Biology subjects

Benes, V.

Publications and source records attributed to Benes, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Lack of centrosomes limits the epithelial-to-mesenchymal transition in human mammary cells

As the main microtubule network organisers of animal cells, centrosomes orchestrate diverse cellular processes. Centrosomes have been linked to cancer when numerically or structurally aberrant, yet their precise roles at different cancer stages remains to be defined. Here, we study centrosome function in the epithelial-to-mesenchymal transition (EMT), a key driver of cancer initiation, progression and metastasis. We first depleted centrosomes in a breast cancer mesenchymal-like model, resulting in insignificant cellular or nuclear rearrangements. We then investigated non-carcinogenic breast cells undergoing the EMT via TGF{beta} signalling, and removed the centrosomes before triggering this transition. Stimulated epithelial cells acquired a mesenchymal phenotype and rearranged their cytoskeleton. By contrast, stimulated cells lacking centrosomes retained epithelial morphology, showed little cytoskeletal remodelling, yet still altered nuclear shape and upregulated key EMT markers. Transcriptome analysis revealed reduced expression of important genes involved in extracellular matrix organisation and a downregulation of cell junction components when lacking centrosomes; further analysis revealed paxillin showing reduced colocalisation with actin focal adhesions. Centrosomes thus prove essential for cytoskeletal remodelling in the EMT of breast cancer cells.

cell biology↗

bioRxiv2026

Single-Cell Mapping of tRNA Expression Dynamics Across Human Hematopoiesis

How transfer RNA (tRNA) expression changes as hematopoietic stem cells are specified into human blood cell types remains unknown. tRNAs translate the 64-codon genetic code into the 21-amino acid protein code, and their relative abundance can influence proteomic output. Here, we introduce simultaneous single-cell tRNA and mRNA sequencing (sc-STM-seq), a scalable, high-throughput approach for profiling tRNAs alongside mRNA transcriptomes in individual cells. Applying sc-STM-seq to human bone marrow, we map tRNA expression and splicing across hematopoietic differentiation trajectories and identify tRNAs associated with stemness, differentiation, and specific cell lineages. Our study provides an atlas of the hematopoietic tRNA landscape and establishes tRNA expression as a previously underappreciated layer of cellular heterogeneity during human blood cell development.

cell biology↗

bioRxiv2026

Ubiquitin selective ribosome profiling reveals systematic principles of co-translational quality control

Protein biogenesis is a stress- and error-sensitive process that can lead to nascent protein misfolding and aggregation, challenging cellular proteostasis.1 Co-translational ubiquitination (CTU) is a critical surveillance mechanism,2,3 yet its regulatory principles remain unclear due to limited known substrates and the lack of translatome-wide methods to query CTU. Here, we introduce UbSeRP, an approach that enables ubiquitin linkage-specific, and translatome-wide mapping of CTU. Focusing on ribosome-associated quality control (RQC), we expand the known endogenous RQC substrates in S. cerevisiae from few4-7 to thousands, reveal that only a subset of all identified disomes undergoes RQC, and uncover biophysical features of nascent chains that predict RQC engagement. We also identify widespread RQC-independent ubiquitination of nascent proteins, implicating broader roles of CTU in protein complex assembly. In a chronological aging model, we show that aging remodels translation and diminishes RQC engagement, favoring RQC-independent proteasomal pathways. Our findings provide systematic insight into the determinants and adaptability of CTU in maintaining proteostasis under changing physiological conditions.

biochemistry↗

bioRxiv2026

Alternative lengthening of telomeres stratifies complex karyotype sarcomas into distinct genomic and transcriptomic states

Complex karyotype sarcomas (CKS) are heterogeneous mesenchymal malignancies that typically lack recurrent actionable oncogenic drivers and remain therapeutically challenging. Loss of ATRX is a recurrent feature of CKS and defines a particularly high-risk subgroup. ATRX loss is also associated with activation of the alternative lengthening of telomeres (ALT) pathway, and ALT-positive sarcomas have been linked to poor clinical outcomes. However, the molecular underpinnings underlying ALT-status-dependent differences in CKS, as well as the therapeutic vulnerabilities associated with ALT, remain poorly defined. By integrating C-circle-based ALT detection across 776 sarcoma samples with multi-modal sequencing of five CKS subtypes, we find that ALT activity is associated with enriched hallmarks of genomic instability. ALT-positive transcriptomes are dominated by a coordinated DNA damage response and mitotic program, in contrast to oncogenic signaling pathways that drive TERT activation in ALT-negative tumors. Long-read sequencing reveals telomere repeat clusters and telomere-mediated healing at structural breakpoints in ALT-positive tumors. These events also occur on extrachromosomal DNA (ecDNA), linking ALT activity to ecDNA biology. Together, our findings position ALT status as an important stratifying feature of CKS and identify ALT-associated transcriptional programs as potential therapeutic targets.

