salvage logging did not decrease dissimilarities of bird communities over time and taxonomic, functional and phylogenetic dissimilarities persisted for over a decade. We recommend resource managers and decision makers to reserve portions of disturbed forest to enable unmanaged post‐disturbance succession of bird communities, particularly to conserve rare species found in unsalvaged disturbed forests.
Apart from some model organisms, the interactome of most organisms is largely unidentified. High-throughput experimental techniques to determine protein-protein interactions (PPIs) are resource intensive and highly susceptible to noise. Computational methods of PPI determination can accelerate biological discovery by identifying the most promising interacting pairs of proteins and by assessing the reliability of identified PPIs. Here we present a first in-depth study describing a global view of the ant Camponotus floridanus interactome. Although several ant genomes have been sequenced in the last eight years, studies exploring and investigating PPIs in ants are lacking. Our study attempts to fill this gap and the presented interactome will also serve as a template for determining PPIs in other ants in future. Our C. floridanus interactome covers 51,866 non-redundant PPIs among 6,274 proteins, including 20,544 interactions supported by domain-domain interactions (DDIs), 13,640 interactions supported by DDIs and subcellular localization, and 10,834 high confidence interactions mediated by 3,289 proteins. These interactions involve and cover 30.6% of the entire C. floridanus proteome.
Background
Preoperative chemoradiotherapy is the recommended standard of care for patients with local advanced rectal cancer. However, it remains unclear, whether a prolonged time interval to surgery results in an increased perioperative morbidity, reduced TME quality or better pathological response. Aim of this study was to determine the time interval for best pathological response and perioperative outcome compared to current recommended interval of 6 to 8 weeks.
Methods
This is a retrospective analysis of the German StuDoQ|Rectalcarcinoma registry. Patients were grouped for the time intervals of "less than 6 weeks", "6 to 8 weeks", "8 to 10 weeks" and "more than 10 weeks". Primary endpoint was pathological response, secondary endpoint TME quality and complications according to Clavien-Dindo classification.
Results
Due to our inclusion criteria (preoperative chemoradiation, surgery in curative intention, M0), 1.809 of 9.560 patients were suitable for analysis. We observed a trend for increased rates of pathological complete response (pCR: ypT0ypN0) and pathological good response (pGR: ypT0-1ypN0) for groups with a prolonged time interval which was not significant. Ultimately, it led to a steady state of pCR (16.5%) and pGR (22.6%) in "8 to 10" and "more than 10" weeks. We were not able to observe any differences between the subgroups in perioperative morbidity, proportion of rectal extirpation (for cancer of the lower third) or difference in TME quality.
Conclusion
A prolonged time interval between neoadjuvant chemoradiation can be performed, as the rate of pCR seems to be increased without influencing perioperative morbidity.
Background
The plant endophytic fungus Serendipita indica colonizes roots of a wide range of plant species and can enhance growth and stress resistance of these plants. Due to its ease of axenic cultivation and its broad host plant range including the model plant Arabidopsis thaliana and numerous crop plants, it is widely used as a model fungus to study beneficial fungus-root interactions. In addition, it was suggested to be utilized for commercial applications, e.g. to enhance yield in barley and other species. To produce inoculum, S. indica is mostly cultivated in a complex Hill-Kafer medium (CM medium), however, growth in this medium is slow, and yield of chlamydospores, which are often used for plant root inoculation, is relatively low.
Results
We tested and optimized a simple vegetable juice-based medium for an enhanced yield of fungal inoculum. The described vegetable juice (VJ) medium is based on commercially available vegetable juice and is easy to prepare. VJ medium was superior to the currently used CM medium with respect to biomass production in liquid medium and hyphal growth on agar plates. Using solid VJ medium supplemented with sucrose (VJS), a high amount of chlamydospores developed already after 8 days of cultivation, producing significantly more spores than on CM medium. Use of VJ medium is not restricted to S. indica, as it also supported growth of two pathogenic fungi often used in plant pathology experiments: the ascomycete Fusarium graminearum, the causal agent of Fusarium head blight disease on wheat and barley, and Verticillium longisporum, the causal agent of verticillium wilt.
Conclusions
The described VJ medium is recommended for streamlined and efficient production of inoculum for the plant endophytic fungus Serendipita indica and might prove superior for the propagation of other fungi for research purposes.
Background
Processing and analysis of DNA sequences obtained from next-generation sequencing (NGS) face some difficulties in terms of the correct prediction of DNA sequencing outcomes without the implementation of bioinformatics approaches. However, algorithms based on NGS perform inefficiently due to the generation of long DNA fragments, the difficulty of assembling them and the complexity of the used genomes. On the other hand, the Sanger DNA sequencing method is still considered to be the most reliable; it is a reliable choice for virtual modeling to build all possible consensus sequences from smaller DNA fragments.
Results
In silico and in vitro experiments were conducted: (1) to implement and test our novel sequencing algorithm, using the standard cloning vectors of different length and (2) to validate experimentally virtual shotgun sequencing using the PCR technique with the number of cycles from 1 to 9 for each reaction.
Conclusions
We applied a novel algorithm based on Sanger methodology to correctly predict and emphasize the performance of DNA sequencing techniques as well as in de novo DNA sequencing and its further application in synthetic biology. We demonstrate the statistical significance of our results.
Background
Phosphorylated histone H2AX, also known as gamma H2AX, forms mu m-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, gamma H2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of gamma H2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of gamma H2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected.
Results
To overcome the limitations of 2D foci counting, we present a freely available ImageJ-based plugin (FocAn) for automated 3D analysis of gamma H2AX foci in z-image stacks acquired by confocal fluorescence microscopy. The image-stack processing algorithm implemented in FocAn is capable of automatic 3D recognition of individual cell nuclei and gamma H2AX foci, as well as evaluation of the total foci number per cell nucleus. The FocAn algorithm consists of two parts: nucleus identification and foci detection, each employing specific sequences of auto local thresholding in combination with watershed segmentation techniques. We validated the FocAn algorithm using fluorescence-labeled gamma H2AX in two glioblastoma cell lines, irradiated with 2 Gy and given up to 24 h post-irradiation for repair. We found that the data obtained with FocAn agreed well with those obtained with an already available software (FoCo) and manual counting. Moreover, FocAn was capable of identifying overlapping foci in 3D space, which ensured accurate foci counting even at high DSB density of up to similar to 200 DSB/nucleus.
Conclusions
FocAn is freely available an open-source 3D foci analyzer. The user-friendly algorithm FocAn requires little supervision and can automatically count the amount of DNA-DSBs, i.e. fluorescence-labeled gamma H2AX foci, in 3D image stacks acquired by laser-scanning microscopes without additional nuclei staining.
Mapping human pressures on biodiversity across the planet uncovers anthropogenic threat complexes
(2020)
Climate change and other anthropogenic drivers of biodiversity change are unequally distributed across the world. Overlap in the distributions of different drivers have important implications for biodiversity change attribution and the potential for interactive effects. However, the spatial relationships among different drivers and whether they differ between the terrestrial and marine realm has yet to be examined.
We compiled global gridded datasets on climate change, land‐use, resource exploitation, pollution, alien species potential and human population density. We used multivariate statistics to examine the spatial relationships among the drivers and to characterize the typical combinations of drivers experienced by different regions of the world.
We found stronger positive correlations among drivers in the terrestrial than in the marine realm, leading to areas with high intensities of multiple drivers on land. Climate change tended to be negatively correlated with other drivers in the terrestrial realm (e.g. in the tundra and boreal forest with high climate change but low human use and pollution), whereas the opposite was true in the marine realm (e.g. in the Indo‐Pacific with high climate change and high fishing).
We show that different regions of the world can be defined by Anthropogenic Threat Complexes (ATCs), distinguished by different sets of drivers with varying intensities. We identify 11 ATCs that can be used to test hypotheses about patterns of biodiversity and ecosystem change, especially about the joint effects of multiple drivers.
Our global analysis highlights the broad conservation priorities needed to mitigate the impacts of anthropogenic change, with different priorities emerging on land and in the ocean, and in different parts of the world.
The fruit fly Drosophila melanogaster is an established model organism in chronobiology, because genetic manipulation and breeding in the laboratory are easy. The circadian clock neuroanatomy in D. melanogaster is one of the best-known clock networks in insects and basic circadian behavior has been characterized in detail in this insect. Another model in chronobiology is the honey bee Apis mellifera, of which diurnal foraging behavior has been described already in the early twentieth century. A. mellifera hallmarks the research on the interplay between the clock and sociality and complex behaviors like sun compass navigation and time-place-learning. Nevertheless, there are aspects of clock structure and function, like for example the role of the clock in photoperiodism and diapause, which can be only insufficiently investigated in these two models. Unlike high-latitude flies such as Chymomyza costata or D. ezoana, cosmopolitan D. melanogaster flies do not display a photoperiodic diapause. Similarly, A. mellifera bees do not go into “real” diapause, but most solitary bee species exhibit an obligatory diapause. Furthermore, sociality evolved in different Hymenoptera independently, wherefore it might be misleading to study the social clock only in one social insect. Consequently, additional research on non-model insects is required to understand the circadian clock in Diptera and Hymenoptera. In this review, we introduce the two chronobiology model insects D. melanogaster and A. mellifera, compare them with other insects and show their advantages and limitations as general models for insect circadian clocks.
