Biology subjects

Nikolic, M.

Publications and source records attributed to Nikolic, M..

16 recordsLinked to original sources

bioRxiv2025

Fibroblast specialisation across microanatomy in a single-cell atlas of healthy human Achilles tendon

Tendons are transitional tissues linking muscle to bone, enabling locomotion and fine motor control. The cellular biology across the Achilles tendon unit is poorly understood, yet critical for interpreting normal function and pathological changes across its microanatomically-defined functional zones. We generated a spatially-resolved transcriptomic atlas of human Achilles tendon, sampling the tendon-bone junction (enthesis), midbody, myotendinous junction, and adjoining muscle. Six fibroblast subtypes were identified, with distinct transcriptional profiles and spatial distributions, suggesting specialised functional roles across the tendon-muscle unit. Two dominant fibroblast types were specifically positioned in the tendon mid-substance and paratenon (vessel-rich region surrounding the tendon fibrils); other populations included perineural, myotendinous junction-specific, muscle-specific, and lining-layer fibroblasts. These findings demonstrate how cellular diversity across a transitional tissue may underlie microanatomical-specific roles. This atlas provides a foundation for understanding cellular functions across the tendon-muscle unit and will be essential for comparisons with diseased tissue, identifying pathogenic mediators and treatment targets for autoimmune and degenerative pathologies of the Achilles tendon.

genomics↗

bioRxiv2025

Variant U1 snRNAs contribute to cell cycle and differentiation control of human iPS cells

Maintenance of stem cell identity as well as differentiation of stem cells into the various lineages requires intricate regulation of gene expression. Despite intensive research, our understanding of these regulatory networks remains incomplete. Here, we focus on the understudied paralogs of the U1 small nuclear RNA (snRNA) gene known as variant U1 (vU1) snRNAs. By generating isogenic knockout lines of human induced pluripotent stem cells for two different vU1s, we show that their loss profoundly changes both hiPSC gene expression and cell cycle profiles. These changes appear to manifest at the level of alternative splicing choices, including those at recursive splicing sites, and lead to the differential availability of stem cell regulators. Together, our results shed new light on the function of vU1 snRNAs and further our understanding of the programs controlling human pluripotency.

genomics↗

bioRxiv2024

Effects of serotonergic psychedelics on synaptogenesis and immediate early genes expression - comparison with ketamine, fluoxetine and lithium

BackgroundRecent evidence suggests that psychedelics are able to induce rapid and long-lasting antidepressant effects. The generally acknowledged explanation for these traits is the phenomenon of neuroplasticity, although exact underlying molecular mechanisms remain unclear. AimsThis study investigates selected neuroplastic effects of psilocin, lysergic acid diethylamide (LSD) and N,N-dimethyltryptamine (DMT) in direct comparison with ketamine, fluoxetine and lithium after acute (1 h) and/or prolonged (24 h) treatment in vitro. MethodsRat primary cortical cultures were treated with 10 {micro}M psilocin, 1 {micro}M lysergic acid diethylamide (LSD), 90 {micro}M N, N-dimethyltryptamine (DMT), 1 {micro}M ketamine, 10 {micro}M fluoxetine and 5 mM lithium. Analysis of synaptic puncta was performed; puncta of presynaptic marker synapsin I/II, postsynaptic density protein 95 (PSD-95), and their co-localization (established synapse) were assessed 24 h after drug treatment. Next, expressions of immediate early genes (IEGs) encoding activity-regulated cytoskeleton-associated protein (Arc), early growth response 1 (Egr1), and neuronal PAS (Per-ArntSim) domain protein 4 (Npas4) were analysed 1 and 24 h after drug treatments. ResultsPsilocin increased synaptic puncta count and induced Arc expression. The effect to promote synaptogenesis was comparable to ketamine and lithium; ketamine additionally increased PSD-95 puncta count. LSD and DMT didnt induce any significant effect. Interestingly, fluoxetine had no effect on synaptic puncta count, but upregulated Egr1 and Npas4. ConclusionsPsilocin demonstrated a significant neuroplastic effect comparable to that of ketamine and lithium, adding another piece of evidence to its profile as a promising therapeutic agent.

