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Drosophila melanogaster is a long-standing model organism in the circadian clock research. A major advantage is the relative small number of about 150 neurons, which built the circadian clock in Drosophila. In our recent work, we focused on the neuroanatomical properties of the lateral neurons of the clock network. By applying the multicolor-labeling technique Flybow we were able to identify the anatomical similarity of the previously described E2 subunit of the evening oscillator of the clock, which is built by the 5th small ventrolateral neuron (5th s-LNv) and one ITP positive dorsolateral neuron (LNd). These two clock neurons share the same spatial and functional properties. We found both neurons innervating the same brain areas with similar pre- and postsynaptic sites in the brain. Here the anatomical findings support their shared function as a main evening oscillator in the clock network like also found in previous studies. A second quite surprising finding addresses the large lateral ventral PDF-neurons (l-LNvs). We could show that the four hardly distinguishable l-LNvs consist of two subgroups with different innervation patterns. While three of the neurons reflect the well-known branching pattern reproduced by PDF immunohistochemistry, one neuron per brain hemisphere has a distinguished innervation profile and is restricted only to the proximal part of the medulla-surface. We named this neuron “extra” l-LNv (l-LNvx). We suggest the anatomical findings reflect different functional properties of the two l-LNv subgroups.
To study delayed genetic and epigenetic radiation effects, which may trigger radiation-induced carcinogenesis, we have established single-cell clones from irradiated and non-irradiated primary human fibroblasts. Stable clones were endowed with the same karyotype in all analyzed metaphases after 20 population doublings (PDs), whereas unstable clones displayed mosaics of normal and abnormal karyotypes. To account for variation in radiation sensitivity, all experiments were performed with two different fibroblast strains. After a single X-ray dose of 2 Gy more than half of the irradiated clones exhibited radiation-induced genome instability (RIGI). Irradiated clones displayed an increased rate of loss of chromosome Y (LOY) and copy number variations (CNVs), compared to controls. CNV breakpoints clustered in specific chromosome regions, in particular 3p14.2 and 7q11.21, coinciding with common fragile sites. CNVs affecting the FHIT gene in FRA3B were observed in independent unstable clones and may drive RIGI. Bisulfite pyrosequencing of control clones and the respective primary culture revealed global hypomethylation of ALU, LINE-1, and alpha-satellite repeats as well as rDNA hypermethylation during in vitro ageing. Irradiated clones showed further reduced ALU and alpha-satellite methylation and increased rDNA methylation, compared to controls. Methylation arrays identified several hundred differentially methylated genes and several enriched pathways associated with in vitro ageing. Methylation changes in 259 genes and the MAP kinase signaling pathway were associated with delayed radiation effects (after 20 PDs). Collectively, our results suggest that both genetic (LOY and CNVs) and epigenetic changes occur in the progeny of exposed cells that were not damaged directly by irradiation, likely contributing to radiation-induced carcinogenesis. We did not observe epigenetic differences between stable and unstable irradiated clones. The fact that the DNA methylation (DNAm) age of clones derived from the same primary culture varied greatly suggests that DNAm age of a single cell (represented by a clone) can be quite different from the DNAm age of a tissue. We propose that DNAm age reflects the emergent property of a large number of individual cells whose respective DNAm ages can be highly variable.
The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.
Patient-tailored therapy based on tumor drivers is promising for lung cancer treatment. For this, we combined in vitro tissue models with in silico analyses. Using individual cell lines with specific mutations, we demonstrate a generic and rapid stratification pipeline for targeted tumor therapy. We improve in vitro models of tissue conditions by a biological matrix-based three-dimensional (3D) tissue culture that allows in vitro drug testing: It correctly shows a strong drug response upon gefitinib (Gef) treatment in a cell line harboring an EGFR-activating mutation (HCC827), but no clear drug response upon treatment with the HSP90 inhibitor 17AAG in two cell lines with KRAS mutations (H441, A549). In contrast, 2D testing implies wrongly KRAS as a biomarker for HSP90 inhibitor treatment, although this fails in clinical studies. Signaling analysis by phospho-arrays showed similar effects of EGFR inhibition by Gef in HCC827 cells, under both 2D and 3D conditions. Western blot analysis confirmed that for 3D conditions, HSP90 inhibitor treatment implies different p53 regulation and decreased MET inhibition in HCC827 and H441 cells. Using in vitro data (western, phospho-kinase array, proliferation, and apoptosis), we generated cell line-specific in silico topologies and condition-specific (2D, 3D) simulations of signaling correctly mirroring in vitro treatment responses. Networks predict drug targets considering key interactions and individual cell line mutations using the Human Protein Reference Database and the COSMIC database. A signature of potential biomarkers and matching drugs improve stratification and treatment in KRAS-mutated tumors. In silico screening and dynamic simulation of drug actions resulted in individual therapeutic suggestions, that is, targeting HIF1A in H441 and LKB1 in A549 cells. In conclusion, our in vitro tumor tissue model combined with an in silico tool improves drug effect prediction and patient stratification. Our tool is used in our comprehensive cancer center and is made now publicly available for targeted therapy decisions.
Sand mines represent anthropogenically impacted habitats found worldwide, which bear potential for bee conservation. Although floral resources can be limited at these habitats, vegetation free patches of open sandy soils and embankments may offer good nesting possibilities for sand restricted and other bees. We compared bee communities as found in three reclaimed sand mines and at adjacent roadside meadows in Maryland, USA, over two years. Both sand mines and roadsides hosted diverse bee communities with 111 and 88 bee species, respectively. Bee abundances as well as richness and Shannon diversity of bee species were higher in sand mines than at roadsides and negatively correlated with the percentage of vegetational ground cover. Species composition also differed significantly between habitats. Sand mines hosted a higher proportion of ground nesters, more uncommon and more ‘sand loving’ bees similar to natural sandy areas of Maryland. Despite the destruction of the original pre-mining habitat, sand mines thus appear to represent a unique habitat for wild bees, particularly when natural vegetation and open sand spots are encouraged. Considering habitat loss, the lack of natural disturbance regimes, and ongoing declines of wild bees, sand mines could add promising opportunities for bee conservation which has hitherto mainly focused on agricultural and urban habitats.