Post-embryonic Development of the Circadian Clock Seems to Correlate With Social Life Style in Bees
(2020)
Social life style can influence many aspects of an animal’s daily life, but it has not yet been clarified, whether development of the circadian clock in social and solitary living bees differs. In a comparative study, with the social honey bee, Apis mellifera, and the solitary mason bee, Osmia bicornis, we now found indications for a differentially timed clock development in social and solitary bees. Newly emerged solitary bees showed rhythmic locomotion right away and the number of neurons in the brain that produce the clock component pigment-dispersing factor (PDF) did not change during aging of the adult solitary bee. Honey bees on the other hand, showed no circadian locomotion directly after emergence and the neuronal clock network continued to grow after emergence. Social bees appear to emerge at an early developmental stage at which the circadian clock is still immature, but bees are already able to fulfill in-hive tasks.
The transcription factor ∆Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ∆Np63 and maintains elevated ∆NP63 levels in SCC by counteracting its proteasome‐mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9‐engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ∆Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.
Bees rely on floral pollen and nectar for food. Therefore, pollinator friendly plantings are often used to enrich habitats in bee conservation efforts. As part of these plantings, non‐native plants may provide valuable floral resources, but their effects on native bee communities have not been assessed in direct comparison with native pollinator friendly plantings. In this study, we performed a common garden experiment by seeding mixes of 20 native and 20 non‐native pollinator friendly plant species at separate neighboring plots at three sites in Maryland, USA, and recorded flower visitors for 2 years. A total of 3,744 bees (120 species) were collected. Bee abundance and species richness were either similar across plant types (midseason and for abundance also late season) or lower at native than at non‐native plots (early season and for richness also late season). The overall bee community composition differed significantly between native and non‐native plots, with 11 and 23 bee species being found exclusively at one plot type or the other, respectively. Additionally, some species were more abundant at native plant plots, while others were more abundant at non‐natives. Native plants hosted more specialized plant–bee visitation networks than non‐native plants. Three species out of the five most abundant bee species were more specialized when foraging on native plants than on non‐native plants. Overall, visitation networks were more specialized in the early season than in late seasons. Our findings suggest that non‐native plants can benefit native pollinators, but may alter foraging patterns, bee community assemblage, and bee–plant network structures.
Ischemic stroke is one of the leading causes of death worldwide. It damages neurons and other supporting cellular elements in the brain. However, the impairment is not only confined to the region of assault but the surrounding area as well. Besides, it also brings about damage to the blood-brain barrier (BBB) which in turn leads to microvascular failure and edema. Hence, this necessitates an on-going, continuous search for intervention strategies and effective treatment. Of late, the natural sweetener stevioside proved to exhibit neuroprotective effects and therapeutic benefits against cerebral ischemia-induced injury. Its injectable formulation, isosteviol sodium (STVNA) also demonstrated favorable results. Nonetheless, its effects on the BBB have not yet been investigated to date. As such, this present study was designed to assess the effects of STVNA in our in vitro stroke model of the BBB.The integrity and permeability of the BBB are governed and maintained by tight junction proteins (TJPs) such as claudin-5 and occludin. Our data show increased claudin-5 and occludin expression in oxygen and glucose (OGD)-deprived murine brain capillary cerebellar endothelial cells (cerebEND) after STVNa treatment. Likewise, the upregulation of the transmembrane protein integrin-αv was also observed. Finally, cell volume was reduced with the simultaneous administration of STVNA and OGD in cerebEND cells. In neuropathologies such as stroke, the failure of cell volume control is a major feature leading to loss of cells in the penumbra as well as adverse outcomes. Our initial findings, therefore, point to the neuroprotective effects of STVNA at the BBB in vitro, which warrant further investigation for a possible future clinical intervention.
Simkania negevensis is a Chlamydia-like bacterium and emerging pathogen of the respiratory tract. It is an obligate intracellular bacterium with a biphasic developmental cycle, which replicates in a wide range of host cells. The life cycle of S. negevensis has been shown to proceed for more than 12 days, but little is known about the mechanisms that mediate the cellular release of these bacteria. This study focuses on the investigation of host cell exit by S. negevensis and its connection to host cell death modulation. We show that Simkania-infected epithelial HeLa as well as macrophage-like THP-1 cells reduce in number during the course of infection. At the same time, the infectivity of the cell culture supernatant increases, starting at the day 3 for HeLa and day 4 for THP-1 cells and reaching maximum at day 5 post infection. This correlates with the ability of S. negevensis to block TNFα-, but not staurosporin-induced cell death up to 3 days post infection, after which cell death is boosted by the presence of bacteria. Mitochondrial permeabilization through Bax and Bak is not essential for host cell lysis and release of S. negevensis. The inhibition of caspases by Z-VAD-FMK, caspase 1 by Ac-YVAD-CMK, and proteases significantly reduces the number of released infectious particles. In addition, the inhibition of myosin II by blebbistatin also strongly affects Simkania release, pointing to a possible double mechanism of exit through host cell lysis and potentially extrusion.
Cooperative Breeding in the Ambrosia Beetle Xyleborus affinis and Management of Its Fungal Symbionts
(2020)
Fungus-farming is known from attine ants, macrotermites, and ambrosia beetles (Scolytinae, Platypodinae). Farming ant and termite societies are superorganismal and grow fungal cultivars in monocultures. Social organization of ambrosia beetle groups and their farming systems are poorly studied, because of their enigmatic life within tunnel systems inside of wood. Ambrosia beetle-fungus symbioses evolved many times independently in both the beetles and their fungal cultivars. Observations suggest that there is evolutionary convergence between these lineages, but also a high variation in the degree of sociality and the modes of fungiculture. Using a laboratory observation technique, I here tried to give insights into the social system and fungus symbiosis of the sugar-cane borer, Xyleborus affinis Eichhoff (Scolytinae: Curculionidae), a currently poorly studied ambrosia beetle. The study revealed a cooperatively breeding system characterized by delayed dispersal of adult daughters, alloparental brood care by larvae and adults, and about half of the totipotent adult daughters laying eggs within the natal nest. Most interesting, there was a tendency of egg-laying females to engage more commonly in mutually beneficial behaviors than non-egg-layers. Fungus gardens covering gallery walls composed of five different filamentous fungi. A Raffaelea isolate was predominant and together with an unidentified fungus likely served as the main food for adults and larvae. Three isolates, a Mucor, a Fusarium and a Phaeoacremonium isolate were most abundant in the oldest gallery part close to the entrance; Mucor, Fusarium and the Raffaelea isolate in diseased individuals. Additionally, there was correlative evidence for some fungal isoaltes influencing beetle feeding and hygienic behaviors. Overall, X. affinis is now the second ambrosia beetle that can be classified as a cooperative breeder with division of labor among and between adults and larvae.
Mycotoxins in agriculturally used plants can cause intoxication in animals and can lead to severe financial losses for farmers. The endophytic fungus Epichloë festucae var. lolii living symbiotically within the cool season grass species Lolium perenne can produce vertebrate and invertebrate toxic alkaloids. Hence, an exact quantitation of alkaloid concentrations is essential to determine intoxication risk for animals. Many studies use different methods to detect alkaloid concentrations, which complicates the comparability. In this study, we showed that alkaloid concentrations of individual plants exceeded toxicity thresholds on real world grasslands in Germany, but not on the population level. Alkaloid concentrations on five German grasslands with high alkaloid levels peaked in summer but were also below toxicity thresholds on population level. Furthermore, we showed that alkaloid concentrations follow the same seasonal trend, regardless of whether plant fresh or dry weight was used, in the field and in a common garden study. However, alkaloid concentrations were around three times higher when detected with dry weight. Finally, we showed that alkaloid concentrations can additionally be biased to different alkaloid detection methods. We highlight that toxicity risks should be analyzed using plant dry weight, but concentration trends of fresh weight are reliable.
Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56\(^{bright}\)CD16\(^{dim}\) cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.
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.
Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae
(2020)
Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.
The lipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEth) are normally asymmetrically localized to the cytosolic face of membrane bilayers, but can both be externalized during diverse biological processes, including cell division, cell fusion, and cell death. Externalized lipids in the plasma membrane are recognized by lipid-binding proteins to regulate the clearance of cell corpses and other cell debris. However, it is unclear whether PtdSer and PtdEth contribute in similar or distinct ways to these processes. We discovered that disruption of the lipid flippases that maintain PtdSer or PtdEth asymmetry in the plasma membrane have opposite effects on phagocytosis in Caenorhabditis elegans embryos. Constitutive PtdSer externalization caused by disruption of the major PtdSer flippase TAT-1 led to increased phagocytosis of cell debris, sometimes leading to two cells engulfing the same debris. In contrast, PtdEth externalization caused by depletion of the major PtdEth flippase TAT-5 or its activator PAD-1 disrupted phagocytosis. These data suggest that PtdSer and PtdEth externalization have opposite effects on phagocytosis. Furthermore, externalizing PtdEth is associated with increased extracellular vesicle release, and we present evidence that the extent of extracellular vesicle accumulation correlates with the extent of phagocytic defects. Thus, a general loss of lipid asymmetry can have opposing impacts through different lipid subtypes simultaneously exerting disparate effects.
Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells.
Downregulation of miR-221-3p expression in prostate cancer (PCa) predicted overall and cancer-specific survival of high-risk PCa patients. Apart from PCa, miR-221-3p expression levels predicted a response to tyrosine kinase inhibitors (TKI) in clear cell renal cell carcinoma (ccRCC) patients. Since this role of miR-221-3p was explained with a specific targeting of VEGFR2, we examined whether miR-221-3p regulated VEGFR2 in PCa. First, we confirmed VEGFR2/KDR as a target gene of miR-221-3p in PCa cells by applying Luciferase reporter assays and Western blotting experiments. Although VEGFR2 was mainly downregulated in the PCa cohort of the TCGA (The Cancer Genome Atlas) database, VEGFR2 was upregulated in our high-risk PCa cohort (n = 142) and predicted clinical progression. In vitro miR-221-3p acted as an escape mechanism from TKI in PC3 cells, as displayed by proliferation and apoptosis assays. Moreover, we confirmed that Sunitinib induced an interferon-related gene signature in PC3 cells by analyzing external microarray data and by demonstrating a significant upregulation of miR-221-3p/miR-222-3p after Sunitinib exposure. Our findings bear a clinical perspective for high-risk PCa patients with low miR-221-3p levels since this could predict a favorable TKI response. Apart from this therapeutic niche, we identified a partially oncogenic function of miR-221-3p as an escape mechanism from VEGFR2 inhibition.
Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.
Mushroom bodies (MBs) are multisensory integration centers in the insect brain involved in learning and memory formation. In the honeybee, the main sensory input region (calyx) of MBs is comparatively large and receives input from mainly olfactory and visual senses, but also from gustatory/tactile modalities. Behavioral plasticity following differential brood care, changes in sensory exposure or the formation of associative long-term memory (LTM) was shown to be associated with structural plasticity in synaptic microcircuits (microglomeruli) within olfactory and visual compartments of the MB calyx. In the same line, physiological studies have demonstrated that MB-calyx microcircuits change response properties after associative learning. The aim of this review is to provide an update and synthesis of recent research on the plasticity of microcircuits in the MB calyx of the honeybee, specifically looking at the synaptic connectivity between sensory projection neurons (PNs) and MB intrinsic neurons (Kenyon cells). We focus on the honeybee as a favorable experimental insect for studying neuronal mechanisms underlying complex social behavior, but also compare it with other insect species for certain aspects. This review concludes by highlighting open questions and promising routes for future research aimed at understanding the causal relationships between neuronal and behavioral plasticity in this charismatic social insect.
Acromyrmex fracticornis grass-cutting ants construct conspicuous chimney-shaped nest turrets made of intermeshed grass fragments. We asked whether turrets are constructed by merely piling up nearby materials around the entrance, or whether ants incorporate different materials as the turret develops. By removing the original nest turrets and following their rebuilding process over three consecutive days, age-dependent changes in wall morphology and inner lining fabrics were characterized. Micromorphological descriptions based on thin sections of turret walls revealed the building behaviors involved. Ants started by collecting nearby twigs and dry grass fragments that are piled up around the nest entrance. Several large fragments held the structure like beams. As a net-like structure grew, soil pellets were placed in between the intermeshed plant fragments from the turret base to the top, reinforcing the structure. Concomitantly, the turret inner wall was lined with soil pellets, starting from the base. Therefore, the consolidation of the turret occurred both over time and from its base upwards. It is argued that nest turrets do not simply arise by the arbitrary deposition of nearby materials, and that workers selectively incorporate large materials at the beginning, and respond to the developing structure by reinforcing the intermeshed plant fragments over time.
Amphibians evolved in the Devonian period about 400 Mya and represent a transition step in tetrapod evolution. Among amphibians, high-throughput sequencing data are very limited for Caudata, due to their largest genome sizes among terrestrial vertebrates. In this paper we present the transcriptome from the fire bellied newt Cynops orientalis. Data here presented display a high level of completeness, comparable to the fully sequenced genomes available from other amphibians. Moreover, this work focused on genes involved in gametogenesis and sexual development. Surprisingly, the gsdf gene was identified for the first time in a tetrapod species, so far known only from bony fish and basal sarcopterygians. Our analysis failed to isolate fgf24 and foxl3, supporting the possible loss of both genes in the common ancestor of Rhipidistians. In Cynops, the expression analysis of genes described to be sex-related in vertebrates singled out an expected functional role for some genes, while others displayed an unforeseen behavior, confirming the high variability of the sex-related pathway in vertebrates.
The current molecular genetic diagnostic rates for hereditary hearing loss (HL) vary considerably according to the population background. Pakistan and other countries with high rates of consanguineous marriages have served as a unique resource for studying rare and novel forms of recessive HL. A combined exome sequencing, bioinformatics analysis, and gene mapping approach for 21 consanguineous Pakistani families revealed 13 pathogenic or likely pathogenic variants in the genes GJB2, MYO7A, FGF3, CDC14A, SLITRK6, CDH23, and MYO15A, with an overall resolve rate of 61.9%. GJB2 and MYO7A were the most frequently involved genes in this cohort. All the identified variants were either homozygous or compound heterozygous, with two of them not previously described in the literature (15.4%). Overall, seven missense variants (53.8%), three nonsense variants (23.1%), two frameshift variants (15.4%), and one splice-site variant (7.7%) were observed. Syndromic HL was identified in five (23.8%) of the 21 families studied. This study reflects the extreme genetic heterogeneity observed in HL and expands the spectrum of variants in deafness-associated genes.
Erfolgreiche räumliche Orientierung ist für viele Tiere eine alltägliche Herausforderung. Cataglyphis‐Wüstenameisen sind bekannt für ihre Navigationsfähigkeiten, mit deren Hilfe sie nach langen Futtersuchläufen problemlos zum Nest zurückfinden. Wie aber nehmen naive Ameisen ihre Navigationssysteme in Betrieb? Nach mehrwöchigem Innendienst im dunklen Nest werden sie zu Sammlerinnen bei hellem Sonnenschein. Dieser Wechsel erfordert einen drastischen Wandel im Verhalten sowie neuronale Veränderungen im Gehirn. Erfahrene Ameisen orientieren sich vor allem visuell, sie nutzen einen Himmelskompass und Landmarkenpanoramen. Daher absolvieren naive Ameisen stereotype Lernläufe, um ihren Kompass zu kalibrieren und die Nestumgebung kennenzulernen. Während der Lernläufe blicken sie wiederholt zum Nesteingang zurück und prägen sich so ihren Heimweg ein. Zur Ausrichtung ihrer Blicke nutzen sie das Erdmagnetfeld als Kompassreferenz. Cataglyphis‐Ameisen besitzen hierfür einen Magnetkompass, der bislang unbekannt war.
Die Entwicklung hochauflösender Fluoreszenzmikroskopiemethoden hat die Lichtmikroskopie revolutioniert. Einerseits ermöglicht die höhere erzielte räumliche Auflösung die Abbildung von Strukturen, die deutlich unterhalb der beugungsbedingten Auflösungsgrenze liegen. Andererseits erhält man durch Einzelmoleküllokalisationsmikroskopiemethoden
wie dSTORM (Direct Stochastic Optical Reconstruction Microscopy) Informationen, welche man für quantitative Analysen heranziehen kann. Aufgrund der sich dadurch bietenden neuen Möglichkeiten, hat sich die hochauflösende Fluoreszenzmikroskopie rasant entwickelt und kommt mittlerweile zur Untersuchung einer Vielzahl biologischer und medizinischer Fragestellungen zum Einsatz. Trotz dieses Erfolgs ist jedoch nicht zu verleugnen, dass auch diese neuen Methoden ihre Nachteile haben. Dazu zählt die Notwendigkeit relativ hoher Laserleistungen, welche Voraussetzung für hohe Auflösung ist und bei lebenden Proben zur Photoschädigung führen kann.
Diese Arbeit widmet sich sowohl dem Thema der Photoschädigung durch Einzelmoleküllokalisationsmikroskopie,
als auch der Anwendung von dSTORM und SIM (Structured Illumination Microscopy) zur Untersuchung neurobiologischer Fragestellungen auf Proteinebene.
Zur Ermittlung der Photoschädigung wurden lebende Zellen unter typischen Bedingungen bestrahlt und anschließend für 20−24 h beobachtet. Als quantitatives Maß für den Grad der Photoschädigung wurde der Anteil sterbender Zellen bestimmt. Neben der zu erwartenden Intensitäts- und Wellenlängenabhängigkeit, zeigte sich, dass die Schwere der Photoschädigung auch von vielen weiteren Faktoren abhängt und dass sich Einzelmoleküllokalisationsmikroskopie bei Berücksichtigung der gewonnenen Erkenntnisse durchaus mit Lebendzellexperimenten vereinbaren lässt.
