Biology subjects

Popovic, M.

Publications and source records attributed to Popovic, M..

32 records · Page 2Linked to original sources

bioRxiv2021

The landscape of high-affinity human antibodies against intratumoral antigens

High expression of immunoglobulin transcripts is often detected in human tumours and correlates with favourable clinical outcomes across different cancer types. However, the antigens recognized by such immunoglobulins (Ig) and their biological significance remain largely unknown. We computationally inferred the paired sequence of thousands of clonally expanded Ig using bulk RNA sequencing data from solid tumors in The Cancer Genome Atlas (TCGA). After expressing 283 Ig as recombinant antibodies in mammalian cells, we individually tested them against a library of twenty thousands full-length human proteins and an additional library of six thousand structurally intact membrane proteins, thus identifying their candidate target antigens. Using surface plasmon resonance we then confirmed 16 high-affinity antibodies that bind to their targets with KD in the nanomolar range. Our work provides insights into the antigens that drive B cell responses in human cancers, while also obtaining fully human, high-affinity recombinant antibodies against them.

immunology↗

bioRxiv2020

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress which is known to induce genomic instability causing chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea induced replicative stress versus normal growth conditions in an equally-mixed cell population of isogenic euploid and 17q+ hESCs. We could show that 17q+ hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

cancer biology↗

bioRxiv2020

Somite deformations buffer imprecise segment lengths to ensure left-right symmetry

The body axis of vertebrate embryos is periodically segmented into bilaterally symmetric pairs of somites. The anteroposterior (AP) length of somites, their position and left-right symmetry are thought to be molecularly determined prior to somite morphogenesis. Here we discover that in zebrafish embryos, initial somite AP lengths and positions are imprecise and consequently many somite pairs form left-right asymmetrically. Strikingly, these imprecisions are not left unchecked and we find that AP lengths adjust within an hour after somite formation, thereby increasing morphological symmetry. We find that AP length adjustments result entirely from changes in somite shape without change in somite volume, with changes in AP length being compensated by corresponding changes in mediolateral length. The AP adjustment mechanism is facilitated by somite surface tension, which we show by comparing in vivo experiments and in vitro single-somite explant cultures with a mechanical model. Length adjustment is inhibited by perturbation of Integrin and Fibronectin, consistent with their involvement in surface tension. In contrast, the adjustment mechanism is unaffected by perturbations to the segmentation clock, thus revealing a distinct process that determines morphological segment lengths. We propose that tissue surface tension provides a general mechanism to adjust shapes and ensure precision and symmetry of tissues in developing embryos.

developmental biology↗

bioRxiv2020

Self-organized patterning of cell morphology via mechanosensitive feedback

Tissue organization is often characterized by specific patterns of cell morphology. How such patterns emerge in developing tissues is a fundamental open question. Here, we investigate the emergence of tissue-scale patterns of cell shape and mechanical tissue stress in the Drosophila wing imaginal disc during larval development. Using quantitative analysis of the cellular dynamics, we reveal a pattern of radially oriented cell rearrangements that is coupled to the buildup of tangential cell elongation. Developing a laser ablation method, we map tissue stresses and extract key parameters of tissue mechanics. We present a continuum theory showing that this pattern of cell morphology and tissue stress can arise via self-organization of a mechanical feedback that couples cell polarity to active cell rearrangements. The predictions of this model are supported by knockdown of MyoVI, a component of mechanosensitive feedback. Our work reveals a mechanism for the emergence of cellular patterns in morphogenesis.

developmental biology↗

bioRxiv2020

Turning old foes into new allies - harnessing drainage canals for biodiversity conservation in desiccated novel ecosystems

