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Institute
- Theodor-Boveri-Institut für Biowissenschaften (2243) (remove)
Schriftenreihe
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ResearcherID
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- N-2030-2015 (1)
Most of the studies in cell biology primarily focus on models from the opisthokont group of eukaryotes. However, opisthokonts do not encompass the full diversity of eukaryotes. Thus, it is necessary to broaden the research focus to other organisms to gain a comprehensive understanding of basic cellular processes shared across the tree of life. In this sense, Trypanosoma brucei, a unicellular eukaryote, emerges as a viable alternative. The collaborative efforts in genome sequencing and protein tagging over the past two decades have significantly expanded our knowledge on this organism and have provided valuable tools to facilitate a more detailed analysis of this parasite. Nevertheless, numerous questions still remain.
The survival of T. brucei within the mammalian host is intricately linked to the endo-lysosomal system, which plays a critical role in surface glycoprotein recycling, antibody clearance, and plasma membrane homeostasis. However, the dynamics of the duplication of the endo-lysosomal system during T. brucei proliferation and its potential relationship with plasma membrane growth remain poorly understood. Thus, as the primary objective, this thesis explores the endo-lysosomal system of T. brucei in the context of the cell cycle, providing insights on cell surface growth, endosome duplication, and clathrin recruitment. In addition, the study revisits ferritin endocytosis to provide quantitative data on the involvement of TbRab proteins (TbRab5A, TbRab7, and TbRab11) and the different endosomal subpopulations (early, late, and recycling endosomes, respectively) in the transport of this fluid-phase marker. Notably, while these subpopulations function as distinct compartments, different TbRabs can be found within the same region or structure, suggesting a potential physical connection between the endosomal subpopulations. The potential physical connection of endosomes is further explored within the context of the cell cycle and, finally, the duplication and morphological plasticity of the lysosome are also investigated. Overall, these findings provide insights into the dynamics of plasma membrane growth and the coordinated duplication of the endo-lysosomal system during T. brucei proliferation. The early duplication of endosomes suggests their potential involvement in plasma membrane growth, while the late duplication of the lysosome indicates a reduced role in this process. The recruitment of clathrin and TbRab GTPases to the site of endosome formation supports the assumption that the newly formed endosomal system is active during cell division and, consequently, indicates its potential role in plasma membrane homeostasis.
Furthermore, considering the vast diversity within the Trypanosoma genus, which includes ~500 described species, the macroevolution of the group was investigated using the combined information of the 18S rRNA gene sequence and structure. The sequence-structure analysis of T. brucei and other 42 trypanosome species was conducted in the context of the diversity of Trypanosomatida, the order in which trypanosomes are placed. An additional analysis focused on Trypanosoma highlighted key aspects of the group’s macroevolution. To explore these aspects further, additional trypanosome species were included, and the changes in the Trypanosoma tree topology were analyzed. The sequence-structure phylogeny confirmed the independent evolutionary history of the human pathogens T. brucei and Trypanosoma cruzi, while also providing insights into the evolution of the Aquatic clade, paraphyly of groups, and species classification into subgenera.
(1) Background: Clear cell renal cell carcinoma extending into the inferior vena cava (ccRCC\(^{IVC}\)) represents a clinical high-risk setting. However, there is substantial heterogeneity within this patient subgroup regarding survival outcomes. Previously, members of our group developed a microRNA(miR)-based risk classifier — containing miR-21-5p, miR-126-3p and miR-221-3p expression — which significantly predicted the cancer-specific survival (CSS) of ccRCC\(^{IVC}\) patients. (2) Methods: Examining a single-center cohort of tumor tissue from n = 56 patients with ccRCC\(^{IVC}\), we measured the expression levels of miR-21, miR-126, and miR-221 using qRT-PCR. The prognostic impact of clinicopathological parameters and miR expression were investigated via single-variable and multivariable Cox regression. Referring to the previously established risk classifier, we performed Kaplan–Meier analyses for single miR expression levels and the combined risk classifier. Cut-off values and weights within the risk classifier were taken from the previous study. (3) Results: miR-21 and miR-126 expression were significantly associated with lymphonodal status at the time of surgery, the development of metastasis during follow-up, and cancer-related death. In Kaplan–Meier analyses, miR-21 and miR-126 significantly impacted CSS in our cohort. Moreover, applying the miR-based risk classifier significantly stratified ccRCC\(^{IVC}\) according to CSS. (4) Conclusions: In our retrospective analysis, we successfully validated the miR-based risk classifier within an independent ccRCC\(^{IVC}\) cohort.
Die Rolle transposabler Elemente in der Genese des malignen Melanom im Fischmodell Xiphophorus
(2023)
Der Name der transposablen Elemente beruht auf ihrer Fähigkeit, ihre genomische Position verändern zu können. Durch Chromosomenaberrationen, Insertionen oder Deletionen können ihre genomischen Transpositionen genetische Instabilität verursachen. Inwieweit sie darüber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterführung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der ältesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des bösartigen schwarzen Hautkrebses, welcher nach wie vor die tödlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit beschäftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus.
DNA methylation acts as a major epigenetic modification in mammals, characterized by the transfer of a methyl group to a cytosine. DNA methylation plays a pivotal role in regulating normal development, and misregulation in cells leads to an abnormal phenotype as is seen in several cancers. Any mutations or expression anomalies of genes encoding regulators of DNA methylation may lead to abnormal expression of critical molecules. A comprehensive genomic study encompassing all the genes related to DNA methylation regulation in relation to breast cancer is lacking. We used genomic and transcriptomic datasets from the Cancer Genome Atlas (TGCA) Pan-Cancer Atlas, Genotype-Tissue Expression (GTEx) and microarray platforms and conducted in silico analysis of all the genes related to DNA methylation with respect to writing, reading and erasing this epigenetic mark. Analysis of mutations was conducted using cBioportal, while Xena and KMPlot were utilized for expression changes and patient survival, respectively. Our study identified multiple mutations in the genes encoding regulators of DNA methylation. The expression profiling of these showed significant differences between normal and disease tissues. Moreover, deregulated expression of some of the genes, namely DNMT3B, MBD1, MBD6, BAZ2B, ZBTB38, KLF4, TET2 and TDG, was correlated with patient prognosis. The current study, to our best knowledge, is the first to provide a comprehensive molecular and genetic profile of DNA methylation machinery genes in breast cancer and identifies DNA methylation machinery as an important determinant of the disease progression. The findings of this study will advance our understanding of the etiology of the disease and may serve to identify alternative targets for novel therapeutic strategies in cancer.