Ein weiteres Projekt diente der Untersuchung der A- und B-Typ-Glutamatrezeptoren an der neuromuskulären Synapse von Drosophila melanogaster mittels dSTORM. Dabei konnte eine veränderte Anordnung beider Rezeptortypen infolge synaptischer Plastizität beobachtet, sowie eine absolute Quantifizierung des A-Typ-Rezeptors durchgeführt werden.
Im Mittelpunkt eines dritten Projekts standen Cadherin-13 (CDH13) sowie der Glucosetransporter Typ 3 (GluT3), welche beide mit der Aufmerksamkeitsdefizit-Hyperaktivitätsstörung in Verbindung gebracht werden. CDH13 konnte mittels SIM in serotonergen Neuronen, sowie radiären Gliazellen der dorsalen Raphekerne des embryonalen Mausgehirns nachgewiesen werden. Die Rolle von GluT3 wurde in aus induzierten pluripotenten Stammzellen differenzierten Neuronen analysiert, welche verschiedene Kopienzahlvariation des für GluT3-codierenden SLC2A3-Gens aufwiesen. Die Proteine GluT3, Bassoon und Homer wurden mittels dSTORM relativ quantifiziert. Während die Deletion des Gens zu einer erwartenden Verminderung von GluT3 auf Proteinebene führte, hatte die Duplikation keinen Effekt auf die GluT3-Menge. Für Bassoon und Homer zeigte sich weder durch die Deletion noch die Duplikation eine signifikante Veränderung.
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.
Mit fortschreitender chronischer Niereninsuffizienz kommt es zur Akkumulation von Urämietoxinen und im Endstadium unbehandelt zum Tod im sogenannten Urämischen Syndrom. Die Blutreinigung erfolgt bei der am häufigsten verwendeten Form der Nierenersatztherapie, der Hämodialyse, nur unzureichend. Die Folge ist eine erhöhte Morbidität und Mortalität der betroffenen Patienten. Bei der Hämodialyse werden nur Urämietoxine bis zu einer Größe von 20 kDa über die im Dialysator eingesetzten Hohlfaserdialysemembranen diffusiv und konvektiv semiselektiv nach Größenausschluss entfernt. Proteingebundene Urämietoxine, deren effektive Größe durch die Bindung an Transportproteine wie beispielsweise Albumin die Trennschärfe der Dialysemembranen übersteigt, werden retiniert. In-vivo werden proteingebundene Urämietoxine im proximalen Tubulus, einem Teil des tubulären Systems des Nephrons, sekretorisch eliminiert.
Im Rahmen der vorliegenden Promotionsarbeit wurden die ersten Entwicklungsschritte auf dem Weg zu einem sogenannten bio-artifiziellen Tubulus evaluiert. Der angedachte biohybride Filter sollte aus einer Ko-Kultur funktionaler humaner proximaler Tubuluszellen und humaner Endothelzellen (HUVEC) auf synthetischen Hohlfasermembranen bestehen und könnte während der Hämodialyse als zusätzlicher Reinigungsschritt angewendet werden, um unter anderem proteingebundene Urämietoxine effektiv durch aktiven Transport aus dem Blut der Patienten zu entfernen. Die Differenzierung der proximalen Tubuluszellen erfolgte dabei aus adulten adipozytären mesenchymalen Stammzellen (ASC), deren Herkunft eine spätere autologe Behandlung ermöglicht. Die Ko-Kultur mit Endothelzellen wurde zur potentiellen Steigerung der Sekretion proteingebundener Urämietoxine verwendet.
In der vorliegenden Arbeit konnten ASCs durch eine Kombination der löslichen Differenzierungsfaktoren All-Trans-Retinoinsäure (ATRA), Aktivin A und BMP-7 erfolgreich in Zytokeratin 18-exprimierende Zellen differenziert werden, wodurch die erwünschte epitheliale Differenzierung bestätigt wurde. Die Expression funktionaler Proteine, wie das für den Wassertransport relevante Aquaporin 1 oder auch der Na+-/K+-ATPase, konnte in dieser Arbeit bereits vor der Differenzierung nachgewiesen werden. Im nächsten Schritt wurde erfolgreich gezeigt, dass eine simultane, qualitativ hochwertige Ko-Kultur von ASCs und HUVECs auf der mit dem extrazellulären Matrixprotein Fibronektin modifizierten Innen- bzw. Außenseite von synthetischen Hohlfasermembranen aus Polypropylen bzw. Polyethersulfon möglich ist. Die Viabilität beider Zelltypen wurde dabei durch die Verwendung eines für die Ko-Kultur entwickelten Nährmediums erreicht, in welchem die Proliferation von ASCs bei gleichzeitiger Aufrechterhaltung ihrer Stammzelleigenschaften deutlich erhöht war.
Die in dieser Arbeit erzielten Ergebnisse stellen eine aussichtsreiche Basis für einen bio-artifiziellen renalen Tubulus dar. Weitere Entwicklungsschritte, wie die Differenzierung der ASCs zu proximalen Tubuluszellen im 3D-Bioreaktor einschließlich ihrer funktionalen Charakterisierung anhand Tubulusepithel-spezifischer Transporter, sind erforderlich, be-vor erste funktionale Experimente vor dem „Upscaling“ auf klinisch verwendbare Module möglich sind.
Cuticular hydrocarbons (CHC) abound on the surface of arthropods. In spite of their simple structure (molecules of carbon and hydrogen atoms), they provide pivotal functions in insects: their hydrophobic properties confer the insects a means to regulate water balance and avoid desiccation, whereas their diversity has enhanced their use as signals and cues in a wide range of communication and recognition processes. Although the study of CHC in insects over the past two decades has provided great insight into the wide range of functions they play, there is still a gap in understanding how they diversify and evolve. In this thesis, I have used members of the family Chrysididae to explore patterns of diversification of CHC. Most of the species of cuckoo wasps in this study are specialized parasitoids or kleptoparasites of mainly solitary hymenopteran hosts. Other hosts of the family include butterflies or stick insects. Cuckoo wasps are a particular interesting model to study the evolution of cuticular hydrocarbons because of their chemical adaptations that allow them to remain unrecognized by their hosts. Chemical insignificance (the reduction of the total amount of CHC on the cuticle) and chemical mimicry (the de novo production of CHC profiles resembling those of their female host) have been described in some representatives of the family and unpublished evidence suggests chemical deception is widespread in Chrysididae (Chapter 2). Nonetheless, to trace the evolution of any trait of interest, a reliable phylogenetic reconstruction of the family is required. Therefore, the first study of this thesis constitutes the largest and to-date most reliable phylogenetic reconstruction of the family Chrysididae, which includes representatives of 186 species of cuckoo wasps. While the results of this phylogenetic reconstruction are consistent with previous ideas on the relationships of subfamilies and tribes, it shows the existence of several non-monophyletic genera (Chapter 3). CHC are involved in intraspecific recognition, often acting as contact sex pheromones. Nevertheless, it is not yet understood to what extent CHC profiles differ between the two sexes and whether some compound classes are more prevalent in one or the other sex. So far, no comparison of CHC profiles of males and females has been done for more than a dozen of related species. In Chapter 4, I describe and compare CHC profiles of females and males of 58 species of cuckoo wasps in order to evaluate whether and to what extent CHC profiles of these species differ between the sexes. I demonstrated that CHC profiles of cuckoo wasps are frequently (more than 90% of the species analyzed) and strongly dimorphic (both sexes of a given species tend to produce very different CHC compounds). Methyl-branched compounds tend to be more prevalent in males (especially dimethyl-branched compounds) and unsaturated compounds prevail in females. Moreover, a sex-specific pattern in the distribution of the double bond position of alkenes was evident: internal double bond positions (> 11) occur predominantly in males, whereas alkenes with the doublé bond at position 9 were more abundant and frequent in females (Chapter4). In Chapter5, I investigated how CHC profiles of cuckoo wasps differ across species. Are CHC profiles of cuckoo wasps species-specific, enabling their use as cues for species recognition? How do CHC profiles resemble phylogenetic relatedness? In Chapter 5, I try to answer these questions by comparing CHC profiles of 59 species of cuckoo wasps. CHC profiles of cuckoo wasps are shown to be species (and sex-) specific. I show that CHC profiles are useful as a complementary tool to help delimiting taxonomically difficult sibling species. Moreover, the evaluation of CHC profiles of five commonly occurring species within a genus, showed little or no geographical variation. However, CHC profiles of closely related species may differ strongly among each other, not being useful to track the evolutionary history of species (Chapter 5). Sexual selection is generally credited for generating striking sexual dimorphism by causing changes in male traits. Most often, sexual selection has a stronger effect on males, who compete for access to and may be selected by females, thus male traits may rapidly evolve. Nevertheless, in cuckoo wasps, it appears that it is the female sex the one evolving faster changes, with females of very closely related species showing extremely divergent profiles. One plausible reason for this disparity is that natural selection acting on female’s CHC profiles may be stronger than sexual selection on males (Chapter 6). Since females of cuckoo wasps are most probably engaged in an evolutionary arms race with their female hosts, CHC profiles of female cuckoo wasps are likely rapidly evolving, thus explaining part of the