O_LIDrainage canals are ubiquitous components of agricultural landscapes worldwide. Although canals have greatly contributed to biodiversity loss by desiccating wetlands, they have recently attracted conservation attention due to their potential to function as refugia for native wetland-dependent species in intensively managed landscapes. However, their conservation role in complex landscapes comprising a mosaic of agricultural and desiccated semi-natural habitats, on which canals still pose a heavy burden, is unknown. Improved understanding of drainage canals and related biodiversity in these landscapes could help unlock their potential and support synergistic land management for nature conservation and water management. C_LIO_LIWe applied a multitaxon approach, including plants, butterflies, true bugs, spiders and birds, to (1) assess the conservation value of drainage canals in a heavily drained European lowland region, (2) to test landscape-level and local canal parameters for aiding prioritization among canal types, and (3) to propose a reconciliation-based management framework that suits the interest of all stakeholders. C_LIO_LIWe found that drainage canals concentrate more species across most taxa than adjacent semi-natural habitats, owing to the micro-environmental heterogeneity and the comparatively low management intensity in the canals. The species-concentrating capacity is particularly high in canals that traverse semi-natural habitats, although agricultural canals also support remarkable species diversity. However, agricultural canals are important dispersal corridors for invasive plants, which may negatively affect native species. Canal size has little effect on biodiversity but habitat stress is an important determinant. The higher the stress (due to sandiness and salinity), the higher is the added value of canals to landscape-wide biodiversity. C_LI Synthesis and applicationsWe provide evidence that drainage canals can harbour surprisingly high levels of biodiversity and should therefore be recognized as important novel ecosystems with high conservation value, even within semi-natural habitats. Canals have previously been considered detrimental to nature conservation due to their association with loss of wetlands. However, by reducing water loss with reversible obstructions, controlling invasive species and applying specific conservation measures, they may be turned into conservation allies without compromising long-term interests of water management and agricultural land use.

ecology↗

bioRxiv2020

Search for top-down and bottom-up drivers of latitudinal trends in insect herbivory in oak trees in Europe

AimThe strength of species interactions is traditionally expected to become stronger toward the Equator. However, recent studies have reported opposite or inconsistent latitudinal trends in the bottom-up (plant quality) and top-down (natural enemies) forces driving insect herbivory, possibly because these forces have rarely been studied concomitantly. This makes previous attempts to understand the effect of large scale climatic gradients on insect herbivory unsuccessful. LocationEurope Time period2018-2019 Major taxa studiedQuercus robur MethodsWe used scholar-based citizen science to simultaneously test for latitudinal variation in plant-herbivore-natural enemy interactions. We further investigated the underlying climatic factors associated with variation in herbivory, leaf chemistry and attack rates in Quercus robur across its complete latitudinal range in Europe. We quantified insect herbivory and the occurrence of specialist herbivores as well as leaf chemistry and bird attack rates on dummy caterpillars on 261 oak trees. ResultsClimatic factors rather than latitude per se were the best predictors of the large-scale (geographical) variation in the incidence of gall-inducers and leaf-miners as well as of leaf nutritional quality. However, insect herbivory, plant chemical defences (leaf phenolics) and bird attack rates were not influenced by latitude or climatic factors. The incidence of leaf-miners increased with increasing concentrations of hydrolysable tannins and decreased with those of condensed tannins, whereas the incidence of gall-inducers increased with increasing leaf soluble sugar concentration and decreased with increasing leaf C:N ratios. However, neither other traits nor bird attack rates varied with insect herbivory. Main conclusionsThese findings help to refine our understanding of the bottom-up and top-down mechanisms driving geographical variation in plant-herbivore interactions, and urge for further examination of the drivers of insect herbivory on trees.

ecology↗

bioRxiv2020

SPRTN protease and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability

DNA-protein crosslinks (DPCs) are a specific type of DNA lesions where proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration and accelerated ageing. DPC proteolysis was recently discovered as a specialised pathway for DPC repair. The DNA-dependent SPRTN protease and 26S proteasome emerged as as two independent proteolytic systems for DPC repair. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the role of ubiquitin and SUMO in DPC repair, we identified mutually exclusive signalling mechanisms associated with DPC repair pathway choice. DPC modification by SUMO-1 favours SPRTN proteolysis as the preferred pathway for DPC repair. DPC SUMOylation counteracts DPC ubiquitination, which promotes DNA breaks and the switch to HR. We propose that modification of DPCs by SUMO-1 promotes SPRTN proteolysis, which is essential for DPC removal to prevent DNA replication defects, chromosomal recombination and genomic instability.