neuroscience↗

bioRxiv2024

Prenatal VEGF Nano-Delivery Reverses Congenital Diaphragmatic Hernia-Associated Pulmonary Abnormalities

RationaleCongenital diaphragmatic hernia (CDH) results in lung hypoplasia. In severe cases, tracheal occlusion (TO) can be offered to promote lung growth. However the benefit is limited, and novel treatments are required to supplement TO. Vascular endothelial growth factor (VEGF) is downregulated in animal models of CDH and could be a therapeutic target, but its role in human CDH is not known. ObjectivesTo investigate whether VEGF supplementation could be a suitable treatment for CDH-associated lung pathology. MethodsFetal lungs from CDH patients were used to determine pulmonary morphology and VEGF expression. A novel human ex vivo model of fetal lung compression recapitulating CDH features was developed and used to determine the effect of exogenous VEGF supplementation (Figure 1A). A nanoparticle-based approach for intra-pulmonary delivery of VEGF was developed by conjugating it on functionalized nanodiamonds (ND-VEGF) and was tested in experimental CDH in vivo. O_FIG O_LINKSMALLFIG WIDTH=138 HEIGHT=200 SRC="FIGDIR/small/581170v1_fig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@2ba882org.highwire.dtl.DTLVardef@4413d2org.highwire.dtl.DTLVardef@17155aborg.highwire.dtl.DTLVardef@1af7c73_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1C_FLOATNO (A) Schematic of the study design based on the analysis of the early-stage CDH fetal lung sections to assess early hypoplasia and VEGF impairment. In parallel, a human compression-based CDH model will be developed to validate the CDH phenotype and test a therapeutical approach to rescue lung hypoplasia. (B) H&E analysis of 18 and 22 pcw post-mortem CDH human lung tissue reveals impaired epithelial development compared to control healthy tissue at matching developmental stages. Scale bar 100m. (C) Morphometric analysis of CDH and healthy lung tissue at 18-22 pcw reveals decreased values of volume densities of air space (Vair%) and corresponding higher values of volume densities of alveolar septa (Vsep%) in CDH tissues compared to healthy tissue at the same developmental stages. A similar trend can be observed for the mean linear intercept of air space (Lma%) and its corresponding mean linear intercept of septal thickness (Lmw%). (D) Histologic analysis of VEGFA, KDR, Ki67 and NKX2-1 markers in normal and CDH fetal lung tissue at 18 and 22 pcw. Scale bar 100m. (E) Quantification of epithelial VEGFA and KDR expression reported in D shows a significant decrease for both markers in CDH compared to healthy samples. Correspondingly, the percentage of Ki67-positive epithelial cells is significantly lower in CDH compared to healthy samples, with no significant differences in the number of NKX2-1-positive cells. ***<p=0.001. (F) Immunofluorescence analysis of ECAD in normal and CDH fetal lung tissue at 18 and 22 pcw. Scale bar 100m. (G) Quantification of epithelial nuclear density based on results in Figure F shows no significant differences between CDH and healthy tissue. C_FIG Measurements and Main ResultsVEGF expression was downregulated in distal pulmonary epithelium of human CDH fetuses in conjunction with attenuated cell proliferation. The compression model resulted in impaired branching morphogenesis similar to CDH and downregulation of VEGF expression in conjunction with reduced proliferation of terminal bud epithelial progenitors; these could be reversed by exogenous supplementation of VEGF. Prenatal delivery of VEGF with the ND-VEGF platform in CDH fetal rats resulted in lung growth and pulmonary arterial remodelling that was complementary to that achieved by TO alone with appearances comparable to healthy controls. ConclusionsThis innovative approach could have a significant impact on the treatment of CDH.