Ambrosia beetles farm ascomycetous fungi in tunnels within wood. These ambrosia fungi are regarded asexual, although population genetic proof is missing. Here we explored the intraspecific genetic diversity of Ambrosiella grosmanniae and Ambrosiella hartigii (Ascomycota: Microascales), the mutualists of the beetles Xylosandrus germanus and Anisandrus dispar. By sequencing five markers (ITS, LSU, TEF1α, RPB2, β-tubulin) from several fungal strains, we show that X. germanus cultivates the same two clones of A. grosmanniae in the USA and in Europe, whereas A. dispar is associated with a single A. hartigii clone across Europe. This low genetic diversity is consistent with predominantly asexual vertical transmission of Ambrosiella cultivars between beetle generations. This clonal agriculture is a remarkable case of convergence with fungus-farming ants, given that both groups have a completely different ecology and evolutionary history.
Recent progress in nanobiotechnology has attracted interest to a biomedical application of the carbon nanostructure C60 fullerene since it possesses a unique structure and versatile biological activity. C60 fullerene potential application in the frame of cancer photodynamic therapy (PDT) relies on rapid development of new light sources as well as on better understanding of the fullerene interaction with cells.
The aim of this study was to analyze C60 fullerene effects on human leukemic cells (CCRF-CEM) in combination with high power single chip light-emitting diodes (LEDs) light irradiation of different wavelengths: ultraviolet (UV, 365 nm), violet (405 nm), green (515 nm) and red (632 nm). The time-dependent accumulation of fullerene C60 in CCRF-CEM cells up to 250 ng/106 cells at 24 h with predominant localization within mitochondria was demonstrated with immunocytochemical staining and liquid chromatography mass spectrometry. In a cell viability assay we studied photoexcitation of the accumulated C60 nanostructures with ultraviolet or violet LEDs and could prove that significant phototoxic effects did arise. A less pronounced C60 fullerene phototoxic effect was observed after irradiation with green, and no effect was detected with red light. A C60 fullerene photoactivation with violet light induced substantial ROS generation and apoptotic cell death, confirmed by caspase3/7 activation and plasma membrane phosphatidylserine externalization. Our work proved C60 fullerene ability to induce apoptosis of leukemic cells after photoexcitation with high power single chip 405 nm LED as a light source. This underlined the potential for application of C60 nanostructure as a photosensitizer for anticancer therapy.
Pathogenic variants in COL9A1 are primarily associated with autosomal recessive Stickler syndrome. Patients with COL9A1-associated Stickler syndrome (STL) present hearing loss (HL), ophthalmic manifestations and skeletal abnormalities. However, the clinical spectrum of patients with COL9A1 variants can also include multiple epiphyseal dysplasia, as well as non-syndromic HL that was observed in one previously reported proband. Exome sequencing was performed on the genomic DNA of an Iranian patient and his affected brother who both report non-syndromic HL. A 44.6 kb homozygous in-frame deletion spanning exons 6 to 33 of COL9A1 was detected via exome-based copy number variation analysis. The deleted exons were confirmed by PCR in the patient and his affected brother, who both have non-syndromic HL. Segregation analysis via qPCR confirmed the parents as heterozygous deletion carriers. Breakpoint analysis mapped the homozygous deletion spanning introns 5 to 33 (g.70,948,188_70,997,277del, NM_001851.4(COL9A1):c.697–3754_2112+769del, p.(Phe233_Ser704del), with an additional 67 bp of inserted intronic sequence that may have originated due to a fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) mechanism. This mechanism has not been previously implicated in HL or STL. This is also the first reported copy number variation in COL9A1 that was identified through an exome data set in an Iranian family with apparent non-syndromic HL. The present study emphasizes the importance of exome-wide copy number variation analysis in molecular diagnosis and provides supporting evidence to associate COL9A1 with autosomal recessive non-syndromic HL.
OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased number of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info.
Meiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis.
“Everything changes and nothing stands still” (Heraclitus). Here we review three major improvements to freshwater aquatic ecosystem models — and ecological models in general — as water quality scenario analysis tools towards a sustainable future. To tackle the rapid and deeply connected dynamics characteristic of the Anthropocene, we argue for the inclusion of eco-evolutionary, novel ecosystem and social-ecological dynamics. These dynamics arise from adaptive responses in organisms and ecosystems to global environmental change and act at different integration levels and different time scales. We provide reasons and means to incorporate each improvement into aquatic ecosystem models. Throughout this study we refer to Lake Victoria as a microcosm of the evolving novel social-ecological systems of the Anthropocene. The Lake Victoria case clearly shows how interlinked eco-evolutionary, novel ecosystem and social-ecological dynamics are, and demonstrates the need for transdisciplinary research approaches towards global sustainability.
Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.