The behavior of honeybees and bumblebees relies on a constant sensory integration of abiotic or biotic stimuli. As eusocial insects, a sophisticated intraspecific communication as well as the processing of multisensory cues during foraging is of utter importance. To tackle the arising challenges, both honeybees and bumblebees have evolved a sophisticated olfactory and visual processing system.
In both organisms, olfactory reception starts at the antennae, where olfactory sensilla cover the antennal surface in a sex-specific manner. These sensilla house olfactory receptor neurons (ORN) that express olfactory receptors. ORNs send their axons via four tracts to the antennal lobe (AL), the prime olfactory processing center in the bee brain. Here, ORNs specifically innervate spheroidal structures, so-called glomeruli, in which they form synapses with local interneurons and projection neurons (PN). PNs subsequently project the olfactory information via two distinct tracts, the medial and the lateral antennal-lobe tract, to the mushroom body (MB), the main center of sensory integration and memory formation. In the honeybee calyx, the sensory input region of the MB, PNs synapse on Kenyon cells (KC), the principal neuron type of the MB. Olfactory PNs mainly innervate the lip and basal ring layer of the calyx. In addition, the basal ring receives input from visual PNs, making it the first site of integration of visual and olfactory information. Visual PNs, carrying sensory information from the optic lobes, send their terminals not only to the to the basal ring compartment but also to the collar of the calyx. Receiving olfactory or visual input, KCs send their axons along the MB peduncle and terminate in the main output regions of the MB, the medial and the vertical lobe (VL) in a layer-specific manner. In the MB lobes, KCs synapse onto mushroom body output neurons (MBON). In so far barely understood processes, multimodal information is integrated by the MBONs and then relayed further into the protocerebral lobes, the contralateral brain hemisphere, or the central brain among others.
This dissertation comprises a dichotomous structure that (i) aims to gain more insight into the olfactory processing in bumblebees and (ii) sets out to broaden our understanding of visual processing in honeybee MBONs.
The first manuscript examines the olfactory processing of Bombus terrestris and specifically investigates sex-specific differences. We used behavioral (absolute conditioning) and electrophysiological approaches to elaborate the processing of ecologically relevant odors (components of plant odors and pheromones) at three distinct levels, in the periphery, in the AL and during olfactory conditioning. We found both sexes to form robust memories after absolute conditioning and to generalize towards the carbon chain length of the presented odors. On the contrary, electroantennographic (EAG) activity showed distinct stimulus and sex-specific activity, e.g. reduced activity towards citronellol in drones. Interestingly, extracellular multi-unit recordings in the AL confirmed stimulus and sex-specific differences in olfactory processing, but did not reflect the differences previously found in the EAG. Here, farnesol and 2,3-dihydrofarnesol, components of sex-specific pheromones, show a distinct representation, especially in workers, corroborating the results of a previous study. This explicitly different representation suggests that the peripheral stimulus representation is an imperfect indication for neuronal representation in high-order neuropils and ecological importance of a specific odor.
The second manuscript investigates MBONs in honeybees to gain more insights into visual processing in the VL. Honeybee MBONs can be categorized into visually responsive, olfactory responsive and multimodal. To clarify which visual features are represented at this high-order integration center, we used extracellular multi-unit recordings in combination with visual and olfactory stimulation. We show for the first time that information about brightness and wavelength is preserved in the VL. Furthermore, we defined three specific classes of visual MBONs that distinctly encode the intensity, identity or simply the onset of a stimulus. The identity-subgroup exhibits a specific tuning towards UV light. These results support the view of the MB as the center of multimodal integration that categorizes sensory input and subsequently channels this information into specific MBON populations.
Finally, I discuss differences between the peripheral representations of stimuli and their distinct processing in high-order neuropils. The unique activity of farnesol in manuscript 1 or the representation of UV light in manuscript 2 suggest that the peripheral representation of a stimulus is insufficient as a sole indicator for its neural activity in subsequent neuropils or its putative behavioral importance. In addition, I discuss the influence of hard-wired concepts or plasticity induced changes in the sensory pathways on the processing of such key stimuli in the peripheral reception as well as in high-order centers like the AL or the MB. The MB as the center of multisensory integration has been broadly examined for its olfactory processing capabilities and receives increasing interest about its visual coding properties. To further unravel its role of sensory integration and to include neglected modalities, future studies need to combine additional approaches and gain more insights on the multimodal aspects in both the input and output region.
Copy number variations (CNVs) of the KITLG gene seem to be involved in the oncogenesis of digital squamous cell carcinoma (dSCC). The aims of this study were (1) to investigate KITLG CNV in giant (GS), standard (SS), and miniature (MS) schnauzers and (2) to compare KITLG CNV between black GS with and without dSCC. Blood samples from black GS (22 with and 17 without dSCC), black SS (18 with and 4 without dSSC; 5 unknown), and 50 MS (unknown dSSC status and coat colour) were analysed by digital droplet PCR. The results are that (1) most dogs had a copy number (CN) value > 4 (range 2.5–7.6) with no significant differences between GS, SS, and MS, and (2) the CN value in black GS with dSCC was significantly higher than in those without dSCC (p = 0.02). CN values > 5.8 indicate a significantly increased risk for dSCC, while CN values < 4.7 suggest a reduced risk for dSCC (grey area: 4.7–5.8). Diagnostic testing for KITLG CNV may sensitise owners to the individual risk of their black GS for dSCC. Further studies should investigate the relevance of KITLG CNV in SS and the protective effects in MS, who rarely suffer from dSCC.
Agricultural abandonment is one of the main land-use changes in Europe, and its consequences on biodiversity are context- and taxa-dependent. While several studies have worked on this topic, few have focused on traditional orchards, especially in different landscapes and under a Mediterranean climate. In this context, we aimed to determine the effects of almond orchard abandonment on the communities of three groups of beneficial arthropods and the role of the landscape context in modulating these effects. Between February and September 2019, four samplings were carried out in twelve almond orchards (three abandoned and three traditional (active orchards under traditional agricultural management) located in simple landscapes as well as three abandoned and three traditional in complex landscapes). Abandoned and traditional almond orchards harbor different arthropod communities and diversity metrics that are strongly conditioned by seasonality. Abandoned orchards can favor pollinators and natural enemies, providing alternative resources in simple landscapes. However, the role that abandoned orchards play in simple landscapes disappears as the percentage of semi-natural habitats in the landscape increases. Our results show that landscape simplification, through the loss of semi-natural habitats, has negative consequences on arthropod biodiversity, even in traditional farming landscapes with small fields and high crop diversity.