strong observed sexual dimorphism of CHC (Chapter 6). In fact, Chapter 7 shows evidence of a possible ongoing evolutionary arms race between five cuckoo wasps of the genus Hedychrum and their hosts. Hedychrum species parasitize either Coleoptera-hunting or Hymenoptera-hunting digger wasps. Since the coleopteran prey of the former digger wasps is naturally better protected against fungus infestation, these wasps do not embalm their prey with alkene-enriched secretions as do the Hymenoptera-hunting digger wasps. Thus, Coleoptera-hunting digger wasps can apparently diversify their profiles to escape chemical mimicry. Interestingly, only female cuckoo wasps of these hosts have started producing the same compound classes and even the same CHC compounds as those of their hosts. Male cuckoo wasps, however retain an alkene-enriched CHC profile that reflects the molecular phylogeny of the genus (Chapter 7). Whereas, a larger number of parasite-host comparisons may be needed to further conclude that an arms race between cuckoo wasps and their hosts is capable of generating sexual dimorphism of cuckoo wasps, this thesis constitutes the first effort towards this, providing a starting point for further studies. Finally, I provide some methodological tools that may help in speeding up the sometimes cumbersome process of analyzing and identifying CHC profiles. One of the most time-demanding steps in the processing of CHC data is the alignment of CHC chromatograms. This process is often done manually, because alignment programs are mostly designed for metabolomics or are just recently being developed. I analyzed CHC profiles using a combined approach with two freely available programs. I used AMDIS (Automated Mass Spectral Deconvolution and Identification System, http://chemdata.nist.gov/mass-spc/amdis/) to deconvolute and automatically identify all CHC of interest present in a chromatogram. I then developed a series of R scripts to correct for potential, unavoidable errors while processing CHC chromatograms with AMDIS. Chapter 8 explains this procedure. In the next chapter, I developed a program that helps in the identification of one commonly occurring class of hydrocarbons. The limited number of linear alkanes (only one per carbon atom) and their characteristic diagnostic ion allows a rapid and unambigous identification of these substances. In opposition, unsaturated and methyl-branched compounds are more difficult to identify, as a result of the much larger diversity of existing compounds. To identify unsaturated compounds a derivatization is necessary to determine the position of the double bond. Methyl-branched alkanes, however can be identified from the original chromatogram if their diagnostic ions are known. Nonetheless, polymethyl-branched alkanes (e.g., compounds with two or more methyl groups along the chain) are often difficult to identify, because they may appear in mixes (e.g., 3,7 diMeC27 and 3,9 diMeC27), and tables containing the diagnostic ions are not easily available. Therefore, I developed a program that creates a table with all possiblemethyl-branched compounds containing up to 4 methyl groups, and that provides their diagnostic ions and a calculated retention index. This may allow a much faster identification of the methyl-branched compound a researcher is dealing with, without having to lose time in the tedious calculations by hand. The program is able to correctly identify, or at least, greatly reduce the number of possible options for the identification of an unknown methyl-branched compound. Thus, using this tool, most methyl-branched compounds can be readily identified (Chapter 9). This thesis ends with a general discussion (Chapter 10). Overall, this work provides a comprehensive overview of the diversity of cuticular hydrocarbons of cuckoo wasps. The analyses presented here shed light on the emergence and evolution of interspecific diversity and intraspecific sexual dimorphism of CHC profiles. In addition, two technical methods have been developed that could greatly facilitate the CHC analysis of insects.
Eine veränderte Expression des Transkriptionsfaktors MYC wird als entscheidender Faktor für Tumorentstehung und -progress im kolorektalen Karzinom gesehen. Somit ist die Hemmung dessen Expression und Funktion ein zentraler Ansatz bei der zielgerichteten Tumortherapie.
Als geeignete Strategie, sowohl die Halbwertszeit als auch die Translation von MYC zu verringern, erschien eine duale PI3K-/mTOR-Hemmung durch den small molecule-Inhibitor BEZ235. Gegenteilig ist jedoch unter Behandlung mit BEZ235 eine verstärkte MYC-Expression in verschiedenen Kolonkarzinom-Zelllinien zu beobachten. Neben verstärkter Transkription, konnte eine verstärkte IRES-abhängige Translation von MYC nach Hemmung der mTOR-/5´Cap-abhängigen Translation durch BEZ235, als Ursache der MYC-Induktion nachgewiesen werden.
Es konnte gezeigt werden, dass die Induktion von MYC nach PI3K-/mTOR-Hemmung durch eine kompensatorische Aktivierung des MAPK-Signalwegs in Folge einer FOXO-abhängigen Induktion von Rezeptortyrosinkinasen, stattfindet.
Eine mögliche Strategie, diese Feedback-Mechanismen zu umgehen, ist die direkte Hemmung der Translationsinitiation. Hierfür wurden Rocaglamid und dessen Derivat Silvestrol als small molecule-Inhibitoren der eIF4A-Helikase verwendet. Im Gegensatz zur PI3K/mTOR-Hemmung, ist durch eIF4A-Inhibition eine Reduktion der MYC-Proteinexpression in verschiedenen Kolonkarzinom-Zelllinien zu erreichen – ohne einhergehende MAPK-Aktivierung.
Anhand der Ergebnisse kann postuliert werden, dass Silvestrol das Potential besitzt, sowohl die Cap-/eIF4F-abhängie als auch die somit eIF4A-abhängige IRES-vermittelte Translation von MYC zu hemmen.
Weiterhin kann eine proliferationshemmende Wirkung durch Silvestrol auf Kolonkarzinom-Zellen in vitro, via Zellzyklusarrest und Induktion von Apoptose, gezeigt werden. Dies stellt die Voraussetzung für eine potentielle Eignung als tumorhemmender Wirkstoff in der Therapie des kolorektalen Karzinoms dar.
Modern agriculture is the basis of human existence, a blessing, but also a curse. It provides nourishment and well-being to the ever-growing human population, yet destroys biodiversity-mediated processes that underpin productivity: ecosystem services such as water filtration, pollination and biological pest control. Ecological intensification is a promising alternative to conventional farming, and aims to sustain yield and ecosystem health by actively managing biodiversity and essential ecosystem services. Here, I investigate opportunities and obstacles for ecological intensification. My research focuses on 1) the relative importance of soil, management and landscape variables for biodiversity and wheat yield (Chapter II); 2) the influence of multi-scale landscape-level crop diversity on biological pest control in wheat (Chapter III) and 3) on overall and functional bird diversity (Chapter IV). I conclude 4) by introducing a guide that helps scientists to increase research impact by acknowledging the role of stakeholder engagement for the successful implementation of ecological intensification (Chapter V).
Ecological intensification relies on the identification of natural pathways that are able to sustain current yields. Here, we crossed an observational field study of arthropod pests and natural enemies in 28 real-life wheat systems with an orthogonal on-field insecticide-fertilizer experiment. Using path analysis, we quantified the effect of 34 factors (soil characteristics, recent and historic crop management, landscape heterogeneity) that directly or indirectly (via predator-prey interactions) contribute to winter wheat yield. Reduced soil preparation and high crop rotation diversity enhanced crop productivity independent of external agrochemical inputs. Concurrently, biological control by arthropod natural enemies could be restored by decreasing average field sizes on the landscape scale, extending crop rotations and reducing soil disturbance. Furthermore, reductions in agrochemical inputs decreased pest abundances, thereby facilitating yield quality.
Landscape-level crop diversity is a promising tool for ecological intensification. However, biodiversity enhancement via diversification measures does not always translate into agricultural benefits due to antagonistic species interactions (intraguild predation). Additionally, positive effects of crop diversity on biological control may be masked by inappropriate study scales or correlations with other landscape variables (e.g. seminatural habitat). Therefore, the multiscale and context-dependent impact of crop diversity on biodiversity and ecosystem services is ambiguous. In 18 winter wheat fields along a crop diversity gradient, insect- and bird-mediated pest control was assessed using a natural enemy exclusion experiment with cereal grain aphids. Although birds did not influence the strength of insect-mediated pest control, crop diversity (rather than seminatural habitat cover) enhanced aphid regulation by up to 33%, particularly on small spatial scales. Crop diversification, an important Greening measure in the European Common Agricultural Policy, can improve biological control, and could lower dependence on insecticides, if the functional identity of crops is taken into account. Simple measures such as ‘effective number of crop types’ help in science communication.