bioengineering↗

bioRxiv2022

Rapid biomechanical imaging at low irradiation level via dual line-scanning Brillouin microscopy

Brillouin microscopy is a promising all-optical technique for biomechanics but is limited by slow acquisition speed and/or large irradiation doses. Here, we introduce multiplexed Brillouin microscopy that overcomes both these limits by over one order of magnitude with selective illumination and single-shot analysis of hundreds of points along the incident beam axis. We demonstrate the enabling capabilities of this method probing rapid response to perturbations and long-term mechanical evolution of tumor spheroids.

bioengineering↗

bioRxiv2022

Multimodal microscale mechanical mapping of cancer cells in complex microenvironments

The mechanical phenotype of the cell is critical for survival following deformations due to confinement and fluid flow. One idea is that cancer cells are plastic and adopt different mechanical phenotypes under different geometries that aid in their survival. Thus, an attractive goal, is to disrupt the cancer cells ability to adopt multiple mechanical states. To begin to address this question, we aimed to quantify the diversity of these mechanical states using in vitro biomimetics to mimic in vivo 2D and 3D extracellular matrix environments. Here, we used two modalities Brillouin microscopy ([~]GHz) and broadband frequency (3-15kHz) optical tweezer microrheology to measure microscale cell mechanics. We measured the response of intracellular mechanics of cancer cells cultured in 2D and 3D environments where we modified substrate stiffness, dimensionality (2D versus 3D), and presence of fibrillar topography. We determined that there was good agreement between two modalities despite the difference in timescale of the two measurements. These findings on cell mechanical phenotype in different environments confirm a correlation between modalities that employ different mechanisms at different temporal scales (Hz-kHz vs. GHz). We also determined that observed heterogeneity in cell shape that is more closely linked to the cells mechanical state. We also determined that individual cells in multicellular spheroids exhibit a lower degree of mechanical heterogeneity when compared to single cells cultured in monodisperse 3D cultures. Moreover, the observed decreased heterogeneity among cells in spheroids suggested that there is mechanical cooperativity between cells that make up a single spheroid.

biophysics↗

bioRxiv2022

Brain responses in aggression-prone individuals: A systematic review and meta-analysis of functional magnetic resonance imaging (fMRI) studies of anger- and aggression-eliciting tasks

BackgroundWhile reactive aggression (in response to a perceived threat or provocation) is part of humans adaptive behavioral repertoire, it can violate social and legal norms. Understanding brain function in individuals with high levels of reactive aggression as they process anger- and aggression-eliciting stimuli is critical for refining interventions. Three neurobiological models of reactive aggression-the limbic hyperactivity, prefrontal hypoactivity, and dysregulated limbic-prefrontal connectivity models-have been proposed. However, these models are based on neuroimaging studies involving mainly healthy individuals, leaving it unclear which model best describes brain function in aggression-prone individuals. MethodsWe conducted a systematic literature search (PubMed and Psycinfo) and Multilevel Kernel Density meta-analysis (MKDA) of nine functional magnetic resonance imaging (fMRI) studies of brain responses to tasks putatively eliciting anger and aggression in aggression-prone individuals alone, and relative to healthy controls. ResultsAggression-prone individuals exhibited greater activity during reactive aggression relative to baseline in the superior temporal gyrus and in regions comprising the cognitive control and default mode networks (right posterior cingulate cortex, precentral gyrus, precuneus, right inferior frontal gyrus). Compared to healthy controls, aggression-prone individuals exhibited increased activity in limbic regions (left hippocampus, left amygdala, left parahippocampal gyrus) and temporal regions (superior, middle, inferior temporal gyrus), and reduced activity in occipital regions (left occipital cortex, left calcarine cortex). ConclusionsThese findings lend support to the limbic hyperactivity model and further indicate altered temporal and occipital activity in anger- and aggression-eliciting situations that involve face and speech processing.

neuroscience↗

bioRxiv2021

Directing multicellular organization by varying the aspect ratio of soft hydrogel microwells