cancer biology↗

bioRxiv2025

Cancer pathway connectivity resolved by drug perturbation and RNA sequencing

Pathway inhibitors are a backbone of cancer treatment. The configuration of pathway dependencies varies from tumour to tumour. Better treatment for individual patients could be designed if the pathway wiring were readily measurable. Here, we characterise the transcriptional responses of 116 lymphoma patient samples exposed to ten drug perturbations. We used factor analysis to decompose individual and shared drug effects, thereby generating a pathway connectivity map of chronic lymphocytic leukemia (CLL). The expression profiles of the major disease subgroups, defined by IGHV mutation status, became more similar to each other after BTK inhibition, consistent with B-cell receptor (BCR) signalling as a driver of their phenotypic difference. An even stronger convergence was observed with combined IRAK4 and BTK inhibition, indicating cooperation of BCR and toll-like receptor (TLR) signalling in CLL. We identified genetic aberrations (BRAF, TP53, deletion 17p, deletion 15q, trisomy 12) that modulated drug effects in CLL and constituted specific interaction patterns. IRAK4 inhibition effects depended on the presence of trisomy 12, a finding that suggests that the trisomy 12 driver event in CLL acts by gene dosage-dependent IRAK4 upregulation and amplification of the BCR/TLR cooperation. Our results highlight the potential of systematic drug perturbation assays with transcriptome readout to map pathway interconnectivity and functionally annotate tumour drivers.

systems biology↗

bioRxiv2025

Integrative spatial and multi-omic profiling in bladder cancer links L1 retrotransposition to extrachromosomal DNA, genomic instability, and viral mimicry response

Bladder cancer is one of the most frequent cancers and shows high recurrence rates. Despite recent advances, key knowledge gaps remain in understanding the molecular mechanisms of disease progression, which would support the development of early detection methods and effective personalized treatments. We apply integrated multi-omics and spatial analyses in a cohort of 49 bladder cancer patients to comprehensively profile genetic, epigenetic, transcriptomic, and spatial features of bladder cancer, alongside cell-free DNA blood analysis. Combining low-pass whole-genome cell-free DNA sequencing, Oxford Nanopore long-read tumor DNA sequencing, RNA-sequencing, and spatial transcriptomics, we provide insights into molecular alterations driving bladder cancer. We show frequent somatic LINE-1 (L1) insertions, with up to more than 500 insertions per tumor. We find that L1 insertions are active and occur early in bladder cancer development. We link aberrant somatic L1 insertion in bladder cancer with downstream genomic rearrangements and chromosomal instability, with an excess of structural variants and extrachromosomal DNA (ecDNA) in patients with particularly high L1 counts. By detecting ecDNA within tissue architecture using spatial transcriptomics, we identify the localization of ecDNA to distinct spatial clusters with differential expression of APOBEC3B and immune response pathways. These results, combined with replication timing analysis and gene set enrichment analysis (GSEA), offer evidence for the previously hypothesized viral mimicry response to L1 retrotransposition, mediated via APOBEC3B-editing, the cGAS-STING pathway, and RIG-I and MDA5 responses.

cancer biology↗

bioRxiv2025

Activation of nuclear receptors correlates with tuberculosis severity and is a target for host-directed therapy

The immune response to Mycobacterium tuberculosis is accompanied by metabolic adaptations that fuel host immunity, but that are exploited by the pathogen to ensure persistence and growth. Nuclear receptors, such as liver-X-receptors (LXR), orchestrate macrophage immunometabolic adaptations to infection and globally associate with tuberculosis (TB) protection. Here, we show that the "signalling by nuclear receptors" (SNR) pathway is detected in the whole blood of TB patients and that its expression correlates with disease severity. Accordingly, we also show that the activation of the LXR pathway progressively increases in the lungs of M. tuberculosis-infected C57BL/6 and C3HeB/FeJ mice. Pharmacologic activation of LXR, specifically at the chronic stage of infection, improved infection outcomes and significantly prolonged the survival of the highly susceptible C3HeB/FeJ mice. Common to both mouse models and to in vitro macrophage infections, LXR activation enhanced bacterial control together with an increase in extracellular cholesterol levels. We propose that progressive LXR activation is required to fine-tune host cholesterol availability during M. tuberculosis infections and restrict access to this nutrient during chronic stages of infections. Collectively, we identify the SNR pathway as a potential biomarker of TB severity and timely LXR activation as a candidate host-directed therapy.

immunology↗

bioRxiv2025

Regulation of input excitability in human and mouse parvalbumin interneurons by Kir potassium channels