Orthopteran diversity in steep slope vineyards: the role of vineyard type and vegetation management
(2023)
The abandonment of traditional agricultural practices and subsequent succession are major threats to many open-adapted species and species-rich ecosystems. Viticulture on steep slopes has recently suffered from strong declines due to insufficient profitability, thus increasing the area of fallow land considerably. Changing cultivation systems from vertically oriented to modern vineyard terraces offers an opportunity to maintain management economically viable and thus reduces further abandonment. Hillside parallel terraces favor mechanization, and their embankments offer large undisturbed areas that could provide valuable habitats. We investigated the effects of vineyard abandonment, different vineyard management types (vertically oriented vs. terraced), and local parameters on Orthoptera diversity in 45 study sites along the Upper Middle Rhine Valley in Germany. Our results show that woody structures and vineyard abandonment reduced Orthoptera diversity at the local and landscape scale due to decreased habitat quality, especially for open-adapted species. In contrast, open inter-rows of actively managed vineyard types supported heat-adapted Caelifera species. On terrace embankments, extensive management and taller vegetation benefited Ensifera species, while short and mulched vegetation in vertically oriented vineyards favored the dominance of one single Caelifera species. Our results highlight the significance of maintaining viticultural management on steep slopes for the preservation of both open-adapted Orthoptera species and the cultural landscape.
Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air–liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.
Dark-haired dogs are predisposed to the development of digital squamous cell carcinoma (DSCC). This may potentially suggest an underlying genetic predisposition not yet completely elucidated. Some authors have suggested a potential correlation between the number of copies KIT Ligand (KITLG) and the predisposition of dogs to DSCC, containing a higher number of copies in those affected by the neoplasm. In this study, the aim was to evaluate a potential correlation between the number of copies of the KITLG and the histological grade of malignancy in dogs with DSCC. For this, 72 paraffin-embedded DSCCs with paired whole blood samples of 70 different dogs were included and grouped according to their haircoat color as follow: Group 0/unknown haircoat color (n = 11); Group 1.a/black non-Schnauzers (n = 15); group 1.b/black Schnauzers (n = 33); group 1.c/black and tan dogs (n = 7); group 2/tan animals (n = 4). The DSCCs were histologically graded. Additionally, KITLG Copy Number Variation (CNV) was determined by ddPCR. A significant correlation was observed between KITLG copy number and the histological grade and score value. This finding may suggest a possible factor for the development of canine DSCC, thus potentially having an impact on personalized veterinary oncological strategies and breeding programs.
Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host.
Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression.
A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts.
The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes.
In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection.
After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host.
In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods.
The increasing availability and variety of global satellite products and the rapid development of new algorithms has provided great potential to generate a new level of data with different spatial, temporal, and spectral resolutions. However, the ability of these synthetic spatiotemporal datasets to accurately map and monitor our planet on a field or regional scale remains underexplored. This study aimed to support future research efforts in estimating crop yields by identifying the optimal spatial (10 m, 30 m, or 250 m) and temporal (8 or 16 days) resolutions on a regional scale. The current study explored and discussed the suitability of four different synthetic (Landsat (L)-MOD13Q1 (30 m, 8 and 16 days) and Sentinel-2 (S)-MOD13Q1 (10 m, 8 and 16 days)) and two real (MOD13Q1 (250 m, 8 and 16 days)) NDVI products combined separately to two widely used crop growth models (CGMs) (World Food Studies (WOFOST), and the semi-empiric Light Use Efficiency approach (LUE)) for winter wheat (WW) and oil seed rape (OSR) yield forecasts in Bavaria (70,550 km\(^2\)) for the year 2019. For WW and OSR, the synthetic products’ high spatial and temporal resolution resulted in higher yield accuracies using LUE and WOFOST. The observations of high temporal resolution (8-day) products of both S-MOD13Q1 and L-MOD13Q1 played a significant role in accurately measuring the yield of WW and OSR. For example, L- and S-MOD13Q1 resulted in an R\(^2\) = 0.82 and 0.85, RMSE = 5.46 and 5.01 dt/ha for WW, R\(^2\) = 0.89 and 0.82, and RMSE = 2.23 and 2.11 dt/ha for OSR using the LUE model, respectively. Similarly, for the 8- and 16-day products, the simple LUE model (R\(^2\) = 0.77 and relative RMSE (RRMSE) = 8.17%) required fewer input parameters to simulate crop yield and was highly accurate, reliable, and more precise than the complex WOFOST model (R\(^2\) = 0.66 and RRMSE = 11.35%) with higher input parameters. Conclusively, both S-MOD13Q1 and L-MOD13Q1, in combination with LUE, were more prominent for predicting crop yields on a regional scale than the 16-day products; however, L-MOD13Q1 was advantageous for generating and exploring the long-term yield time series due to the availability of Landsat data since 1982, with a maximum resolution of 30 m. In addition, this study recommended the further use of its findings for implementing and validating the long-term crop yield time series in different regions of the world.
The cancer stem cell hypothesis is a cancer development model which elicited great interest in the last decades stating that cancer heterogeneity arises from a stem cell through asymmetrical division. The Cancer Stem Cell subset is described as the only population to be tumorigenic and having the potential to renew. Conventional therapy often fails to eradicate CSC resulting in tumor relapse. Consequently, it is of great inter-est to eliminate this subset of cells to provide the best patient outcome. In the last years several approaches to target CSC were developed, one of them being immunotherapeu-tic targeting with antibodies. Since markers associated with CSC are also expressed on normal stem cells or healthy adjacent tissue in colorectal cancer, dual targeting strate-gies are preferred over targeting only a single antigen. Subsequently, the idea of dual targeting two CSC markers in parallel by a newly developed split T cell-engaging anti-body format termed as Hemibodies emerged. In a preliminary single cell RNA sequenc-ing analysis of colorectal cancer cells CD133, CD24, CD166 and CEA were identified as suitable targets for the combinatorial targeting strategy. Therefore, this study focused on trispecific and trivalent Hemibodies comprising a split binding moiety against CD3 and a binding moiety against either CD133, CD24, CD166 or CEA to overcome the occurrence of resistance and to efficiently eradicate all tumor cells including the CSC compartment. The study showed that the Hemibody combinations CD133xCD24, CD133xCD166 and CD133xCEA are able to eliminate double positive CHO cells with high efficacy while having a high specificity indicated by no killing of single antigen positive cells. A thera-peutic window ranging between one to two log levels could be achieved for all combina-tions mentioned above. The combinations CD133xCD24 and CD133xCD166 further-more proved its efficacy and specificity on established colorectal cancer cell lines. Be-sides the evaluation of specificity and efficacy the already introduced 1st generation of Hemibodies could be improved into a 2nd generation Hemibody format with increased half-life, stability and production yield. In future experiments the applicability of above-mentioned Hemibodies will be proven on patient-derived micro tumors to also include variables like tumor microenvironment and infiltration.