Pflanzen sind verschiedenen Umweltbedingungen ausgesetzt, die zu suboptimalen Wachstumsbedingungen führen können. Dies gilt für eine Vielzahl von biotischen und abiotischen Faktoren. In der hier vorgelegten Arbeit wird der Effekt von erhöhten Temperaturen und Hitze genauer analysiert. Hitze ist einer der wichtigsten abiotischen Stressfaktoren, der das Pflanzenwachstum und die Reproduktion beeinflusst. Viele wichtige Kulturpflanzen zeigen immense Ertragseinbußen, die durch Hitze hervorgerufen werden. Durch den fortschreitenden Klimawandel werden jedoch Hitzeperioden immer häufiger und somit die Folgen für die Nahrungsproduktion immer gravierender. Zur Züchtung von Pflanzen die hitzetolerant sind und weniger hohe Ertragseinbußen unter diesem Stress aufweisen, ist es essenziell die grundlegenden molekularen Mechanismen der Hitzetoleranz zu verstehen. Es müssen die verschiedenen physiologischen und biochemischen Prozesse identifiziert werden, die es Pflanzen ermöglichen, sich anzupassen. Es ist bekannt, dass die Anpassungsmechanismen von Pflanzen komplex sind und sowohl Veränderungen auf zellulärer wie auch auf organismischer Ebene beinhalten. Ziel dieser Arbeit war es, weitere Erkenntnisse zu gewinnen, wie diese Anpassung vonstattengeht und welche molekularen Prozesse an ihr beteiligt sind. Ein Hauptaugenmerk lag dabei auf dem Einfluss des Lipidmetabolismus und den daran beteiligten Enzymen. Es konnte bereits gezeigt werden, dass die Akkumulation von Triacylglycerolen bei hohen Temperaturen die basale Thermotoleranz bei Arabidopsis thaliana erhöht. Wie jedoch der genaue Mechanismus dieser durch Triacylglycerole vermittelten Thermotoleranz funktioniert, war bis dato nicht bekannt. Ich konnte zeigen, dass die angesammelten Triacylglycerole genutzt werden können, um die Stomata während des Hitzestress zu öffnen. Dies führt zu einer erhöhten Transpiration und somit einer Kühlung der Blätter. Der Abbau von Triacylglycerolen und Stärke am Morgen ist notwendig, um die Stomata zu öffnen. Zusätzlich dient der Abbau der Aufrechterhaltung des Citratzyklus und somit der Energieversorgung. In weiteren Experimenten konnte ich durch Fütterung mit stabil markierter Laurinsäure zeigen, dass die Triacylglycerole auch dem Aufbau neuer Aminosäuren unter Stressbedingungen dienen. Die hier vorgestellten Arbeiten bieten die Grundlage, um den Mechanismus der Thermotoleranz besser zu verstehen. Das Verständnis der in dieser Arbeit beschriebenen molekularen Signalwege und Enzyme kann langfristig dazu beitragen hitzeresistentere Nutzpflanzen zu züchten.
Tropical peat swamp forests sequester globally significant stores of carbon in deep layers of waterlogged, anoxic, acidic and nutrient-depleted peat. The roles of microbes in supporting these forests through the formation of peat, carbon sequestration and nutrient cycling are virtually unknown. This study investigated physicochemical peat properties and microbial diversity between three dominant tree species: Shorea uliginosa (Dipterocarpaceae), Koompassia malaccensis (legumes associated with nitrogen-fixing bacteria), Eleiodoxa conferta (palm) and depths (surface, 45 and 90 cm) using microbial 16S rRNA gene amplicon sequencing. Water pH, oxygen, nitrogen, phosphorus, total phenolic contents and C/N ratio differed significantly between depths, but not tree species. Depth also strongly influenced microbial diversity and composition, while both depth and tree species exhibited significant impact on the archaeal communities. Microbial diversity was highest at the surface, where fresh leaf litter accumulates, and nutrient supply is guaranteed. Nitrogen was the core parameter correlating to microbial communities, but the interactive effects from various environmental variables displayed significant correlation to relative abundance of major microbial groups. Proteobacteria was the dominant phylum and the most abundant genus, Rhodoplanes, might be involved in nitrogen fixation. The most abundant methanogens and methanotrophs affiliated, respectively, to families Methanomassiliicoccaceae and Methylocystaceae. Our results demonstrated diverse microbial communities and provide valuable insights on microbial ecology in these extreme ecosystems.
Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently (p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD.
Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities. However, the dependency of most methods on reference genomes, which are currently unavailable for a substantial fraction of microbial species, introduces estimation biases. We present an updated and functionally extended tool based on universal (i.e., reference-independent), phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) enabling the profiling of >7700 microbial species. As more than 30% of them could not previously be quantified at this taxonomic resolution, relative abundance estimates based on mOTUs are more accurate compared to other methods. As a new feature, we show that mOTUs, which are based on essential housekeeping genes, are demonstrably well-suited for quantification of basal transcriptional activity of community members. Furthermore, single nucleotide variation profiles estimated using mOTUs reflect those from whole genomes, which allows for comparing microbial strain populations (e.g., across different human body sites).
Thermal melanism theory states that dark-colored ectotherm organisms are at an advantage at low temperature due to increased warming. This theory is generally supported for ectotherm animals, however, the function of colors in the fungal kingdom is largely unknown. Here, we test whether the color lightness of mushroom assemblages is related to climate using a dataset of 3.2 million observations of 3,054 species across Europe. Consistent with the thermal melanism theory, mushroom assemblages are significantly darker in areas with cold climates. We further show differences in color phenotype between fungal lifestyles and a lifestyle differentiated response to seasonality. These results indicate a more complex ecological role of mushroom colors and suggest functions beyond thermal adaption. Because fungi play a crucial role in terrestrial carbon and nutrient cycles, understanding the links between the thermal environment, functional coloration and species’ geographical distributions will be critical in predicting ecosystem responses to global warming.
How insects promote crop pollination remains poorly understood in terms of the contribution of functional trait differences between species. We used meta-analyses to test for correlations between community abundance, species richness and functional trait metrics with oilseed rape yield, a globally important crop. While overall abundance is consistently important in predicting yield, functional divergence between species traits also showed a positive correlation. This result supports the complementarity hypothesis that pollination function is maintained by non-overlapping trait distributions. In artificially constructed communities (mesocosms), species richness is positively correlated with yield, although this effect is not seen under field conditions. As traits of the dominant species do not predict yield above that attributed to the effect of abundance alone, we find no evidence in support of the mass ratio hypothesis. Management practices increasing not just pollinator abundance, but also functional divergence, could benefit oilseed rape agriculture.
Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors.
On the basis of the results of previous national and international trials and studies, the Renal Tumour Study Group of the International Society of Paediatric Oncology (SIOP–RTSG) has developed a new study protocol for paediatric renal tumours: the UMBRELLA SIOP–RTSG 2016 protocol (the UMBRELLA protocol). Currently, the overall outcomes of patients with Wilms tumour are excellent, but subgroups with poor prognosis and increased relapse rates still exist. The identification of these subgroups is of utmost importance to improve treatment stratification, which might lead to reduction of the direct and late effects of chemotherapy. The UMBRELLA protocol aims to validate new prognostic factors, such as blastemal tumour volume and molecular markers, to further improve outcome. To achieve this aim, large, international, high-quality databases are needed, which dictate optimization and international harmonization of specimen handling and comprehensive sampling of biological material, refine definitions and improve logistics for expert review. To promote broad implementation of the UMBRELLA protocol, the updated SIOP–RTSG pathology and molecular biology protocol for Wilms tumours has been outlined, which is a consensus from the SIOP–RTSG pathology panel.