cell biology↗

bioRxiv2019

Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

DNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of terminally differentiated gametes to the pluripotent cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, thereby allowing us to systematically probe the dynamics of global demethylation. First, when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, using scMspJI-seq in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, we found that human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse preimplantation embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells, thereby enabling quantitative investigation of methylation dynamics in developmental systems.

genomics↗

bioRxiv2019

Can school children support ecological research? Lessons from the ‘Oak bodyguard’ citizen science project

Scientific knowledge in the field of ecology is increasingly enriched by data acquired by the general public participating in citizen science (CS) programs. Yet, doubts remain about the reliability of such data, in particular when acquired by school children. We built upon an ongoing CS program - Oak bodyguards - to assess the ability of European schoolchildren to accurately estimate the strength of biotic interactions in terrestrial ecosystems. We used standardized protocols to estimate predation rates on artificial caterpillars and insect herbivory on oak leaves and compared estimates made by school children, trained and untrained professional scientists (with no or limited expertise in predation or herbivory assessment). Compared to trained scientists, both schoolchildren and untrained professional scientists overestimated predation rates, but assessments made by the latter were more consistent. School children overestimated insect herbivory, as did untrained professional scientists. Thus, raw data acquired by school children participating in CS programs cannot be used and require several quality checks. However, such data are of no less value than data collected by untrained professional scientists and can be calibrated for bias.

scientific communication and education↗

bioRxiv2018

SPRTN Protease and Checkpoint Kinase 1 Cross-Activation Loop Safeguards DNA Replication

The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease activity exhibit severe DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer in humans and mice. Strikingly, SPRTN-deficient cells also show a severe G2/M checkpoint defect and fail to activate a robust checkpoint kinase 1 (CHK1) signalling cascade normally triggered in response to replication stress. Here, we show that SPRTN activates the CHK1 signalling cascade during physiological (steady-state) DNA replication by proteolysis-dependent eviction of CHK1 from chromatin. The N-terminal CHK1 fragments cleaved by SPRTN protease still possess kinase activity and phosphorylate SPRTN. This CHK1-dependent phosphorylation stimulates SPRTN recruitment to chromatin to further promote CHK1 eviction from chromatin, DPC removal and unperturbed DNA replication. Our data suggest that a SPRTN-CHK1 cross-activation loop is essential for steady-state DNA replication and protection from DNA replication stress, a major source of genome instability in diseases that cause premature ageing and cancer. In addition, we disclose a mechanism of CHK1 activation during physiological DNA replication, when long stretches of single stranded (ss) DNA that are induced by genotoxic stress and activate canonical and robust CHK1 signalling are not present.

cell biology↗

bioRxiv2017

Development of cross-orientation suppression and size tuning and the role of experience