Single-molecule dynamics at a bottleneck: a systematic study of the narrow escape problem in a disc
(2023)
Diffusion facilitates numerous reactions within the biological context of a cell. It is remarkable how the cost-efficient random process of Brownian motion promotes fast reactions. From the narrow escape theory, it is possible to determine the mean first passage time of such processes based on their reaction space and diffusion coefficient. The narrow escape theory of Brownian particles is characterized by a confining domain with reflective boundaries and a small reaction site. In this thesis, the mean first passage time was systematically tested in a disc as a function of the escape opening size in vitro and in silico. For the in vitro experiments, a model system of patterned supported-lipid bilayers (SLB) was established. Such a model is prepared by a combined colloid metalization approach, where a gold scaffold on glass facilitates assembly of SLB patches of distinct sizes through vesicle fusion. The model setup was evaluated and found to match all necessary requirements to test the nar- row escape problem in vitro. In particular, the reflectivity of the boundaries, the unhindered, free diffusion of the tracer lipids, and the distinct area were assessed. Observed results of the mean first passage time agreed with the theory of the narrow escape problem. There was excellent agreement in both absolute values and across a range of small escape opening sizes. Additionally, I developed a straightforward method, a correction factor, to calculate the mean first passage time from incomplete experimental traces. By re-scaling the mean first passage time to the fraction of particles that escaped, I was able to overcome the lifetime limitations of fluorescent probes. Previously inaccessible measurements of the mean first passage time relying on fluorescent probes will be made possible through this approach. The in vitro experiments were complemented with various in silico experiments. The latter were based on random walk simulations in discs, mimicking the in vitro situation with its uncertainties. The lifetime of single particles was either set sufficiently long to allow all particles to escape, or was adjusted to meet the lifetime limitations observed in the in vitro experiments. A comparison of the mean first passage time from lifetime-unlimited particles to the corrected, lifetime-limited particles did support the use of the correction factor. In agreement with the narrow escape theory, it was experimentally found that the mean first passage time is independent of the start point of the particle within the domain. This is when the particle adheres to a minimum distance to the escape site. In general, the presented random walk simulations do accurately represent the in vitro experiments in this study. The required hardware for the establishment of an astigmatism-based 3D system was installed in the existing microscope. The first attempts to analyze the obtained 3D imaging data gave insight into the potential of the method to investigate molecule dynamics in living trypanosome cells. The full functionality will be realized with the ongoing improvement of image analysis outside of this thesis.
Outdoor dust covers a shattered range of microbial agents from land over transportation, human microbial flora, which includes pathogen and commensals, and airborne from the environment. Dust aerosols are rich in bacterial communities that have a major impact on human health and living environments. In this study, outdoor samples from roadside barricades, safety walls, and fences (18 samples) were collected from Abu Dhabi, UAE and bacterial diversity was assessed through a 16S rRNA amplicon next generation sequencing approach. Clean data from HiSeq produced 1,099,892 total reads pairs for 18 samples. For all samples, taxonomic classifications were assigned to the OTUs (operational taxonomic units) representative sequence using the Ribosomal Database Project database. Analysis such as alpha diversity, beta diversity, differential species analysis, and species relative abundance were performed in the clustering of samples and a functional profile heat map was obtained from the OTUs by using bioinformatics tools. A total of 2814 OTUs were identified from those samples with a coverage of more than 99%. In the phylum, all 18 samples had most of the bacterial groups such as Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Twelve samples had Propionibacteria acnes and were mainly found in RD16 and RD3. Major bacteria species such as Propionibacteria acnes, Bacillus persicus, and Staphylococcus captis were found in all samples. Most of the samples had Streptococcus mitis, Staphylococcus capitis. and Nafulsella turpanensis and Enhydrobacter aerosaccus was part of the normal microbes of the skin. Salinimicrobium sp., Bacillus alkalisediminis, and Bacillus persicus are halophilic bacteria found in sediments. The heat map clustered the samples and species in vertical and horizontal classification, which represents the relationship between the samples and bacterial diversity. The heat map for the functional profile had high properties of amino acids, carbohydrate, and cofactor and vitamin metabolisms of all bacterial species from all samples. Taken together, our analyses are very relevant from the perspective of out-door air quality, airborne diseases, and epidemics, with broader implications for health safety and monitoring.
Background: The cabbage moth, Mamestra brassicae, is a polyphagous pest that attacks several crops. Here, the sublethal and lethal effects of chlorantraniliprole and indoxacarb were investigated on the developmental stages, detoxification enzymes, reproductive activity, calling behavior, peripheral physiology, and pheromone titer of M. brasssicae. Methods: To assess pesticide effects, the second instar larvae were maintained for 24 h on a semi-artificial diet containing insecticides at their LC\(_{10}\), LC\(_{30}\), and LC\(_{50}\) concentrations. Results: M. brassicae was more susceptible to chlorantraniliprole (LC\(_{50}\) = 0.35 mg/L) than indoxacarb (LC\(_{50}\) = 1.71 mg/L). A significantly increased developmental time was observed with both insecticides at all tested concentrations but decreases in pupation rate, pupal weight, and emergence were limited to the LC50 concentration. Reductions in both the total number of eggs laid per female and the egg viability were observed with both insecticides at their LC\(_{30}\) and LC\(_{50}\) concentrations. Both female calling activity and the sex pheromone (Z11-hexadecenyl acetate and hexadecenyl acetate) titer were significantly reduced by chlorantraniliprole in LC\(_{50}\) concentration. Antennal responses of female antennae to benzaldehyde and 3-octanone were significantly weaker than controls after exposure to the indoxocarb LC\(_{50}\) concentration. Significant reductions in the enzymatic activity of glutathione S-transferases, mixed-function oxidases, and carboxylesterases were observed in response to both insecticides.
Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux.