Compared to rodents, inhibitory interneurons in the human neocortex exhibit high input excitability because of reduced passive ion leakage across their extracellular membrane. However, the regulation of intrinsic excitability by voltage-gated ion channels activated over a wide range of membrane potentials in human interneurons remains poorly understood. We performed whole-cell patch-clamp microelectrode recordings in mouse and human neocortical slices obtained from surgically resected non-pathological brain tissue finding that Kir channels control the electrical resistance of parvalbumin (Pvalb) neurons in an identical manner in the human and mouse. Molecular analyses revealed predominantly Kir3.1 and Kir3.2 channels in Pvalb neurons in both species. Using whole-cell recordings from synaptically connected neuron pairs and a computational model, we demonstrated that physiological Kir activation inhibits human Pvalb interneurons during postsynaptic potentials evoked by presynaptic neurogliaform cells. The similarity of Kir-mediated inhibition across species suggests that it is an archetypal property of Pvalb neurons.

neuroscience↗

bioRxiv2025

Glucocorticoid receptors mediate reprogramming of astrocytes in depression.

Psychiatric disorders are among the most pressing problems of the modern society, with various forms of depression affecting more than 300 millions of people worldwide. Dysfunction of glial cells has consistently been reported in major depressive disorder (MDD); however, no comprehensive resource detailing glial dysfunction is available. To provide insight into neurobiological mechanisms behind severe psychiatric symptoms, we performed transcriptional analysis of post-mortem samples from a subpopulation of suicide completers with previously reported glial abnormalities. We focused on BA25, a subregion of the prefrontal cortex prioritized for targeted medical interventions, due to its metabolic aberrations in disease. We found that a significant portion of genes deregulated in MDD is enriched in glia, with astrocyte-specific genes representing the highest fraction. Then we employed a novel protocol for enriching astrocytic nuclei to provide a detailed molecular signature of astrocytes in MDD. The analysis of the gene set revealed the glucocorticoid receptor (GR) as a key regulatory transcription factor. We found that astrocyte-specific elimination of the GR in mice largely prevented transcriptional, metabolic and behavioral changes elicited by chronic stress. We also demonstrated that regional manipulation of glutamate turnover in astrocytes suffices to elicit discrete traits of depressive-like behavior. Our data points to astrocytes as a key cellular site of convergence of multiple traits of depression and provide a resource for exploring novel targets for glia-focused therapeutic approaches.

neuroscience↗

bioRxiv2025

Impact of low-calorie sweeteners on gut bacteria is modulated by common xenobiotics

The gut microbiota is implicated in adverse effects associated with low-calorie sweeteners. Yet, the direct impact of sweeteners on gut bacteria remains largely uncharacterized. Here we report interactions between 25 phylogenetically diverse gut bacterial strains and 39 commercially used sweeteners. We tested these sweeteners individually and in combination with four commonly co-consumed compounds, viz., advantame, caffeine, vanillin, and duloxetine. Three quarters of the tested sweeteners individually impacted growth of at least one bacterial strain. Further, over 100 interactions were found between sweeteners and the four co-consumed compounds. Isosteviol, a commonly used sweetener, and duloxetine, an antidepressant, synergistically inhibited Roseburia intestinalis, a bacterium previously linked to glucose homeostasis, and Parabacteroides merdae, a prevalent commensal linked to healthy microbiota. Proteomic, metabolomic, and genetic analyses indicate altered small molecule transport underpinning this sweetener-drug synergy. The isosteviol-duloxetine combination also modulated metabolism of a synthetic gut bacterial community leading to increased toxicity to HeLa cells and altered secretion of inflammation modulatory cytokines IL-6 and IL-8 by Caco-2 cells. Together, our data bring forward the prevalence of interactions between low-calorie sweeteners and common xenobiotics.

microbiology↗

bioRxiv2025

Obligate cross-feeding of metabolites is common in soil microbial communities

Many microorganisms are refractory to laboratory cultivation. One possible explanation, known as the great plate count anomaly, is metabolic dependencies among community members. However, systematic studies of these interactions in communities like soil are missing, hindering advances in understanding these ecologically important ecosystems. Here, we address this issue by systematically analysing 6,931 bacterial isolates of 27 soil microbial communities. We find that the growth of up to 50% of all community members depended essentially on supplementation with amino acids, vitamins, or nucleotides. In 73% of cases, supplementation with multiple amino acids was necessary. Genomic analysis of 62 strains revealed that accumulation of insertion sequences and specific gene loss was associated with the observed auxotrophies. Finally, genome-scale metabolic models, computational analyses, and cocultivation experiments demonstrated that other co-occurring genotypes complemented the metabolic needs of auxotrophs, thus facilitating their growth. Our results demonstrate that soil bacteria exist within integrated metabolic networks, which hampers their cultivation.

microbiology↗

bioRxiv2025

Germline-restricted chromosome of songbirds has different centromere compared to regular chromosomes