Increasing evidence indicates that forest disturbances are changing in response to global change, yet local variability in disturbance remains high. We quantified this considerable variability and analyzed whether recent disturbance episodes around the globe were consistently driven by climate, and if human influence modulates patterns of forest disturbance. We combined remote sensing data on recent (2001–2014) disturbances with in-depth local information for 50 protected landscapes and their surroundings across the temperate biome. Disturbance patterns are highly variable, and shaped by variation in disturbance agents and traits of prevailing tree species. However, high disturbance activity is consistently linked to warmer and drier than average conditions across the globe. Disturbances in protected areas are smaller and more complex in shape compared to their surroundings affected by human land use. This signal disappears in areas with high recent natural disturbance activity, underlining the potential of climate-mediated disturbance to transform forest landscapes.
The metabolic rewiring that occurs during cell transformation is a hallmark of cancer. It is diverse in different cancers as it reflects different combinations of oncogenic drivers, tumor suppressors, and the microenvironment. Metabolic rewiring is essential to cancer as it enables uncontrolled proliferation and adaptation to the fluctuating availability of nutrients and oxygen caused by poor access to the vasculature due to tumor growth and a foreign microenvironment encountered during metastasis. Increasing evidence now indicates that the metabolic state in cancer cells also plays a causal role in tumor growth and metastasis, for example through the action of oncometabolites, which modulate cell signaling and epigenetic pathways to promote malignancy. In addition to altering the metabolic state in cancer cells, some multifunctional enzymes possess non-metabolic functions that also contribute to cell transformation. Some multifunctional enzymes that are highly expressed in cancer, such as pyruvate kinase M2 (PKM2), have non-canonical functions that are co-opted by oncogenic signaling to drive proliferation and inhibit apoptosis. Other multifunctional enzymes that are frequently downregulated in cancer, such as fructose-bisphosphatase 1 (FBP1), are tumor suppressors, directly opposing mitogenic signaling via their non-canonical functions. In some cases, the enzymatic and non-canonical roles of these enzymes are functionally linked, making the modulation of non-metabolic cellular processes dependent on the metabolic state of the cell.
The Drosophila microbiome has a limited influence on sleep, activity, and courtship behaviors
(2018)
In animals, commensal microbes modulate various physiological functions, including behavior. While microbiota exposure is required for normal behavior in mammals, it is not known how widely this dependency is present in other animal species. We proposed the hypothesis that the microbiome has a major influence on the behavior of the vinegar fly (Drosophila melanogaster), a major invertebrate model organism. Several assays were used to test the contribution of the microbiome on some well-characterized behaviors: defensive behavior, sleep, locomotion, and courtship in microbe-bearing, control flies and two generations of germ-free animals. None of the behaviors were largely influenced by the absence of a microbiome, and the small or moderate effects were not generalizable between replicates and/or generations. These results refute the hypothesis, indicating that the Drosophila microbiome does not have a major influence over several behaviors fundamental to the animal’s survival and reproduction. The impact of commensal microbes on animal behaviour may not be broadly conserved.
Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.
Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.
Periodontal treatment prevents arthritis in mice and methotrexate ameliorates periodontal bone loss
(2019)
Recent studies indicate a causal relationship between the periodontal pathogen P. gingivalis and rheumatoid arthritis involving the production of autoantibodies against citrullinated peptides. We therefore postulated that therapeutic eradication P. gingivalis may ameliorate rheumatoid arthritis development and here turned to a mouse model in order to challenge our hypothesis. F1 (DBA/1 x B10.Q) mice were orally inoculated with P. gingivalis before collagen-induced arthritis was provoked. Chlorhexidine or metronidazole were orally administered either before or during the induction phase of arthritis and their effects on arthritis progression and alveolar bone loss were compared to intraperitoneally injected methotrexate. Arthritis incidence and severity were macroscopically scored and alveolar bone loss was evaluated via microcomputed tomography. Serum antibody titres against P. gingivalis were quantified by ELISA and microbial dysbiosis following oral inoculation was monitored in stool samples via microbiome analyses. Both, oral chlorhexidine and metronidazole reduced the incidence and ameliorated the severity of collagen-induced arthritis comparable to methotrexate. Likewise, all three therapies attenuated alveolar bone loss. Relative abundance of Porphyromonadaceae was increased after oral inoculation with P. gingivalis and decreased after treatment. This is the first study to describe beneficial effects of non-surgical periodontal treatment on collagen-induced arthritis in mice and suggests that mouthwash with chlorhexidine or metronidazole may also be beneficial for patients with rheumatoid arthritis and a coexisting periodontitis. Methotrexate ameliorated periodontitis in mice, further raising the possibility that methotrexate may also positively impact on the tooth supporting tissues of patients with rheumatoid arthritis.