Land-use intensification and climate change threaten ecosystem functions. A fundamental, yet often overlooked, function is decomposition of necromass. The direct and indirect anthropogenic effects on decomposition, however, are poorly understood. We measured decomposition of two contrasting types of necromass, rat carrion and bison dung, on 179 study sites in Central Europe across an elevational climate gradient of 168–1122 m a.s.l. and within both local and regional land uses. Local land-use types included forest, grassland, arable fields, and settlements and were embedded in three regional land-use types (near-natural, agricultural, and urban). The effects of insects on decomposition were quantified by experimental exclusion, while controlling for removal by vertebrates. We used generalized additive mixed models to evaluate dung weight loss and carrion decay rate along elevation and across regional and local land-use types. We observed a unimodal relationship of dung decomposition with elevation, where greatest weight loss occurred between 600 and 700 m, but no effects of local temperature, land use, or insects. In contrast to dung, carrion decomposition was continuously faster with both increasing elevation and local temperature. Carrion reached the final decomposition stage six days earlier when insect access was allowed, and this did not depend on land-use effect. Our experiment identified different major drivers of decomposition on each necromass form. The results show that dung and carrion decomposition are rather robust to local and regional land use, but future climate change and decline of insects could alter decomposition processes and the self-regulation of ecosystems.
Efficient spatial orientation in the natural environment is crucial for the survival of most animal species. Cataglyphis desert ants possess excellent navigational skills. After far-ranging foraging excursions, the ants return to their inconspicuous nest entrance using celestial and panoramic cues. This review focuses on the question about how naïve ants acquire the necessary spatial information and adjust their visual compass systems. Naïve ants perform structured learning walks during their transition from the dark nest interior to foraging under bright sunlight. During initial learning walks, the ants perform rotational movements with nest-directed views using the earth’s magnetic field as an earthbound compass reference. Experimental manipulations demonstrate that specific sky compass cues trigger structural neuronal plasticity in visual circuits to integration centers in the central complex and mushroom bodies. During learning walks, rotation of the sky-polarization pattern is required for an increase in volume and synaptic complexes in both integration centers. In contrast, passive light exposure triggers light-spectrum (especially UV light) dependent changes in synaptic complexes upstream of the central complex. We discuss a multisensory circuit model in the ant brain for pathways mediating structural neuroplasticity at different levels following passive light exposure and multisensory experience during the performance of learning walks.
Grading, immunohistochemistry and c-kit mutation status are criteria for assessing the prognosis and therapeutic options of canine cutaneous mast cell tumours (MCTs). As a subset, canine digital MCTs have rarely been explored in this context. Therefore, in this retrospective study, 68 paraffin-embedded canine digital MCTs were analysed, and histological grading was assessed according to Patnaik and Kiupel. The immunohistochemical markers KIT and Ki67 were used, as well as polymerase chain reaction (PCR) for mutational screening in c-kit exons 8, 9, 11 and 14. Patnaik grading resulted in 22.1% grade I, 67.6% grade II and 10.3% grade III tumours. Some 86.8% of the digital MCTs were Kiupel low-grade. Aberrant KIT staining patterns II and III were found in 58.8%, and a count of more than 23 Ki67-positive cells in 52.3% of the cases. Both parameters were significantly associated with an internal tandem duplication (ITD) in c-kit exon 11 (12.7%). French Bulldogs, which tend to form well-differentiated cutaneous MCTs, had a higher proportion of digital high-grade MCTs and ITD in c-kit exon 11 compared with mongrels. Due to its retrospective nature, this study did not allow for an analysis of survival data. Nevertheless, it may contribute to the targeted characterisation of digital MCTs.
PRO-Simat is a simulation tool for analysing protein interaction networks, their dynamic change and pathway engineering. It provides GO enrichment, KEGG pathway analyses, and network visualisation from an integrated database of more than 8 million protein-protein interactions across 32 model organisms and the human proteome. We integrated dynamical network simulation using the Jimena framework, which quickly and efficiently simulates Boolean genetic regulatory networks. It enables simulation outputs with in-depth analysis of the type, strength, duration and pathway of the protein interactions on the website. Furthermore, the user can efficiently edit and analyse the effect of network modifications and engineering experiments. In case studies, applications of PRO-Simat are demonstrated: (i) understanding mutually exclusive differentiation pathways in Bacillus subtilis, (ii) making Vaccinia virus oncolytic by switching on its viral replication mainly in cancer cells and triggering cancer cell apoptosis and (iii) optogenetic control of nucleotide processing protein networks to operate DNA storage. Multilevel communication between components is critical for efficient network switching, as demonstrated by a general census on prokaryotic and eukaryotic networks and comparing design with synthetic networks using PRO-Simat. The tool is available at https://prosimat.heinzelab.de/ as a web-based query server.
Formic acid is the main component of the ant’s major weapon against enemies. Being mainly used as a chemical defense, the acid is also exploited for recruitment and trail marking. The repelling effect of the organic acid is used by some mammals and birds which rub themselves in the acid to eliminate ectoparasites. Beekeepers across the world rely on this effect to control the parasitic mite Varroa destructor. Varroa mites are considered the most destructive pest of honey bees worldwide and can lead to the loss of entire colonies. Formic acid is highly effective against Varroa mites but can also kill the honeybee queen and worker brood. Whether formic acid can also affect the behavior of honey bees is unknown. We here study the effect of formic acid on sucrose responsiveness and cognition of honey bees treated at different live stages in field-relevant doses. Both behaviors are essential for survival of the honey bee colony. Rather unexpectedly, formic acid clearly improved the learning performance of the bees in appetitive olfactory conditioning, while not affecting sucrose responsiveness. This exciting side effect of formic acid certainly deserves further detailed investigations.
Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.
The neuronal RNA-binding protein Ptbp2 regulates neuronal differentiation by modulating alternative splicing programs in the nucleus. Such programs contribute to axonogenesis by adjusting the levels of protein isoforms involved in axon growth and branching. While its functions in alternative splicing have been described in detail, cytosolic roles of Ptbp2 for axon growth have remained elusive. Here, we show that Ptbp2 is located in the cytosol including axons and growth cones of motoneurons, and that depletion of cytosolic Ptbp2 affects axon growth. We identify Ptbp2 as a major interactor of the 3’ UTR of Hnrnpr mRNA encoding the RNA-binding protein hnRNP R. Axonal localization of Hnrnpr mRNA and local synthesis of hnRNP R protein are strongly reduced when Ptbp2 is depleted, leading to defective axon growth. Ptbp2 regulates hnRNP R translation by mediating the association of Hnrnpr with ribosomes in a manner dependent on the translation factor eIF5A2. Our data thus suggest a mechanism whereby cytosolic Ptbp2 modulates axon growth by fine-tuning the mRNA transport and local synthesis of an RNA-binding protein.
Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection, but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary and sufficient to induce downstream open chromatin regions (dOCRs) when transcription termination is disrupted by the viral ICP27 protein. This is accompanied by a marked ICP22-dependent loss of histones downstream of affected genes consistent with impaired histone repositioning in the wake of Pol II. Efficient knock-down of the ICP22-interacting histone chaperone FACT is not sufficient to induce dOCRs in ΔICP22 infection but increases dOCR induction in wild-type HSV-1 infection. Interestingly, this is accompanied by a marked increase in chromatin accessibility within gene bodies. We propose a model in which allosteric changes in Pol II composition downstream of genes and ICP22-mediated interference with FACT activity explain the differential impairment of histone repositioning downstream of genes in the wake of Pol II in HSV-1 infection.
Tropical forest recovery is fundamental to addressing the intertwined climate and biodiversity loss crises. While regenerating trees sequester carbon relatively quickly, the pace of biodiversity recovery remains contentious. Here, we use bioacoustics and metabarcoding to measure forest recovery post-agriculture in a global biodiversity hotspot in Ecuador. We show that the community composition, and not species richness, of vocalizing vertebrates identified by experts reflects the restoration gradient. Two automated measures – an acoustic index model and a bird community composition derived from an independently developed Convolutional Neural Network - correlated well with restoration (adj-R² = 0.62 and 0.69, respectively). Importantly, both measures reflected composition of non-vocalizing nocturnal insects identified via metabarcoding. We show that such automated monitoring tools, based on new technologies, can effectively monitor the success of forest recovery, using robust and reproducible data.
Neural processing of a desired moving direction requires the continuous comparison between the current heading and the goal direction. While the neural basis underlying the current heading is well-studied, the coding of the goal direction remains unclear in insects. Here, we used tetrode recordings in tethered flying monarch butterflies to unravel how a goal direction is represented in the insect brain. While recording, the butterflies maintained robust goal directions relative to a virtual sun. By resetting their goal directions, we found neurons whose spatial tuning was tightly linked to the goal directions. Importantly, their tuning was unaffected when the butterflies changed their heading after compass perturbations, showing that these neurons specifically encode the goal direction. Overall, we here discovered invertebrate goal-direction neurons that share functional similarities to goal-direction cells reported in mammals. Our results give insights into the evolutionarily conserved principles of goal-directed spatial orientation in animals.
Rapid and accurate yield estimates at both field and regional levels remain the goal of sustainable agriculture and food security. Hereby, the identification of consistent and reliable methodologies providing accurate yield predictions is one of the hot topics in agricultural research. This study investigated the relationship of spatiotemporal fusion modelling using STRAFM on crop yield prediction for winter wheat (WW) and oil-seed rape (OSR) using a semi-empirical light use efficiency (LUE) model for the Free State of Bavaria (70,550 km\(^2\)), Germany, from 2001 to 2019. A synthetic normalised difference vegetation index (NDVI) time series was generated and validated by fusing the high spatial resolution (30 m, 16 days) Landsat 5 Thematic Mapper (TM) (2001 to 2012), Landsat 7 Enhanced Thematic Mapper Plus (ETM+) (2012), and Landsat 8 Operational Land Imager (OLI) (2013 to 2019) with the coarse resolution of MOD13Q1 (250 m, 16 days) from 2001 to 2019. Except for some temporal periods (i.e., 2001, 2002, and 2012), the study obtained an R\(^2\) of more than 0.65 and a RMSE of less than 0.11, which proves that the Landsat 8 OLI fused products are of higher accuracy than the Landsat 5 TM products. Moreover, the accuracies of the NDVI fusion data have been found to correlate with the total number of available Landsat scenes every year (N), with a correlation coefficient (R) of +0.83 (between R\(^2\) of yearly synthetic NDVIs and N) and −0.84 (between RMSEs and N). For crop yield prediction, the synthetic NDVI time series and climate elements (such as minimum temperature, maximum temperature, relative humidity, evaporation, transpiration, and solar radiation) are inputted to the LUE model, resulting in an average R\(^2\) of 0.75 (WW) and 0.73 (OSR), and RMSEs of 4.33 dt/ha and 2.19 dt/ha. The yield prediction results prove the consistency and stability of the LUE model for yield estimation. Using the LUE model, accurate crop yield predictions were obtained for WW (R\(^2\) = 0.88) and OSR (R\(^2\) = 0.74). Lastly, the study observed a high positive correlation of R = 0.81 and R = 0.77 between the yearly R\(^2\) of synthetic accuracy and modelled yield accuracy for WW and OSR, respectively.
Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.
Infected wounds pose a major mortality risk in animals. Injuries are common in the ant Megaponera analis, which raids pugnacious prey. Here we show that M. analis can determine when wounds are infected and treat them accordingly. By applying a variety of antimicrobial compounds and proteins secreted from the metapleural gland to infected wounds, workers reduce the mortality of infected individuals by 90%. Chemical analyses showed that wound infection is associated with specific changes in the cuticular hydrocarbon profile, thereby likely allowing nestmates to diagnose the infection state of injured individuals and apply the appropriate antimicrobial treatment. This study demonstrates that M. analis ant societies use antimicrobial compounds produced in the metapleural glands to treat infected wounds and reduce nestmate mortality.
Seasonal plasticity in insects is often triggered by temperature and photoperiod changes. When climatic conditions become sub-optimal, insects might undergo reproductive diapause, a form of seasonal plasticity delaying the development of reproductive organs and activities. During the reproductive diapause, the cuticular hydrocarbon (CHC) profile, which covers the insect body surface, might also change to protect insects from desiccation and cold temperature. However, CHCs are often important cues and signals for mate recognition and changes in CHC composition might affect mate recognition. In the present study, we investigated the CHC profile composition and the mating success of Drosophila suzukii in 1- and 5-day-old males and females of summer and winter morphs. CHC compositions differed with age and morphs. However, no significant differences were found between the sexes of the same age and morph. The results of the behavioral assays show that summer morph pairs start to mate earlier in their life, have a shorter mating duration, and have more offspring compared to winter morph pairs. We hypothesize that CHC profiles of winter morphs are adapted to survive winter conditions, potentially at the cost of reduced mate recognition cues.
T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy.