Although avian pest control did not respond to landscape-level crop diversity, birds may still benefit from increased crop resources in the landscape, depending on their functional grouping (feeding guild, conservation status, habitat preference, nesting behaviour). Observational studies of bird functional diversity on 14 wheat study fields showed that non-crop landscape heterogeneity rather than crop diversity played a key role in determining the richness of all birds. Insect-feeding, non-farmland and non-threatened birds increased across multiple spatial scales (up to 3000 m). Only crop-nesting farmland birds declined in heterogeneous landscapes. Thus, crop diversification may be less suitable for conserving avian diversity, but abundant species benefit from overall habitat heterogeneity. Specialist farmland birds may require more targeted management approaches.
Identifying ecological pathways that favour biodiversity and ecosystem services provides opportunities for ecological intensification that increase the likelihood of balancing conservation and productivity goals. However, change towards a more sustainable agriculture will be slow to come if research findings are not implemented on a global scale. During dissemination activities within the EU project Liberation, I gathered information on the advantages and shortcomings of ecological intensification and its implementation. Here, I introduce a guide (‘TREE’) aimed at scientists that want to increase the impact of their research. TREE emphasizes the need to engage with stakeholders throughout the planning and research process, and actively seek and promote science dissemination and knowledge implementation. This idea requires scientists to leave their comfort zone and consider socioeconomic, practical and legal aspects often ignored in classical research.
Ecological intensification is a valuable instrument for sustainable agriculture. Here, I identified new pathways that facilitate ecological intensification. Soil quality, disturbance levels and spatial or temporal crop diversification showed strong positive correlations with natural enemies, biological pest control and yield, thereby lowering the dependence on agrochemical inputs. Differences between functional groups caused opposing, scale-specific responses to landscape variables. Opposed to our predictions, birds did not disturb insect-mediated pest control in our study system, nor did avian richness relate to landscape-level crop diversity. However, dominant functional bird groups increased with non-crop landscape heterogeneity. These findings highlight the value of combining different on-field and landscape approaches to ecological intensification. Concurrently, the success of ecological intensification can be increased by involving stakeholders throughout the research process. This increases the quality of science and reduces the chance of experiencing unscalable obstacles to implementation.
Das zentrale Paradigma der Systembiologie zielt auf ein möglichst umfassendes Ver-ständnis der komplexen Zusammenhänge biologischer Systeme. Die in dieser Arbeit angewandten Methoden folgen diesem Grundsatz.
Am Beispiel von drei auf Basis von Datenbanken und aktueller Literatur rekonstruier-ten Netzwerkmodellen konnte in der hier vorliegenden Arbeit die Gültigkeit analyti-scher und prädiktiver Algorithmen nachgewiesen werden, die in Form der Analy-sesoftware Jimena angewandt wurden. Die daraus resultierenden Ergebnisse sowohl für die Berechnung von stabilen Systemzuständen, der dynamischen Simulation, als auch der Identifikation zentraler Kontrollknoten konnten experimentell validiert wer-den. Die Ergebnisse wurden in einem iterativen Prozess verwendet werden um das entsprechende Netzwerkmodell zu optimieren.
Beim Vergleich des Verhaltens des semiquantitativ ausgewerteten regulatorischen Netzwerks zur Kontrolle der Differenzierung humaner mesenchymaler Stammzellen in Chondrozyten (Knorpelbildung), Osteoblasten (Knochenbildung) und Adipozyten (Fett-zellbildung) konnten 12 wichtige Faktoren (darunter: RUNX2, OSX/SP7, SOX9, TP53) mit Hilfe der Berechnung der Bedeutung (Kontrollzentralität der Netzwerkknoten identifi-ziert werden). Der Abgleich des simulierten Verhaltens dieses Netzwerkes ergab eine Übereinstimmung mit experimentellen Daten von 47,2%, bei einem widersprüchlichen Verhalten von ca. 25%, dass unter anderem durch die temporäre Natur experimentel-ler Messungen im Vergleich zu den terminalen Bedingungen des Berechnung der stabilen Systemzustände erklärt werden kann.
Bei der Analyse des Netzwerkmodells der menschlichen Immunantwort auf eine Infek-tion durch A. fumigatus konnten vier Hauptregulatoren identifiziert werden (A. fumi-gatus, Blutplättchen, hier Platelets genannt, und TNF), die im Zusammenspiel mit wei-teren Faktoren mit hohen Zentralitätswerten (CCL5, IL1, IL6, Dectin-1, TLR2 und TLR4) fähig sind das gesamte Netzwerkverhalten zu beeinflussen. Es konnte gezeigt werden, dass sich das Aktivitätsverhalten von IL6 in Reaktion auf A. fumigatus und die regulato-rische Wirkung von Blutplättchen mit den entsprechenden experimentellen Resultaten deckt.
Die Simulation, sowie die Berechnung der stabilen Systemzustände der Immunantwort von A. thaliana auf eine Infektion durch Pseudomonas syringae konnte zeigen, dass die in silico Ergebnisse mit den experimentellen Ergebnissen übereinstimmen. Zusätzlich konnten mit Hilfe der Analyse der Zentralitätswerte des Netzwerkmodells fünf Master-regulatoren identifiziert werden: TGA Transkriptionsfaktor, Jasmonsäure, Ent-Kaurenoate-Oxidase, Ent-kaurene-Synthase und Aspartat-Semialdehyd-Dehydrogenase.
Während die ersteren beiden bereits lange als wichtige Regulatoren für die Gib-berellin-Synthese bekannt sind, ist die immunregulatorische Funktion von Aspartat-Semialdehyd-Dehydrogenase bisher weitgehend unbekannt.
Die Schistosomiasis ist nach wie vor eine der häufigsten parasitären Erkrankungen der Welt und verursacht erhebliche gesundheitliche und wirtschaftliche Folgen, insbesondere in ärmeren, ländlichen Regionen. Durch Immunreaktionen auf die im Wirt abgelegten Eier des Parasiten können sich chronische Verlaufsformen manifestieren. Dabei kann es zu irreversiblen Schäden kommen. Um dies zu verhindern sind eine frühe und sichere Diagnose sowie eine Behandlung mit Praziquantel (PZQ) unabdingbar. Zudem spielt der zuverlässige Nachweis der Schistosomiasis eine Schlüsselrolle bei der Überwachung, Prävention und Kontrolle der Erkrankung. In epidemiologischen Studien findet am häufigsten die mikroskopische Kato-Katz (KK)-Methode zum Nachweis von Schistosoma mansoni Eiern im Stuhl Anwendung. Dieses Verfahren ist äußerst spezifisch und bietet die Möglichkeit der Quantifizierung, wodurch die Intensität der vorliegenden Infektion bestimmt werden kann. Die Sensitivität der Testmethode ist jedoch nur moderat, insbesondere bei einer niedrigen Infektionsintensität. Zudem kann eine Infektion erst nach der Präpatenzzeit nachgewiesen werden. Der ebenfalls häufig eingesetzte urinbasierte Point-of-Care Circulating Cathodic Antigen (POC-CCA)-Test weist zwar eine höhere Sensitivität aber geringere Spezifität als das KK-Verfahren auf. Als hochsensitive und sehr spezifische Methode zur Diagnose der Schistosomiasis hat sich der Nachweis von Schistosoma-spezifischer DNA mittels Real-Time PCR herausgestellt. Allerdings wird für die Durchführung dieser Technik ein gut ausgestattetes Labor benötigt, das sich in der Regel nicht in unmittelbarer Nähe zum Patienten im Feld befindet. Daher ist es besonders wichtig, über praktikable und schnelle Konservierungsmethoden zu verfügen, die bevor die Extraktion und Amplifikation der DNA stattfindet, einen einfachen Transport und eine einfache Lagerung des Probenmaterials ermöglichen.
Das Ziel des ersten Teils der vorliegenden Arbeit war, die Sensitivität und Spezifität der klassischerweise verwendeten KK-Methode und des POC-CCA-Tests mit der
Real-Time PCR- Methode unter Verwendung von Stuhlproben, Urinproben, Serumproben sowie auf Filterpapier getrocknete Blutproben (dried blood spots – DBSs) zu vergleichen. Zudem wurde die Anwendbarkeit der Real-Time PCR aus Serum- und Urinproben zur Therapiekontrolle überprüft. Die dazu notwendigen Studien wurden alle in der Region Mwanza in Tansania durchgeführt, welche als hochendemisch für S. mansoni gilt. Für die Untersuchungen zur stuhlbasierten Real-Time PCR wurden als Studienteilnehmer Schulkinder gewählt. Aufgrund der erforderlichen Blutabnahme wurden die anderen Teilstudien nur mit erwachsenen Probanden durchgeführt. Unter Verwendung der KK-Methode als Goldstandard erzielten die Real-Time PCR aus Stuhlproben und der POC-CCA-Test sehr hohe Sensitivitäten von 99,5% bzw. 89,7%, jedoch nur geringe Spezifitäten von 29,55% und 22,73%. Die KK-Methode weist bekanntermaßen nur eine geringe bis moderate Sensitivität auf und ist daher nicht gut als Referenz geeignet. Deshalb wurde zusätzlich eine latente Klassenanalyse angewandt, um die tatsächlich Erkrankten zu ermitteln und anhand dieser die diagnostische Güte der verwendeten Tests zu bestimmen. Hier zeigte der POC-CCA-Test die höchste Sensitivität (99,5%) sowie eine Spezifität von 63,4%. Der Real-Time PCR-Test hatte eine Sensitivität von 98,7% und die höchste Spezifität (81,2%). Die Spezifität der KK-Technik betrug 72,8%, die Sensitivität war signifikant niedriger (89,7%) als bei den anderen beiden Methoden. Diese Ergebnisse verdeutlichen, dass der POC-CCA-Schnelltest empfindlicher ist als die KK-Methode und zum Screening von S. mansoni-Infektionen eingesetzt werden kann. Die Stuhl-PCR war zwar ebenfalls hochsensitiv und zeigte unter den drei getesteten Diagnoseverfahren die höchste Spezifität, aber aufgrund der höheren Kosten und der komplizierten Anwendung sollte für epidemiologische Untersuchungen in Hochprävalenzregionen der POC-CCA-Test bevorzugt werden. Bei unklaren Diagnosen kann die Real-Time PCR-Methode als Bestätigungstest Anwendung finden.