Multicellular organization with precise spatial definition is an essential step in a wide range of biological processes, including morphogenesis, development, and healing. Gradients and patterns of chemoattractants are well-described guides of multicellular organization, but the influences of three-dimensional geometry of soft hydrogels on multicellular organization are less well defined. Here, we report the discovery of a new mode of self-organization of endothelial cells in ring-like patterns on the perimeters of hydrogel microwells that is independent of protein or chemical patterning and is driven only by geometry and substrate stiffness. We observe quantitatively striking influences of both the microwell aspect ratio ({varepsilon} = perimeter/depth) and the hydrogel modulus. We systematically investigate the physical factors of cells and substrates that drive this multicellular behavior and present a mathematical model that explains the multicellular organization based upon balancing extracellular and cytoskeletal forces. These forces are determined in part by substrate stiffness, geometry, and cell density. The force balance model predicts the direction and distance of translational cell migration based on the dynamic interaction between tangential cytoskeletal tension and cell-cell and cell-substrate adhesion. We further show that the experimental observations can be leveraged to drive customized multicellular self-organization. Our observation of this multicellular behavior demonstrates the importance of the combinatorial effects of geometry and stiffness in complex biological processes. It also provides a new methodology for direction of cell organization that may facilitate the engineering of bionics and integrated model organoid systems.

bioengineering↗

bioRxiv2021

Mapping interindividual dynamics of innate immune response at single-cell resolution

Common genetic variants modulate the cellular response to viruses and are implicated in a range of immune pathologies, including infectious and autoimmune diseases. The transcriptional antiviral response is known to vary between infected cells from a single individual, yet how genetic variants across individuals modulate the antiviral response (and its cell-to-cell variability) is not well understood. Here, we triggered the antiviral response in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-seq. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), the first statistical approach designed to identify dynamic eQTLs across a transcriptional trajectory of cell populations, without aggregating single-cell data into pseudo-bulk. This allows us to uncover the underlying architecture and variability of antiviral response across responding cells, and to identify more than two thousands eQTLs modulating the dynamic changes during this response. Many of these eQTLs colocalise with risk loci identified in GWAS of infectious and autoimmune diseases. As a case study, we focus on a COVID-19 susceptibility locus, colocalised with the antiviral OAS1 splicing QTL. We validated it in blood cells from a patient cohort and in the infected nasal cells of a patient with the risk allele, demonstrating the utility of GASPACHO to fine-map and functionally characterise a genetic locus. In summary, our novel analytical approach provides a new framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

genetics↗

bioRxiv2020

Scalable Analysis of Multi-Modal Biomedical Data

Targeted diagnosis and treatment options are dependent on insights drawn from multi-modal analysis of large-scale biomedical datasets. Advances in genomics sequencing, image processing, and medical data management have supported data collection and management within medical institutions. These efforts have produced large-scale datasets and have enabled integrative analyses that provide a more thorough look of the impact of a disease on the underlying system. The integration of large-scale biomedical data commonly involves several complex data transformation steps, such as combining datasets to build feature vectors for learning analysis. Thus, scalable data integration solutions play a key role in the future of targeted medicine. Though large-scale data processing frameworks have shown promising performance for many domains, they fail to support scalable processing of complex datatypes. To address these issues and achieve scalable processing of multi-modal biomedical data, we present TraNCE, a framework that automates the difficulties of designing distributed analyses with complex biomedical data types. We outline research and clinical applications for the platform, including data integration support for building feature sets for classification. We show that the system is capable of outperforming the common alternative, based on "flattening" complex data structures, and runs efficiently when alternative approaches are unable to perform at all. Key PointsO_LIModern biomedical analyses are integrated pipelines of data access mechanisms and analysis components that operate on and produce datasets in a variety of complex, domain specific formats. C_LIO_LIScalable data integration and aggregation solutions that support joint inference on such large-scale datasets play a key role advancing biomedical analysis. C_LIO_LIQuery compilation techniques that optimize nested data processing are essential for scaling multi-modal, biomedical analysis. C_LI

bioinformatics↗

bioRxiv2020

Epigenetic age-predictions in mice using pyrosequencing, droplet digital PCR or barcoded bisulfite amplicon sequencing