Machine learning techniques are excellent to analyze expression data from single cells. These techniques impact all fields ranging from cell annotation and clustering to signature identification. The presented framework evaluates gene selection sets how far they optimally separate defined phenotypes or cell groups. This innovation overcomes the present limitation to objectively and correctly identify a small gene set of high information content regarding separating phenotypes for which corresponding code scripts are provided. The small but meaningful subset of the original genes (or feature space) facilitates human interpretability of the differences of the phenotypes including those found by machine learning results and may even turn correlations between genes and phenotypes into a causal explanation. For the feature selection task, the principal feature analysis is utilized which reduces redundant information while selecting genes that carry the information for separating the phenotypes. In this context, the presented framework shows explainability of unsupervised learning as it reveals cell-type specific signatures. Apart from a Seurat preprocessing tool and the PFA script, the pipeline uses mutual information to balance accuracy and size of the gene set if desired. A validation part to evaluate the gene selection for their information content regarding the separation of the phenotypes is provided as well, binary and multiclass classification of 3 or 4 groups are studied. Results from different single-cell data are presented. In each, only about ten out of more than 30000 genes are identified as carrying the relevant information. The code is provided in a GitHub repository at https://github.com/AC-PHD/Seurat_PFA_pipeline.
In the fast-evolving landscape of biomedical research, the emergence of big data has presented researchers with extraordinary opportunities to explore biological complexities. In biomedical research, big data imply also a big responsibility. This is not only due to genomics data being sensitive information but also due to genomics data being shared and re-analysed among the scientific community. This saves valuable resources and can even help to find new insights in silico. To fully use these opportunities, detailed and correct metadata are imperative. This includes not only the availability of metadata but also their correctness. Metadata integrity serves as a fundamental determinant of research credibility, supporting the reliability and reproducibility of data-driven findings. Ensuring metadata availability, curation, and accuracy are therefore essential for bioinformatic research. Not only must metadata be readily available, but they must also be meticulously curated and ideally error-free. Motivated by an accidental discovery of a critical metadata error in patient data published in two high-impact journals, we aim to raise awareness for the need of correct, complete, and curated metadata. We describe how the metadata error was found, addressed, and present examples for metadata-related challenges in omics research, along with supporting measures, including tools for checking metadata and software to facilitate various steps from data analysis to published research.
Highlights
• Data awareness and data integrity underpins the trustworthiness of results and subsequent further analysis.
• Big data and bioinformatics enable efficient resource use by repurposing publicly available RNA-Sequencing data.
• Manual checks of data quality and integrity are insufficient due to the overwhelming volume and rapidly growing data.
• Automation and artificial intelligence provide cost-effective and efficient solutions for data integrity and quality checks.
• FAIR data management, various software solutions and analysis tools assist metadata maintenance.
Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.
CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.
Alpine bumble bees are the most important pollinators in temperate mountain ecosystems. Although they are used to encounter small-scale successions of very different climates in the mountains, many species respond sensitively to climatic changes, reflected in spatial range shifts and declining populations worldwide. Cuticular hydrocarbons (CHCs) mediate climate adaptation in some insects. However, whether they predict the elevational niche of bumble bees or their responses to climatic changes remains poorly understood. Here, we used three different approaches to study the role of bumble bees’ CHCs in the context of climate adaptation: using a 1,300 m elevational gradient, we first investigated whether the overall composition of CHCs, and two potentially climate-associated chemical traits (proportion of saturated components, mean chain length) on the cuticle of six bumble bee species were linked to the species’ elevational niches. We then analyzed intraspecific variation in CHCs of Bombus pascuorum along the elevational gradient and tested whether these traits respond to temperature. Finally, we used a field translocation experiment to test whether CHCs of Bombus lucorum workers change, when translocated from the foothill of a cool and wet mountain region to (a) higher elevations, and (b) a warm and dry region. Overall, the six species showed distinctive, species-specific CHC profiles. We found inter- and intraspecific variation in the composition of CHCs and in chemical traits along the elevational gradient, but no link to the elevational distribution of species and individuals. According to our expectations, bumble bees translocated to a warm and dry region tended to express longer CHC chains than bumble bees translocated to cool and wet foothills, which could reflect an acclimatization to regional climate. However, chain lengths did not further decrease systematically along the elevational gradient, suggesting that other factors than temperature also shape chain lengths in CHC profiles. We conclude that in alpine bumble bees, CHC profiles and traits respond at best secondarily to the climate conditions tested in this study. While the functional role of species-specific CHC profiles in bumble bees remains elusive, limited plasticity in this trait could restrict species’ ability to adapt to climatic changes.
Xiphophorus fish exhibit a clear phenotypic polymorphism in puberty onset and reproductive strategies of males. In X. nigrensis and X. multilineatus, puberty onset is genetically determined and linked to a melanocortin 4 receptor (Mc4r) polymorphism of wild-type and mutant alleles on the sex chromosomes. We hypothesized that Mc4r mutant alleles act on wild-type alleles by a dominant negative effect through receptor dimerization, leading to differential intracellular signaling and effector gene activation. Depending on signaling strength, the onset of puberty either occurs early or is delayed. Here, we show by Förster Resonance Energy Transfer (FRET) that wild-type Xiphophorus Mc4r monomers can form homodimers, but also heterodimers with mutant receptors resulting in compromised signaling which explains the reduced Mc4r signaling in large males. Thus, hetero- vs. homo- dimerization seems to be the key molecular mechanism for the polymorphism in puberty onset and body size in male fish.
Fungal infections are a major global health burden where Candida albicans is among the most common fungal pathogen in humans and is a common cause of invasive candidiasis. Fungal phenotypes, such as those related to morphology, proliferation and virulence are mainly driven by gene expression, which is primarily regulated by kinase signaling cascades. Serine-arginine (SR) protein kinases are highly conserved among eukaryotes and are involved in major transcriptional processes in human and S. cerevisiae. Candida albicans harbors two SR protein kinases, while Sky2 is important for metabolic adaptation, Sky1 has similar functions as in S. cerevisiae. To investigate the role of these SR kinases for the regulation of transcriptional responses in C. albicans, we performed RNA sequencing of sky1Δ and sky2Δ and integrated a comprehensive phosphoproteome dataset of these mutants. Using a Systems Biology approach, we study transcriptional regulation in the context of kinase signaling networks. Transcriptomic enrichment analysis indicates that pathways involved in the regulation of gene expression are downregulated and mitochondrial processes are upregulated in sky1Δ. In sky2Δ, primarily metabolic processes are affected, especially for arginine, and we observed that arginine-induced hyphae formation is impaired in sky2Δ. In addition, our analysis identifies several transcription factors as potential drivers of the transcriptional response. Among these, a core set is shared between both kinase knockouts, but it appears to regulate different subsets of target genes. To elucidate these diverse regulatory patterns, we created network modules by integrating the data of site-specific protein phosphorylation and gene expression with kinase-substrate predictions and protein-protein interactions. These integrated signaling modules reveal shared parts but also highlight specific patterns characteristic for each kinase. Interestingly, the modules contain many proteins involved in fungal morphogenesis and stress response. Accordingly, experimental phenotyping shows a higher resistance to Hygromycin B for sky1Δ. Thus, our study demonstrates that a combination of computational approaches with integration of experimental data can offer a new systems biological perspective on the complex network of signaling and transcription. With that, the investigation of the interface between signaling and transcriptional regulation in C. albicans provides a deeper insight into how cellular mechanisms can shape the phenotype.