Cell culture and protein target-based compound screening strategies, though broadly utilized in selecting candidate compounds, often fail to eliminate candidate compounds with non-target effects and/or safety concerns until late in the drug developmental process. Phenotype screening using intact research animals is attractive because it can help identify small molecule candidate compounds that have a high probability of proceeding to clinical use. Most FDA approved, first-in-class small molecules were identified from phenotypic screening. However, phenotypic screening using rodent models is labor intensive, low-throughput, and very expensive. As a novel alternative for small molecule screening, we have been developing gene expression disease profiles, termed the Transcriptional Disease Signature (TDS), as readout of small molecule screens for therapeutic molecules. In this concept, compounds that can reverse, or otherwise affect known disease-associated gene expression patterns in whole animals may be rapidly identified for more detailed downstream direct testing of their efficacy and mode of action. To establish proof of concept for this screening strategy, we employed a transgenic strain of a small aquarium fish, medaka (Oryzias latipes), that overexpresses the malignant melanoma driver gene xmrk, a mutant egfr gene, that is driven by a pigment cell-specific mitf promoter. In this model, melanoma develops with 100% penetrance. Using the transgenic medaka malignant melanoma model, we established a screening system that employs the NanoString nCounter platform to quantify gene expression within custom sets of TDS gene targets that we had previously shown to exhibit differential transcription among xmrk-transgenic and wild-type medaka. Compound-modulated gene expression was identified using an internet-accessible custom-built data processing pipeline. The effect of a given drug on the entire TDS profile was estimated by comparing compound-modulated genes in the TDS using an activation Z-score and Kolmogorov-Smirnov statistics. TDS gene probes were designed that target common signaling pathways that include proliferation, development, toxicity, immune function, metabolism and detoxification. These pathways may be utilized to evaluate candidate compounds for potential favorable, or unfavorable, effects on melanoma-associated gene expression. Here we present the logistics of using medaka to screen compounds, as well as, the development of a user-friendly NanoString data analysis pipeline to support feasibility of this novel TDS drug-screening strategy.
Despite their popularity as enzyme engineering targets structural information about Sucrose Phosphorylases remains scarce. We recently clarified that the Q345F variant of Bifidobacterium adolescentis Sucrose Phosphorylase is able to accept large polyphenolic substrates like resveratrol via a domain shift. Here we present a crystal structure of this variant in a conformation suitable for the accommodation of the donor substrate sucrose in excellent agreement with the wild type structure. Remarkably, this conformation does not feature the previously observed domain shift which is therefore reversible and part of a dynamic process rather than a static phenomenon. This crystallographic snapshot completes our understanding of the catalytic cycle of this useful variant and will allow for a more rational design of further generations of Sucrose Phosphorylase variants.
In most organisms, ribosomal RNA (rRNA) contributes to >85% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available.
Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.
Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.
Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models.
Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2,3,4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.
Colorectal cancer (CRC) is the second most common tumour disease in Germany, with the sequential accumulation of certain mutations playing a decisive role in the transition from adenoma to carcinoma. In particular, deregulation of the Wnt signalling pathway and the associated deregulated expression of the MYC oncoprotein play a crucial role. Targeting MYC thus represents an important therapeutic approach in the treatment of tumours. Since direct inhibition of MYC is challenging, various approaches have been pursued to date to target MYC indirectly. The MYC 5' UTR contains an internal ribosomal entry site (IRES), which has a particular role in the initiation of MYC translation, especially in multiple myeloma. As basis for this work, it was hypothesised on the basis of previous data that translation of MYC potentially occurs via its IRES in CRC as well. Based on this, two IRES inhibitors were tested for their potential to regulate MYC expression in CRC cells. In addition, alternative, 5’ UTR-dependent translation of MYC and interacting factors were investigated. EIF3D was identified as a MYC 5' UTR binding protein which has the potential to regulate MYC expression in CRC. The results of this work suggest that there is a link between eIF3D and MYC expression/translation, rendering eIF3D a potential therapeutic target for MYC-driven CRCs.
Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7–8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.
The remarkable diversity of sex determination mechanisms known in fish may be fuelled by exceptionally high rates of sex chromosome turnovers or transitions. However, the evolutionary causes and genomic mechanisms underlying this variation and instability are yet to be understood. Here we report on an over 30-year evolutionary experiment in which we tested the genomic consequences of hybridisation and selection between two Xiphophorus fish species with different sex chromosome systems. We find that introgression and imposing selection for pigmentation phenotypes results in the retention of an unexpectedly large maternally derived genomic region. During the hybridisation process, the sex-determining region of the X chromosome from one parental species was translocated to an autosome in the hybrids leading to the evolution of a new sex chromosome. Our results highlight the complexity of factors contributing to patterns observed in hybrid genomes, and we experimentally demonstrate that hybridisation can catalyze rapid evolution of a new sex chromosome.
Colon carcinomas (CRC) are statistically among the most fatal cancer types and hence one of the top reasons for premature mortality in the developed world. CRC cells are characterized by high proliferation rates caused by deregulation of gene transcription of proto-oncogenes and general chromosomal instability. On macroscopic level, CRC cells show a strongly altered nutrient and energy metabolism.
This work presents research to understand general links between the metabolism and transcription alteration. Mainly focussing on glutamine dependency, shown in colon carcinoma cells and expression pathways of the pro-proliferation protein c-MYC.
Previous studies showed that a depletion of glutamine in the cultivation medium of colon carcinoma cell lines caused a proliferation arrest and a strong decrease of overall c-MYC levels. Re-addition of glutamine quickly replenished c-MYC levels through an unknown mechanism. Several proteins altering this regulation mechanism were identified and proposed as possible starting point for further in detail studies to unveil the precise biochemical pathway controlling c-MYC translation repression and reactivation in a rapid manner.
On a transcriptional level the formation of RNA:DNA hybrids, so called R-loops, was observed under glutamine depleted conditions. The introduction and overexpression of RNaseH1, a R-loop degrading enzyme, in combination with an ectopically expressed c-MYC variant, independent of cellular regulation mechanisms by deleting the regulatory 3’-UTR of the c-MYC gene, lead to a high rate of apoptotic cells in culture. Expression of a functionally inactive variant of RNaseH1 abolished this effect. This indicates a regulatory function of R-loops formed during glutamine starvation in the presence of c-MYC protein in a cell. Degradation of R-loops and high c-MYC levels in this stress condition had no imminent effect on the cell cycle progression is CRC cells but disturbed the nucleotide metabolism. Nucleotide triphosphates were strongly reduced in comparison to starving cells without R-loop degradation and proliferating cells.
This study proposes a model of a terminal cycle of transcription termination, unregulated initiation and elongation of transcription leading to a depletion of energy resources of cells. This could finally lead to high apoptosis of the cells. Sequencing experiments to determine a coinciding of termination sites and R-loop formation sides failed so far but show a starting point for further studies in this essential survival mechanism involving R-loop formation and c-MYC downregulation.