Age-associated DNA methylation reflects aspects of biological aging - therefore epigenetic clocks for mice can help to elucidate the impact of treatments or genetic background on the aging process in this model organism. Initially, age-predictors for mice were trained on genome-wide DNA methylation profiles, whereas we have recently described a targeted assay based on pyrosequencing of DNA methylation at only three CG dinucleotides (CpGs). Here, we have re-evaluated pyrosequencing approaches in comparison to droplet digital PCR (ddPCR) and barcoded bisulfite amplicon sequencing (BBA-seq). At individual CpGs the correlation of DNA methylation with chronological age was slightly higher for pyrosequencing and ddPCR as compared to BBA-seq. On the other hand, BBA-seq revealed that neighboring CpGs tend to be stochastically modified in murine age-associated regions. Furthermore, the binary sequel of methylated and non-methylated CpGs in individual reads can be used for single-read predictions, which may reflect heterogeneity in epigenetic aging. In comparison to C57BL/6 mice the epigenetic age-predictions using BBA-seq were also accelerated in the shorter-lived DBA/2 mice, and in C57BL/6 mice with a lifespan quantitative trait locus of DBA/2 mice. Taken together, we describe further optimized and alternative targeted methods to determine epigenetic clocks in mice.

genomics↗

bioRxiv2020

Loss of TP53 mediates suppression of Macrophage Effector Function via Extracellular Vesicles and PDL1 towards Resistance against Chemoimmunotherapy in B-cell malignancies

Chemoimmunotherapy (CIT) is the standard of care in B-cell malignancies. It is relying on synergistic effects of alkylating chemotherapy and monoclonal antibodies via secretory crosstalk with effector macrophages. Here, we observed that loss of p53 function mediates resistance to CIT by suppressing macrophage phagocytic function. Loss of p53 leads to an upregulation of PDL1 and an increased formation of extracellular vesicles (EVs). EVs directly inhibit macrophage phagocytosis by PDL1 surface expression. Suppression of phagocytic function by lymphoma cell-derived EVs could be abrogated by pre-incubation of EVs with anti-PDL1 antibodies, CRISPR-KO of PDL1 and abrogation of EV formation by RAB27A-KO in lymphoma cells. Immune checkpoint inhibition represents a viable strategy to overcome EV-mediated resistance to chemoimmunotherapy in lymphoma. SignificanceLoss of TP53 mediates cell autonomous resistance to genotoxic chemotherapy, moreover non-cell autonomous effects may cause therapy resistance mediated by the tumor microenvironment. We identify a TP53-dependent mechanism that mediates resistance to synergistic chemoimmunotherapy by increasing formation of EVs and expression of the PDL1 immune checkpoint. PDL1 on EVs is directly responsible for macrophage suppression, preventing the exertion of the essential effector function of antibody-dependent cellular phagocytosis. This novel mechanism of resistance is in turn targetable by PDL1 checkpoint inhibition. Enhanced EV-release and immune checkpoint expression in lymphoma are novel mechanisms of macrophage modulation in the lymphoma microenvironment. We provide a novel principle of resistance to chemoimmunotherapy (CIT) representing of immediate relevance to treatment of refractory B-cell lymphoma. HighlightsO_LILoss of TP53 in B-cell lymphoma induces resistance towards chemoimmunotherapy (CIT) by inhibition of macrophage effector function through PDL1 upregulation C_LIO_LILoss of TP53 increases formation of extracellular vesicles (EVs) carrying PDL1 C_LIO_LIEVs inhibit antibody-mediated cellular phagocytosis (ADCP), a key macrophage effector function in CIT C_LIO_LITargeting PDL1 on EVs with immune checkpoint inhibitors overcomes TP53-mediated resistance to CIT C_LI