Many sensory neural circuits exhibit response normalization, which occurs when the response of a neuron to a combination of multiple stimuli is less than the sum of the responses to the individual stimuli presented alone. In the visual cortex, normalization takes the forms of cross-orientation suppression and surround suppression. At the onset of visual experience, visual circuits are partially developed and exhibit some mature features such as orientation selectivity, but it is unknown whether cross-orientation suppression or surround suppression are present at the onset of visual experience or require visual experience for their emergence. We characterized the development of these properties and their dependence on visual experience in ferrets. Visual experience was varied across three conditions: typical rearing, dark rearing, and dark rearing with daily exposure to simple sinusoidal gratings (14-16 hours total). Cross-orientation suppression and surround suppression were noted in the earliest observations, and did not vary considerably with experience. We also observed evidence of continued maturation of receptive field properties in the second month of visual experience: substantial length summation was observed only in the oldest animals (postnatal day 90); evoked firing rates were greatly increased in older animals; and direction selectivity required experience, but declined slightly in older animals. These results constrain the space of possible circuit implementations of these features.\n\nSignificance StatementThe development of the brain depends on both nature - factors that are independent of the experience of an individual animal - and nurture - factors that depend on experience. While orientation selectivity, one of the major response properties of neurons in visual cortex, is already present at the onset of visual experience, it is unknown if response properties that depend on interactions among multiple stimuli develop without experience. We find that the properties of crossorientation suppression and surround suppression are present at eye opening, and do not depend on visual experience. Our results are consistent with the idea that a majority of the basic properties of sensory neurons in primary visual cortex are derived independent of the experience of an individual animal.

neuroscience↗

bioRxiv2017

Fast and Accurate Genomic Analyses using Genome Graphs

The human reference genome serves as the foundation for genomics by providing a scaffold for alignment of sequencing reads, but currently only reflects a single consensus haplotype, which impairs read alignment and downstream analysis accuracy. Reference genome structures incorporating known genetic variation have been shown to improve the accuracy of genomic analyses, but have so far remained computationally prohibitive for routine large-scale use. Here we present a graph genome implementation that enables read alignment across 2,800 diploid genomes encompassing 12.6 million SNPs and 4.0 million indels. Our Graph Genome Pipeline requires 6.5 hours to process a 30x coverage WGS sample on a system with 36 CPU cores compared with 11 hours required by the GATK Best Practices pipeline. Using complementary benchmarking experiments based on real and simulated data, we show that using a graph genome reference improves read mapping sensitivity and produces a 0.5% increase in variant calling recall, or about 20,000 additional variants being detected per sample, while variant calling specificity is unaffected. Structural variations (SVs) incorporated into a graph genome can be genotyped accurately under a unified framework. Finally, we show that iterative augmentation of graph genomes yields incremental gains in variant calling accuracy. Our implementation is a significant advance towards fulfilling the promise of graph genomes to radically enhance the scalability and accuracy of genomic analyses.

bioinformatics↗

bioRxiv2017

Cell Dynamics Underlying Oriented Growth Of The Drosophila Imaginal Wing Disc

Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding our ability to understand how morphogen patterns regulate tissue shape. Such analysis requires imaging explants under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. Here, we demonstrate a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. We find that low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. We use 20E-containing media to directly observe growth and apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. We discover that while cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc.

developmental biology↗

bioRxiv2016

Perfluorooctane sulfonate affects proliferation and differentiation of pluripotent human teratocarcinoma cells

Perfluorinated compounds have raised concern due to their potential association with detrimental postnatal outcomes in animals and humans. We tested the effects of perfluorooctane sulfonate (PFOS) on a human pluripotent teratocarcinoma (known as NCCIT) cells as an in vitro prototype for developmental toxicity in mammals. NCCIT contains stem-cells able to differentiate into endoderm, mesoderm and ectoderm. We tested our cell model using a teratogenic compound, retinoic acid (RA), a cytotoxin, nocodazole (ND), and PFOS. We assayed cells proliferation, morphology and expression of stem cell and germ layer marker genes. PFOS reduced NCCIT cell proliferation in a concentration-dependent manner and induced morphological changes in cell cultures that resembled ectodermal phenotypes. A tendency towards a differentiated state in NCCIT was confirmed by real-time gene expression. PFOS triggered up-regulation of the gene nestin, indicative of ectodermal lineage differentiation, and interfered with the expression of the pluripotency stem-cell marker TERT. PFOS produced effects on both cells proliferation and differentiation, although not as severe as those observed for RA and ND, at levels that fall within the range of concentrations found in animal and human plasma. We discuss our findings in the context of possible interference of PFOS with the processes governing the early development of mammalian tissues.

pharmacology and toxicology↗