In der Teilstudie zur serum- und urinbasierten Real-Time PCR in einer endemischen Region vor und nach der Behandlung mit PZQ wurden folgende Ergebnisse erzielt: Unter Verwendung einer kombinierten Referenz aus den Ergebnissen des parasitologischen KK-Tests und / oder der serumbasierten PCR konnte zu Studienbeginn eine Prävalenz von S. mansoni von 77,1% ermittelt werden. In Bezug auf die Sensitivität zeigte der DNA-Nachweis aus Serum (96,3%) und der POC-CCA-Assay (77,8%) die höchsten Ergebnisse. Die urinbasierte Real-Time PCR zeigte die geringste Empfindlichkeit (33,3%). Durch die Behandlung mit Praziquantel wurde eine signifikante Reduktion der S. mansoni Prävalenz erreicht. Zwanzig Wochen nach Therapie konnte durch die KK-Methode keine, mit dem POC-CCA-Test 33,3% und mit der serumbasierten Real-Time PCR 58,3% Infektionen festgestellt werden. Die Analyse der mittels der serumbasierten PCR bestimmten mittleren Ct-Werte im zeitlichen Verlauf zeigte, dass dieser eine Woche nach der Behandlung signifikant abnahm (von 30,3 auf 28) und 20 Wochen später über den Basiswert (34,9) anstieg. Der Ct-Wert ist umgekehrt proportional zur DNA-Ausgangsmenge, die in die PCR eingesetzt wurde. Dies deutet darauf hin, dass kurz nach der Therapie ein DNA-Anstieg zu verzeichnen war und 20 Wochen später weniger DNA als zu Beginn der Studie nachweisbar war. Dieser DNA-Verlauf lässt verschiedene Interpretationsmöglichkeiten zu. Die Daten zeigen jedoch, dass die serumbasierte Real-Time PCR eine ausgezeichnete diagnostische Genauigkeit aufweist. Da die nachgewiesene DNA jedoch keine Rückschlüsse auf das Parasitenstadium zulässt und es sich hierbei auch um DNA aus im Gewebe verbliebenen Eiern oder Reinfektionen handeln könnte, ist diese Methode in Hochprävalenz- Regionen nicht zur Therapiekontrolle geeignet. Die Verwendung von Urin zum DNA-Nachweis erzielte keine vielversprechenden Ergebnisse. Die Sensitivität der Real-Time PCR aus DBSs war ebenfalls sehr gering (45,4%) und kann ohne weitere ausführliche Testung hinsichtlich Lagertemperatur, Lagerdauer, verschiedener Filterpapierarten und Extraktionsmethoden nicht empfohlen werden.
Zusammenfassend zeigten die Ergebnisse dieser Studien, dass sowohl die stuhl- als auch die serumbasierte Real-Time PCR bei der Erkennung und Bewertung der Infektionsprävalenz, einem wichtigen Aspekt epidemiologischer Studien, deutlich empfindlicher ist als das mikroskopische KK-Verfahren. Aufgrund des hohen Kosten- und Personalaufwandes und der Notwendigkeit eines gut ausgestatteten Labors wird sich diese Methode aber nicht zum Screening in hochendemischen Ländern durchsetzen. Sie kann jedoch einen Mehrwert bei der Diagnose der Schistosomiasis bieten, vor allem bei frühen oder leichten Infektionen. Zudem kann diese hochsensitive und spezifische Methode als Bestätigungstest bei unklaren Diagnosen herangezogen werden.
Im zweiten Teil dieser Arbeit wurden malakologische Untersuchungen zur Identifizierung potenzieller Übertragungsorte für die Schistosomiasis rund um die im Viktoriasee gelegene Insel Ijinga durchgeführt. Diese Analysen fanden innerhalb eines Pilotprojektes zur Eliminierung der Erkrankung auf der Insel Ijinga statt, wobei ein intensiviertes Behandlungsprotokoll, welches die gesamte Inselbevölkerung einschloss, Anwendung fand. Die Kontrolle der Praziquanteleffektivität nach mehreren Behandlungsrunden bringt eine Reihe diagnostischer Herausforderungen mit sich. Hier könnte die Beurteilung der Schistosoma-Infektion in den Zwischenwirtschnecken vor und nach der Therapie als Indikator für den Erfolg der Maßnahme dienen. Zu diesem Zweck erfolgte zunächst eine Baseline-Untersuchung, bei der Schnecken an Uferregionen gesammelt wurden, an denen die Inselbewohner häufigen Wasserkontakt hatten. Die Schnecken wurden anhand morphologischer Merkmale identifiziert und mithilfe der Real-Time PCR-Methode auf Infektionen mit S. mansoni untersucht. Insgesamt wurden 35,4% (279/788) S. mansoni- positive Zwischenwirtschnecken (Biomphalaria) detektiert. Dies verdeutlicht, dass an den meisten Wasserkontaktstellen um die Insel Ijinga ein potentielles Risiko für die Übertragung der Schistosomiasis besteht. Die mithilfe der KK-Methode ermittelte Gesamtprävalenz von S. mansoni in der humanen Bevölkerung betrug 68,9%. Nachdem die Bewohner der Insel viermal mit PZQ behandelt wurden, zeigte sich in der kontinuierlich überwachten Sentinelgruppe eine Reduktion der Prävalenz auf 28,7%. Zu diesem Zeitpunkt wurde ebenfalls die Analyse der Schnecken wiederholt und es konnten 16,8% (57/350) Schnecken mit einer S. mansoni Infektion nachgewiesen werden. Die Reduktion der Infektionshäufigkeit in den Schnecken vor und nach der viermaligen Behandlung der Bevölkerung war signifikant (χ² = 74.335, p < 0,001). Dies deutet darauf hin, dass die intermediären Wirtsschnecken zur Überwachung von Kontrollmaßnahmen verwendet werden können.
Dispersal is a life-history trait affecting dynamics and persistence of populations; it evolves under various known selective pressures. Theoretical studies on dispersal typically assume 'natal dispersal', where individuals emigrate right after birth. But emigration may also occur during a later moment within a reproductive season ('breeding dispersal'). For example, some female butterflies first deposit eggs in their natal patch before migrating to other site(s) to continue egg-laying there. How breeding compared to natal dispersal influences the evolution of dispersal has not been explored. To close this gap we used an individual-based simulation approach to analyze (i) the evolution of timing of breeding dispersal in annual organisms, (ii) its influence on dispersal (compared to natal dispersal). Furthermore, we tested (iii) its performance in direct evolutionary contest with individuals following a natal dispersal strategy. Our results show that evolution should typically result in lower dispersal under breeding dispersal, especially when costs of dispersal are low and population size is small. By distributing offspring evenly across two patches, breeding dispersal allows reducing direct sibling competition in the next generation whereas natal dispersal can only reduce trans-generational kin competition by producing highly dispersive offspring in each generation. The added benefit of breeding dispersal is most prominent in patches with small population sizes. Finally, the evolutionary contests show that a breeding dispersal strategy would universally out-compete natal dispersal.
Endophytes live in partial symbiosis inside a plant and have been detected in all tested plants. They belong to the group of fungi or bacteria and their ecological function is mostly unknown. The fungal endophytes of the genus Epichloë belong to a special group of endophytes. Epichloë endophytes live symbiotically inside cool season grass species and some of them are able to produce alkaloids toxic to vertebrates and insects. Their symbiosis is seen as mutualistic for the following reasons: the fungus provides the plant herbivore resistance by producing alkaloids, and it increases the plant’s drought tolerance as well as its biomass production. In return, the grass provides the fungus shelter, nutrients and dispersal. Epichloë endophytes are host specific and the ability to produce alkaloids differs between species. In order to estimate intoxication risks in grasslands, it is necessary to detect infection rates of different grass species with Epichloë endophytes, and to determine the genotypes and chemotypes of the Epichloë species as well as the produced alkaloid concentrations. Factors like land-use intensity or season may have an influence on infection rates and alkaloid concentrations. Also, different methodological approaches may lead to different results. In this doctoral thesis my general aim was to evaluate intoxication risks in German grasslands caused by Epichloë endophytes. For that I investigated infection rates of different grass species and the genotypes and chemotypes of their Epichloë endophytes in German grasslands (Chapter II). Furthermore, I compared alkaloid concentrations detected with dry and fresh plant weight and different analytical methods. I also detected possible changes on the influence of season or land-use intensity (Chapter III). Additionally, I examined infections with Epichloë endophytes and alkaloid concentrations in commercially available grass seed mixtures and determined how that influences the intoxication risk of grazing animals in Europe (Chapter IV).