cell biology↗

bioRxiv2019

New Targeted Approaches for Epigenetic Age Predictions

Aging causes epigenetic modifications, which are utilized as a biomarker for the aging process. While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective high-throughput analysis. In this study, we utilized 4,650 Illumina BeadChip datasets of blood to select the best suited CpG sites for targeted analysis. DNA methylation analysis at these sites with either pyrosequencing or droplet digital PCR (ddPCR) revealed a high correlation with chronological age. In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower precision at individual CpGs. However, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often accompanied by a CTCF binding site at the peak. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified but reveals a stochastic pattern. Based on this, we have developed an alternative model for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. Thus, the stochastic evolution of age-associated DNA methylation patterns, which seems to resemble epigenetic drift, enables epigenetic clocks for individual DNA strands.

genetics↗

bioRxiv2019

HMGB1 as a rheostat of chromatin topology and RNA homeostasis on the path to senescence

Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is however disturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from senescent cell nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin or about its roles in the nucleus. To address this, we mapped HMGB1 binding genome-wide in different primary cells using a tailored protocol. We integrated ChIP-seq and Hi-C data with a graph theory approach to uncover HMGB1 demarcation of a subset of topologically-associating domains (TADs) that harbor genes required for paracrine senescence. Moreover, using sCLIP, knock-down and overexpression experiments, we now show that HMGB1 is a bona fide RNA-binding protein (RBP) bound to senescence-relevant mRNAs and affecting splicing. HMGB1 also has an interactome rich in RBPs, many of which are implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and concertedly regulate the availability of SASP-relevant transcripts. Our findings highlight a broader than hitherto assumed role for HMGB1. It coordinates chromatin folding and RNA homeostasis as part of a feedforward loop controlling both cell-autonomous and paracrine senescence inside and outside of cells.

genomics↗

bioRxiv2019

Long-term Brillouin imaging of live cells with reduced absorption-mediated damage at 660nm wavelength

In Brillouin microscopy, absorption-induced photodamage of incident light is the primary limitation on signal-to-noise ratio in many practical scenarios. Here we show that 660 nm may represent an optimal wavelength for Brillouin microscopy as it offers minimal absorption-mediated photodamage at high Brillouin scattering efficiency and the possibility to use a pure and narrow laser line from solid-state lasing medium. We demonstrate that live cells are ~80 times less susceptible to the 660 nm incident light compared to 532 nm light, which overall allows Brillouin imaging of up to more than 30 times higher SNR. We show that this improvement enables Brillouin imaging of live biological samples with improved accuracy, higher speed and/or larger fields of views with denser sampling.

biophysics↗

bioRxiv2017

Topological Demarcation By HMGB2 Is Disrupted Early Upon Senescence Entry Across Cell Types And Induces CTCF Clustering

Ageing-relevant processes, like cellular senescence, are characterized by complex, often stochastic, events giving rise to heterogeneous cell populations. We hypothesized that entry into senescence of different primary human cells can be triggered by one early molecular event affecting the spatial organization of chromosomes. To test this, we combined whole-genome chromosome conformation capture, population and single-cell transcriptomics, super-resolution imaging, and functional analyses applied on proliferating and replicatively-senescent populations from three distinct human cell types. We found a number of genes involved in DNA conformation maintenance being suppressed upon senescence across cell types. Of these, the abundant high mobility group (HMG) B1 and B2 nuclear factors are quantitatively removed from cell nuclei before typical senescence markers appear, and mark a subset of topologically-associating domain (TAD) boundaries. Their loss coincides with obvious reorganization of chromatin interactions via the dramatic spatial clustering of CTCF foci. HMGB2 knock-down recapitulates this senescence-induced CTCF clustering, while also affecting insulation at TAD boundaries. We accordingly propose that HMGB-mediated deregulation of chromosome conformation constitutes a primer for the ensuing senescent program across cell types.

genomics↗