Highlights
• The integrated stress response leads to a general ATF4-dependent activation of NRF2
• ATF4 causes a CHAC1-dependent GSH depletion, resulting in NRF2 stabilization
• An elevation of NRF2 transcript levels fosters this effect
• NRF2 supports the ISR/ATF4 pathway by improving cystine and antioxidant supply
Summary
The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.
The increasing loss of pollinators over the last decades has become more and more evident. Intensive use of plant protection products is one key factor contributing to this decline. Especially the mixture of different plant protection products can pose an increased risk for pollinators as synergistic effects may occur. In this study we investigated the effect of the fungicide Cantus® Gold (boscalid/dimoxystrobin), the neonicotinoid insecticide Mospilan® (acetamiprid) and their mixture on honeybees. Since both plant protection products are frequently applied sequentially to the same plants (e.g. oilseed rape), their combination is a realistic scenario for honeybees. We investigated the mortality, the sucrose responsiveness and the differential olfactory learning performance of honeybees under controlled conditions in the laboratory to reduce environmental noise. Intact sucrose responsiveness and learning performance are of pivotal importance for the survival of individual honeybees as well as for the functioning of the entire colony. Treatment with two sublethal and field relevant concentrations of each plant protection product did not lead to any significant effects on these behaviors but affected the mortality rate. However, our study cannot exclude possible negative sublethal effects of these substances in higher concentrations. In addition, the honeybee seems to be quite robust when it comes to effects of plant protection products, while wild bees might be more sensitive.
Highlights
• Mix of SBI fungicides and neonicotinoids can lead to synergistic effects for bees.
• Combination of non-SBI fungicide and neonicotinoid in field-realistic doses tested.
• Synergistic effect on mortality of honeybees.
• No effects on sucrose responsiveness and learning performance of honeybees.
• Synergistic effects by other pesticide mixtures or on wild bees cannot be excluded.
Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality.
To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca\(^{2+}\). In our search for species-dependent functional TPC1 channel variants with different luminal Ca\(^{2+}\) sensitivity, we found in total three acidic residues present in Ca\(^{2+}\) sensor sites 2 and 3 of the Ca\(^{2+}\)-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca\(^{2+}\). When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca\(^{2+}\) sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca\(^{2+}\) sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.
Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.
Recent studies link increased ozone (O\(_3\)) and carbon dioxide (CO\(_2\)) levels to alteration of plant performance and plant-herbivore interactions, but their interactive effects on plant-pollinator interactions are little understood. Extra floral nectaries (EFNs) are essential organs used by some plants for stimulating defense against herbivory and for the attraction of insect pollinators, e.g., bees. The factors driving the interactions between bees and plants regarding the visitation of bees to EFNs are poorly understood, especially in the face of global change driven by greenhouse gases. Here, we experimentally tested whether elevated levels of O\(_3\) and CO\(_2\) individually and interactively alter the emission of Volatile Organic Compound (VOC) profiles in the field bean plant (Vicia faba, L., Fabaceae), EFN nectar production and EFN visitation by the European orchard bee (Osmia cornuta, Latreille, Megachilidae). Our results showed that O\(_3\) alone had significant negative effects on the blends of VOCs emitted while the treatment with elevated CO\(_2\) alone did not differ from the control. Furthermore, as with O\(_3\) alone, the mixture of O\(_3\) and CO\(_2\) also had a significant difference in the VOCs’ profile. O\(_3\) exposure was also linked to reduced nectar volume and had a negative impact on EFN visitation by bees. Increased CO\(_2\) level, on the other hand, had a positive impact on bee visits. Our results add to the knowledge of the interactive effects of O\(_3\) and CO\(_2\) on plant volatiles emitted by Vicia faba and bee responses. As greenhouse gas levels continue to rise globally, it is important to take these findings into consideration to better prepare for changes in plant-insect interactions.
Natural DNA storage allows cellular differentiation, evolution, the growth of our children and controls all our ecosystems. Here, we discuss the fundamental aspects of DNA storage and recent advances in this field, with special emphasis on natural processes and solutions that can be exploited. We point out new ways of efficient DNA and nucleotide storage that are inspired by nature. Within a few years DNA-based information storage may become an attractive and natural complementation to current electronic data storage systems. We discuss rapid and directed access (e.g. DNA elements such as promotors, enhancers), regulatory signals and modulation (e.g. lncRNA) as well as integrated high-density storage and processing modules (e.g. chromosomal territories). There is pragmatic DNA storage for use in biotechnology and human genetics. We examine DNA storage as an approach for synthetic biology (e.g. light-controlled nucleotide processing enzymes). The natural polymers of DNA and RNA offer much for direct storage operations (read-in, read-out, access control). The inbuilt parallelism (many molecules at many places working at the same time) is important for fast processing of information. Using biology concepts from chromosomal storage, nucleic acid processing as well as polymer material sciences such as electronical effects in enzymes, graphene, nanocellulose up to DNA macramé , DNA wires and DNA-based aptamer field effect transistors will open up new applications gradually replacing classical information storage methods in ever more areas over time (decades).
The narrow escape theory (NET) predicts the escape time distribution of Brownian particles confined to a domain with reflecting borders except for one small window. Applications include molecular activation events in cell biology and biophysics. Specifically, the mean first passage time τ can be analytically calculated from the size of the domain, the escape window, and the diffusion coefficient of the particles. In this study, we systematically tested the NET in a disc by variation of the escape opening. Our model system consisted of micro-patterned lipid bilayers. For the measurement of τ, we imaged diffusing fluorescently-labeled lipids using single-molecule fluorescence microscopy. We overcame the lifetime limitation of fluorescent probes by re-scaling the measured time with the fraction of escaped particles. Experiments were complemented by matching stochastic numerical simulations. To conclude, we confirmed the NET prediction in vitro and in silico for the disc geometry in the limit of small escape openings, and we provide a straightforward solution to determine τ from incomplete experimental traces.
The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.
Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly’s bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites’ development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.