African trypanosomes are unicellular parasites that cause nagana and sleeping sickness in livestock and man, respectively. The major pathogens for the animal disease include Trypanosoma vivax, T. congolense, and T. brucei brucei, whereas T. b. gambiense and T. b. rhodesiense are responsible for human infections. Given that the bloodstream form (BSF) of African trypanosomes is exclusively extracellular, its cell surface forms a critical boundary with the host environment. The cell surface of the BSF African trypanosomes is covered by a dense coat of immunogenic variant surface glycoproteins (VSGs). This surface protein acts as an impenetrable shield that protects the cells from host immune factors and is also involved in antibody clearance and antigenic variation, which collectively ensure that the parasite stays ahead of the host immune system. Gene expression in T. brucei is markedly different from other eukaryotes: most genes are transcribed as long polycistronic units, processed by trans-splicing a 39-nucleotide mini exon at the 5′ and polyadenylation at the 3′ ends of individual genes to generate the mature mRNA.
Therefore, gene expression in T. brucei is regulated post-transcriptionally, mainly by the action of RNA binding proteins (RBPs) and conserved elements in the 3′ untranslated regions (UTR) of transcripts. The expression of VSGs is highly regulated, and only a single VSG gene is expressed at a time from one of the ~15 subtelomeric domains termed bloodstream expression sites (BES). When cells are engineered to simultaneously express two VSGs, the total VSG mRNA do not exceed the wild type amounts. This suggests that a robust VSG mRNA balancing mechanism exists in T. brucei. The present study uses inducible and constitutive expression of ectopic VSG genes to show that the endogenous VSG mRNA is regulated only if the second VSG is properly targeted to the ER. Additionally, the endogenous VSG mRNA response is triggered when high amounts of the GFP reporter with a VSG 3′UTR is targeted to the ER. Further evidence that non-VSG ER import signals can efficiently target VSGs to the ER is presented. This study suggests that a robust trans-regulation of the VSG mRNA is elicited at the ER through a feedback loop to keep the VSG transcripts in check and avoid overshooting the secretory pathway capacity.
Further, it was shown that induction of expression of the T. vivax VSG ILDat1.2 in T. brucei causes a dual cell cycle arrest, with concomitant upregulation of the protein associated with differentiation (PAD1) expression. It could be shown that T. vivax VSG ILDat1.2 can only be sufficiently expressed in T. brucei after replacing its native GPI signal peptide with that of a T. brucei VSG. Taken together, these data indicate that inefficient VSG GPI anchoring and expression of low levels of the VSG protein can trigger differentiation from slender BSF to stumpy forms. However, a second T. vivax VSG, ILDat2.1, is not expressed in T. brucei even after similar modifications to its GPI signals. An X-ray crystallography approach was utilized to solve the N-terminal domain (NTD) structure of VSG ILDat1.2. This is first structure of a non-T. brucei VSG, and the first of a surface protein of T. vivax to be solved. VSG ILDat1.2 NTD maintains the three-helical bundle scaffold conserved in T. brucei surface proteins. However, it is likely that there are variations in the architecture of the membrane proximal region of the ILDat1.2 NTD and its CTD from T. brucei VSGs. The tractable T. brucei system is presented as a model that can be used to study surface proteins of related trypanosome species, thus creating avenues for further characterization of trypanosome surface coats.
Cognition refers to the ability to of animals to acquire, process, store and use vital information from the environment. Cognitive processes are necessary to predict the future and reduce the uncertainty of the ever-changing environment. Classically, research on animal cognition focuses on decisive cognitive tests to determine the capacity of a species by the testing the ability of a few individuals. This approach views variability between these tested key individuals as unwanted noise and is thus often neglected. However, inter-individual variability provides important insights to behavioral plasticity, cognitive specialization and brain modularity. Honey bees Apis mellifera are a robust and traditional model for the study of learning, memory and cognition due to their impressive capabilities and rich behavioral repertoire. In this thesis I have applied a novel view on the learning abilities of honey bees by looking explicitly at individual differences in a variety of learning tasks. Are some individual bees consistently smarter than some of her sisters? If so, will a smart individual always perform good independent of the time, the context and the cognitive requirements or do bees show distinct isolated ‘cognitive modules’?
My thesis presents the first comprehensive investigation of consistent individual differences in the cognitive abilities of honey bees. To speak of an individual as behaving consistently, a crucial step is to test the individual multiple times to examine the repeatability of a behavior. I show that free-flying bees remain consistent in a visual discrimination task for three consecutive days. Successively, I explored individual consistency in cognitive proficiency across tasks involving different sensory modalities, contexts and cognitive requirements. I found that free-flying bees show a cognitive specialization between visual and olfactory learning but remained consistent across a simple discrimination task and a complex concept learning task. I wished to further explore individual consistency with respect to tasks of different cognitive complexity, a question that has never been tackled before in an insect. I thus performed a series of four experiments using either visual or olfactory stimuli and a different training context (free-flying and restrained) and tested bees in a discrimination task, reversal learning and negative patterning. Intriguingly, across all these experiments I evidenced the same results: The bees’ performances were consistent across the discrimination task and reversal learning and negative patterning respectively. No association was evidenced between reversal learning and negative patterning. After establishing the existence of consistent individual differences in the cognitive proficiency of honey bees I wished to determine factors which could underlie these differences. Since genetic components are known to underlie inter-individual variability in learning abilities, I studied the effects of genetics on consistency in cognitive proficiency by contrasting bees originating from either from a hive with a single patriline (low genetic diversity) or with multiple patrilines (high genetic diversity). These two groups of bees showed differences in the patterns of individually correlated performances, indicating a genetic component accounts for consistent cognitive individuality. Another major factor underlying variability in learning performances is the individual responsiveness to sucrose solution and to visual stimuli, as evidenced by many studies on restrained bees showing a positive correlation between responsiveness to task relevant stimuli and learning performances. I thus tested whether these relationships between sucrose/visual responsiveness and learning performances are applicable for free-flying bees. Free-flying bees were again subjected to reversal learning and negative patterning and subsequently tested in the laboratory for their responsiveness to sucrose and to light. There was no evidence of a positive relationship between sucrose/visual responsiveness and neither performances of free-flying bees in an elemental discrimination, reversal learning and negative patterning. These findings indicate that relationships established between responsiveness to task relevant stimuli and learning proficiency established in the laboratory with restrained bees might not hold true for a completely different behavioral context i.e. for free-flying bees in their natural environment.
