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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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
While our knowledge about the roles of microbes and viruses in the ocean has increased tremendously due to recent advances in genomics and metagenomics, research on marine microbial eukaryotes and zooplankton has benefited much less from these new technologies because of their larger genomes, their enormous diversity, and largely unexplored physiologies. Here, we use a metatranscriptomics approach to capture expressed genes in open ocean Tara Oceans stations across four organismal size fractions. The individual sequence reads cluster into 116 million unigenes representing the largest reference collection of eukaryotic transcripts from any single biome. The catalog is used to unveil functions expressed by eukaryotic marine plankton, and to assess their functional biogeography. Almost half of the sequences have no similarity with known proteins, and a great number belong to new gene families with a restricted distribution in the ocean. Overall, the resource provides the foundations for exploring the roles of marine eukaryotes in ocean ecology and biogeochemistry.
HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi
(2018)
The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.