It is of agricultural interest to estimate intoxication risks for grazing livestock on German grasslands due to Epichloë infected grass species. Therefore, it is important to investigate which grasses are infected with the Epichloë endophyte, if the endophytes have the ability to produce vertebrate and invertebrate toxic alkaloids and if the alkaloids are indeed produced. I showed that Epichloë festucae var. lolii infecting agriculturally important Lolium perenne lacked the starting gene for ergovaline biosynthesis. Hence, vertebrate toxic ergovaline was not detected in the majority of the collected L. perenne plants. The detection of alkaloid concentrations is an important tool to estimate intoxication risk for vertebrates, but also invertebrates. My studies showed that the usage of dry plant material is crucial to quantify the correct alkaloid concentrations, and that alkaloid concentrations can vary depending on the detection method. Hence, the usage of validated, similar detection methods is important to be able to compare alkaloid concentrations from different studies. Nevertheless, the trends of seasonal changes and the influence of land-use intensity stayed the same, regardless if dry or fresh plant weight was used. Also, alkaloid concentrations were below toxicity thresholds on population level, regardless of the method used. Two commercially available forage grass and two commercially available turf grass seed mixtures were infected with Epichloë endopyhtes and alkaloids were detected. This might contribute to the spreading of Epichloë endopyhtes in Germany, therefore seed mixtures should be tested for Epichloë infections. My results indicate that the intoxication risk is generally low in Germany at the moment, although that might change due to climate change, an increase of monocultural land-use, or the seeding of Epichloë infected grass seeds.
Fungal endophytes of the genus Epichloë live symbiotically in cool season grass species and can produce alkaloids toxic to insects and vertebrates, yet reports of intoxication of grazing animals have been rare in Europe in contrast to overseas. However, due to the beneficial resistance traits observed in Epichloë infected grasses, the inclusion of Epichloë in seed mixtures might become increasingly advantageous. Despite the toxicity of fungal alkaloids, European seed mixtures are rarely tested for Epichloë infection and their infection status is unknown for consumers. In this study, we tested 24 commercially available seed mixtures for their infection rates with Epichloë endophytes and measured the concentrations of the alkaloids ergovaline, lolitrem B, paxilline, and peramine. We detected Epichloë infections in six seed mixtures, and four contained vertebrate and insect toxic alkaloids typical for Epichloë festucae var. lolii infecting Lolium perenne. As Epichloë infected seed mixtures can harm livestock, when infected grasses become dominant in the seeded grasslands, we recommend seed producers to test and communicate Epichloë infection status or avoiding Epichloë infected seed mixtures.
To foster sustainable environmentally friendly behavior in children it is important to provide an effective form of environmental education. In this context we studied three important factors: Attitude towards nature, environmental knowledge and advanced expert knowledge.
Concerning attitude towards nature our first question was: “Is it possible to affect primary school children’s environmental values during a one-day visit at a wildlife park?”
As a control, the program was also conducted in schools, leading to two different learning settings- wildlife park and school.
Regarding environmental knowledge, in our second question we wanted to know, if our modified teaching approach “guided learning at workstations” (G) combining instructional and constructivist elements would lead to good cognitive learning results of primary school children. Additionally, we compared it to a stronger teacher-centered (T) as well as to a stronger student-centered (S) approach.
The third question we asked was “Is it possible to convey fascinating expert knowledge on a more advanced subject to primary school children using conceptual change theory?” After gathering primary school children’s preconceptions, we defined different groups due to the heterogeneity of their pre-existing conceptions and the change in conceptions. Based on this research we designed a program along with an instrument to measure the impact of the conceptual change teaching method.
After years of building a strong cooperation between the section Didactics of Biology at the Julius-Maximilians University Würzburg, the nearby schools and the wildlife park “Wild-Park Klaushof” near Bad Kissingen in northern Bavaria it was time to evaluate the environmental education programs prepared and applied by undergraduate university students. As a model species we chose the European wildcat (Felis silvestris silvestris) which represents endangered wildlife in Europe and the need for human interaction for the sake of preserving a species by restoring or recreating the habitat conditions needed while maintaining current infrastructure. Drawing from our own as well as teachers’ and university students’ experiences, we built, implemented and evaluated a hands-on program following several workstations between the wildcat enclosure and the wildlife park’s green classroom.
The content of our intervention was presented as a problem-oriented lesson, where children were confronted with the need for human interaction in order to preserve the European wildcat. Not only on a theoretical basis, but very specific to their hometowns they were told where and when nature conservation groups met or where to donate money.
692 Bavarian third grade primary school children in 35 classes participated in the one-day intervention that took place between the months of april, 2014 and november, 2015 in the wildlife park or in their respective classrooms. The ages varied between 8 and 11 years with the mean age being 8.88 ± 0.56 years old. 48.6 % of them were boys, 51.4 % were girls.
(1) To measure primary school children’s environmental attitudes a questionnaire on two major environmental values- preservation and utilization of nature- was administered in a pre, post- and retention test design. It was possible to affect primary school children’s environmental preservation values during our one-day program. This result could be found not only at the wildlife park but unexpectedly also in school, where we educated classes for control purposes. We also found this impact consistent in all used teaching approaches and were surprised to see the preservation values change in a way we did not expect from higher tendency towards preservation of nature to a lower one.
We presume that children of this age group reflected on the contents of our intervention. This had an influence on their own values towards preservation which led to a more realistic marking behavior in the questionnaire. We therefore conclude that it is possible to affect primary school children’s environmental values with a one-day program on environmental content.
(2) We were interested in conveying environmental knowledge about the European wildcat; its morphology, ecology and behavior. We designed and applied a knowledge questionnaire also in a pre-, post- and retention test design, to find out, whether different forms of instruction made a difference in learning success of primary school children.
We used two approaches with a teacher in the role of a didactic leader- our modified guided approach (G) as well as a stronger teacher-centered one (T) with a higher focus on instruction. The third approach was presented as a strong student-centered learning at workstations (S) without a didactic leader we also called “free learning at workstations”.
Overall, all children’s knowledge scores changed significantly from pre- to post-test and from pre- to retention test, indicating learning success. Differences could only be found between the posttest values of both approaches with a didactic leader (G, T) in comparison to the strong student-centered (S) form.
It appears that these primary school children gained knowledge at the out of school learning setting regardless of the used teaching approach.
On the subject of short-term differences, we discuss, that the difference in learning success might have been consistent from post to retention test if a consolidation phase had been added in the days following the program as should be common practice after a visit to an out-of- school learning setting but was not part of our intervention.
When comparing both approaches with a didactic leader (G, T), we prefer our modified guided learning at workstations (G) since constructivist phases can be implemented without losses concerning learning success. Moreover, the (at least temporary) presence of a teacher in the role of a didactic leader ensures maintained discipline and counteracts off-task behavior.
To make sure, different emotional states did not factor in our program, we measured children’s situational emotions directly after the morning intervention using a short scale that evaluated interest, wellbeing and boredom. We found, that these emotions remained consistent over both learning settings as well as different forms of instruction. While interest and wellbeing remained constantly high, boredom values remained low.
We take this as a sign of high quality designing and conducting the intervention.
(3) In the afternoon of the one-day intervention, children were given the opportunity to investigate the wildcat further, this time using the conceptual change theory in combination with a more complex and fascinating content: cats’ vision in dusk and dawn.
Children were confronted with their preconceptions which had been sampled prior to the study and turned into three distinctive topics reflected in a special questionnaire.
In a pre-, post and retention test design we included the most common alternative conceptions, the scientifically correct conceptions as well as other preconceptions.
We gathered a high heterogeneity of preconceptions and defined three groups based on conceptual change literature: “Conceptual change”, “Synthetic Models” and “Conceptual Growth”. In addition to these we identified two more groups after our data analysis: “Knowledge” and “Non-addressed Concepts”.
We found that instruction according to the conceptual change theory did not work with primary school children in our intervention. The conceptual change from the addressed alternative conceptions as well as from other preconceptions towards the scientifically correct conceptions was successfully achieved only on occasion.
In our case and depending on the topic only one third to one fourth of the children actually held the addressed conception while the rest was not targeted by the instruction. Moreover, we conclude children holding other conceptions were rather confused than educated by the confrontation. We assume that children of this age group may be overchallenged by the conceptual change method.