These results show that the honey bee is an excellent insect model to study consistency in cognitive proficiency and to identify the underlying factors. I mainly discuss the results with respect to the question of brain modularity in insects and the adaptive significance of individuality in cognitive abilities for honey bee colonies. I also provide a proposition of research questions which tie in this theme of consistent cognitive proficiency and could provide fruitful areas for future research.
SMART (Simple Modular Architecture Research Tool) is a web resource (https://smart.embl.de) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version 9 contains manually curatedmodels formore than 1300 protein domains, with a topical set of 68 new models added since our last update article (1). All the new models are for diverse recombinase families and subfamilies and as a set they provide a comprehensive overview of mobile element recombinases namely transposase, integrase, relaxase, resolvase, cas1 casposase and Xer like cellular recombinase. Further updates include the synchronization of the underlying protein databases with UniProt (2), Ensembl (3) and STRING (4), greatly increasing the total number of annotated domains and other protein features available in architecture analysis mode. Furthermore, SMART's vector-based protein display engine has been extended and updated to use the latest web technologies and the domain architecture analysis components have been optimized to handle the increased number of protein features available.
In 2020, cancer was the leading cause of death worldwide, accounting for nearly 10 million deaths. Lung cancer was the most common cancer, with 2.21 million cases per year in both sexes. This non-homogeneous disease is further subdivided into small cell lung cancer (SCLC, 15%) and non-small cell lung cancer (NSCLC, 85%). By 2023, the American Cancer Society estimates that NSCLC will account for 13% of all new cancer cases and 21% of all estimated cancer deaths. In recent years, the treatment of patients with NSCLC has improved with the development of new therapeutic interventions and the advent of targeted and personalised therapies. However, these advances have only marginally improved the five-year survival rate, which remains alarmingly low for patients with NSCLC. This observation highlights the importance of having more appropriate experimental and preclinical models to recapitulate, identify and test novel susceptibilities in NSCLC. In recent years, the Trp53fl/fl KRaslsl-G12D/wt mouse model developed by Tuveson, Jacks and Berns has been the main in vivo model used to study NSCLC. This model mimics ADC and SCC to a certain extent. However, it is limited in its ability to reflect the genetic complexity of NSCLC. In this work, we use CRISPR/Cas9 genome editing with targeted mutagenesis and gene deletions to recapitulate the conditional model. By comparing the Trp53fl/fl KRaslsl- G12D/wt with the CRISPR-mediated Trp53mut KRasG12D, we demonstrated that both showed no differences in histopathological features, morphology, and marker expression. Furthermore, next-generation sequencing revealed a very high similarity in their transcriptional profile. Adeno-associated virus-mediated tumour induction and the modular design of the viral vector allow us to introduce additional mutations in a timely manner. CRISPR-mediated mutation of commonly mutated tumour suppressors in NSCLC reliably recapitulated the phenotypes described in patients in the animal model. Lastly, the dual viral approach could induce the formation of lung tumours not only in constitutive Cas9 expressing animals, but also in wildtype animals. Thus, the implementation of CRISPR genome editing can rapidly advance the repertoire of in vivo models for NSCLC research. Furthermore, it can reduce the necessity of extensive breeding.
Across Europe, calcareous grasslands become increasingly fragmented and their quality deteriorates through abandonment and land use intensification, both affecting biodiversity. Here, we investigated local and landscape effects on diversity patterns of several taxonomic groups in a landscape of highly fragmented calcareous grassland remnants. We surveyed 31 grassland fragments near Göttingen, Germany, in spring and summer 2017 for vascular plants, butterflies and birds, with sampling effort adapted to fragment area. Through regression modelling, we tested relationships between species richness and fragment size (from 314 to 51,395 m\(^2\)), successional stage, habitat connectivity and the per cent cover of arable land in the landscape at several radii. We detected 283 plant species, 53 butterfly species and 70 bird species. Of these, 59 plant species, 19 butterfly species and 9 bird species were grassland specialists. Larger fragments supported twice the species richness of plants than small ones, and hosted more species of butterflies, but not of birds. Larger grassland fragments contained more grassland specialist plants, but not butterfly or bird specialists. Increasing amounts of arable land in the landscape from 20 to 90% was related to the loss of a third of species of plants, and less so, of butterflies, but not of birds. Per cent cover of arable land negatively correlated to richness of grassland specialist plants and butterflies, but positively to grassland specialist birds. We found no effect by successional stages and habitat connectivity. Our multi-taxa approach highlights the need for conservation management at the local scale, complemented by measures at the landscape scale.
Quantitative information is omnipresent in the world and a wide range of species has been shown to use quantities to optimize their decisions. While most studies have focused on vertebrates, a growing body of research demonstrates that also insects such as honeybees possess basic quantitative abilities that might aid them in finding profitable flower patches. However, it remains unclear if for insects, quantity is a salient feature relative to other stimulus dimensions, or if it is only used as a “last resort” strategy in case other stimulus dimensions are inconclusive. Here, we tested the stingless bee Trigona fuscipennis, a species representative of a vastly understudied group of tropical pollinators, in a quantity discrimination task. In four experiments, we trained wild, free-flying bees on stimuli that depicted either one or four elements. Subsequently, bees were confronted with a choice between stimuli that matched the training stimulus either in terms of quantity or another stimulus dimension. We found that bees were able to discriminate between the two quantities, but performance differed depending on which quantity was rewarded. Furthermore, quantity was more salient than was shape. However, quantity did not measurably influence the bees' decisions when contrasted with color or surface area. Our results demonstrate that just as honeybees, small-brained stingless bees also possess basic quantitative abilities. Moreover, invertebrate pollinators seem to utilize quantity not only as "last resort" but as a salient stimulus dimension. Our study contributes to the growing body of knowledge on quantitative cognition in invertebrate species and adds to our understanding of the evolution of numerical cognition.
Meiotic chromosomes undergo rapid prophase movements, which are thought to facilitate the formation of inter-homologue recombination intermediates that underlie synapsis, crossing over and segregation. The meiotic telomere complex (MAJIN, TERB1, TERB2) tethers telomere ends to the nuclear envelope and transmits cytoskeletal forces via the LINC complex to drive these rapid movements. Here, we report the molecular architecture of the meiotic telomere complex through the crystal structure of MAJIN-TERB2, together with light and X-ray scattering studies of wider complexes. The MAJIN-TERB2 2:2 hetero-tetramer binds strongly to DNA and is tethered through long flexible linkers to the inner nuclear membrane and two TRF1-binding 1:1 TERB2-TERB1 complexes. Our complementary structured illumination microscopy studies and biochemical findings reveal a telomere attachment mechanism in which MAJIN-TERB2-TERB1 recruits telomere-bound TRF1, which is then displaced during pachytene, allowing MAJIN-TERB2-TERB1 to bind telomeric DNA and form a mature attachment plate.
Fanconi anemia (FA) is a genetically heterogeneous disorder with 22 disease-causing genes reported to date. In some FA genes, monoallelic mutations have been found to be associated with breast cancer risk, while the risk associations of others remain unknown. The gene for FA type C, FANCC, has been proposed as a breast cancer susceptibility gene based on epidemiological and sequencing studies. We used the Oncoarray project to genotype two truncating FANCC variants (p.R185X and p.R548X) in 64,760 breast cancer cases and 49,793 controls of European descent. FANCC mutations were observed in 25 cases (14 with p.R185X, 11 with p.R548X) and 26 controls (18 with p.R185X, 8 with p.R548X). There was no evidence of an association with the risk of breast cancer, neither overall (odds ratio 0.77, 95%CI 0.44–1.33, p = 0.4) nor by histology, hormone receptor status, age or family history. We conclude that the breast cancer risk association of these two FANCC variants, if any, is much smaller than for BRCA1, BRCA2 or PALB2 mutations. If this applies to all truncating variants in FANCC it would suggest there are differences between FA genes in their roles on breast cancer risk and demonstrates the merit of large consortia for clarifying risk associations of rare variants.
MYC paralogs are frequently activated in small cell lung cancer (SCLC) but represent poor drug targets. Thus, a detailed mapping of MYC-paralog-specific vulnerabilities may help to develop effective therapies for SCLC patients. Using a unique cellular CRISPR activation model, we uncover that, in contrast to MYCN and MYCL, MYC represses BCL2 transcription via interaction with MIZ1 and DNMT3a. The resulting lack of BCL2 expression promotes sensitivity to cell cycle control inhibition and dependency on MCL1. Furthermore, MYC activation leads to heightened apoptotic priming, intrinsic genotoxic stress and susceptibility to DNA damage checkpoint inhibitors. Finally, combined AURK and CHK1 inhibition substantially prolongs the survival of mice bearing MYC-driven SCLC beyond that of combination chemotherapy. These analyses uncover MYC-paralog-specific regulation of the apoptotic machinery with implications for genotype-based selection of targeted therapeutics in SCLC patients.
Animals must slow or halt locomotion to integrate sensory inputs or to change direction. In Caenorhabditis elegans, the GABAergic and peptidergic neuron RIS mediates developmentally timed quiescence. Here, we show RIS functions additionally as a locomotion stop neuron. RIS optogenetic stimulation caused acute and persistent inhibition of locomotion and pharyngeal pumping, phenotypes requiring FLP-11 neuropeptides and GABA. RIS photoactivation allows the animal to maintain its body posture by sustaining muscle tone, yet inactivating motor neuron oscillatory activity. During locomotion, RIS axonal Ca2+ signals revealed functional compartmentalization: Activity in the nerve ring process correlated with locomotion stop, while activity in a branch correlated with induced reversals. GABA was required to induce, and FLP-11 neuropeptides were required to sustain locomotion stop. RIS attenuates neuronal activity and inhibits movement, possibly enabling sensory integration and decision making, and exemplifies dual use of one cell across development in a compact nervous system.
Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a thus far unknown cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the lytic-latent switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily drugable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.
The expression of the MYC proto-oncogene is elevated in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC). Previous findings in PDAC have shown that this increased MYC expression mediates immune evasion and promotes S-phase progression. How these functions are mediated and whether a downstream factor of MYC mediates these functions has remained elusive. Recent studies identifying the MYC interactome revealed a complex network of interaction partners, highlighting the need to identify the oncogenic pathway of MYC in an unbiased manner.
In this work, we have shown that MYC ensures genomic stability during S-phase and prevents transcription-replication conflicts. Depletion of MYC and inhibition of ATR kinase showed a synergistic effect to induce DNA damage. A targeted siRNA screen targeting downstream factors of MYC revealed that PAF1c is required for DNA repair and S-phase progression. Recruitment of PAF1c to RNAPII was shown to be MYC dependent. PAF1c was shown to be largely dispensable for cell proliferation and regulation of MYC target genes.
Depletion of CTR9, a subunit of PAF1c, caused strong tumor regression in a pancreatic ductal adenocarcinoma model, with long-term survival in a subset of mice. This effect was not due to induction of DNA damage, but to restoration of tumor immune surveillance.
Depletion of PAF1c resulted in the release of RNAPII with transcription elongation factors, including SPT6, from the bodies of long genes, promoting full-length transcription of short genes. This resulted in the downregulation of long DNA repair genes and the concomitant upregulation of short genes, including MHC class I genes. These data demonstrate that a balance between long and short gene transcription is essential for tumor progression and that interference with PAF1c levels shifts this balance toward a tumor-suppressive transcriptional program. It also directly links MYC-mediated S-phase progression to immune evasion. Unlike MYC, PAF1c has a stable, known folded structure; therefore, the development of a small molecule targeting PAF1c may disrupt the immune evasive function of MYC while sparing its physiological functions in cellular growth.