Centromeres are an important part of chromosomes which direct chromosome segregation during cell division. Their modifications can therefore explain the unusual mitotic and meiotic behaviour of certain chromosomes, such as the germline-restricted chromosome (GRC) of songbirds. This chromosome is eliminated from somatic cells during early embryogenesis and later also from male germ cells during spermatogenesis. Although the mechanism of elimination is not yet known, it is possible that it involves a modification of the centromeric sequence on the GRC, resulting in problems with the attachment of this chromosome to the mitotic or meiotic spindle and its lagging during anaphase, which eventually leads to its elimination from the nucleus. However, the repetitive nature and rapid evolution of centromeres make their identification and comparative analysis across species and chromosomes challenging. Here, we used a combination of cytogenetic and genomic approaches to identify the centromeric sequence of two closely related songbird species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (L. luscinia). We found a 436-bp satellite repeat present in the centromeric regions of all regular chromosomes, making it a strong candidate for the centromeric repeat. This centromeric repeat was highly similar between the two nightingale species. Interestingly, hybridization of the probe to this satellite repeat on meiotic spreads suggested that this repeat is missing on the GRC. Our results indicate that the change of the centromeric sequence may underlie the unusual inheritance and programmed DNA elimination of the GRC in songbirds.

genomics↗

bioRxiv2024

Embryonic Signatures of Intergenerational Inheritance across Paternal Environments and Genetic Backgrounds

The paternal preconception environment has been implicated as a modulator of phenotypic traits and disease risk in F1 offspring. However, the prevalence and mechanisms of such intergenerational epigenetic inheritance (IEI) in mammals remain poorly defined. Moreover, the interplay between paternal exposure, genetics, and age on emergent offspring features is unexplored. Here, we measure the quantitative impact of three paternal environments on early embryogenesis across genetic backgrounds. Using in vitro fertilisation (IVF) at scale, we capture batch-robust transcriptomic signatures of IEI with single-blastocyst resolution. Amongst these, paternal gut microbiota dysbiosis is linked with aberrant expression of lineage regulators in blastocysts, particularly affecting extra-embryonic tissues. Conversely, paternal low-protein high-sugar diet associates with subtle preimplantation developmental delay. We further identify gene expression variability as a paternally-induced F1 phenotype, and highlight confounding issues for IEI such as batch-effects and under-sampling. Finally, paternal genetic background and age exert a dominant influence over the inherited environmental signature. This study systematically characterises how paternal conditioning programmes subtle but detectable molecular responses in early embryos, and suggests guiding principles to dissect intergenerational phenomenology.

genomics↗

bioRxiv2024

Mechanisms and purpose of lowered action potential firing threshold in fast-spiking interneurons in the human neocortex

The mammalian brain exhibits notable interspecies variation. Microanatomical and molecular differences in homologous neurons, those with similar locations and developmental origins across species, are best characterized in the neocortical mantle, the center of complex brain functions; however, the purpose of these differences remains unclear. We performed whole-cell microelectrode recordings along with microanatomical and molecular analyses of human fast-spiking parvalbumin (pvalb)-expressing interneurons in neocortical tissue resected during brain surgery, comparing them with similar data obtained from the mouse neocortex. The action potential (AP) firing threshold was lower in human neurons than in mouse neurons. This was due to a deficiency in low-voltage-activated inhibitory Kv1.1 and Kv1.2 potassium channels in the axon initial segment (AIS), a specialized axonal region that determines AP threshold and initiation, in human cells. In contrast, Kv1 ion channels were prominent in mouse neurons. The AIS was also elongated in humans. Computational simulations of fast-spiking interneurons revealed that the human-type AIS lowers the AP threshold and shortens the time lag for AP initiation. We found that the low membrane AP firing threshold in pvalb neurons is closely linked to slow membrane potential kinetics in the soma. Thus, the human AIS supports fast in-fast out circuit function in human pvalb neurons, compensating for electrically slow somatic membrane responses. When formulating therapeutic strategies that involve fast- spiking neurons, it is crucial to take into account the molecular and functional species differences. Short blurb: Fast-spiking neurons in the human neocortex feature structural and molecular specializations in the axon initial segment that lower firing thresholds and minimize input- output delay.

neuroscience↗

bioRxiv2024

Extensive PFAS accumulation by human gut bacteria

Per- and polyfluoroalkyl Substances (PFAS) - the so-called forever chemicals - are a major cause of environmental and health concern due to their toxicity and long-term persistence1,2. Yet, no efficient mechanisms for their removal have been identified. Here we report bioaccumulation of PFAS by several gut bacterial species over a wide range of concentrations from nanomolar up to 500 M. For bioaccumulating Bacteroides uniformis, a highly prevalent species, we estimate intracellular PFAS concentration in the mM range - above that of most native metabolites. Despite this high bioaccumulation, B. uniformis cells could grow appreciably up to 250 M perfluorononanoic acid (PFNA) exposure. Escherichia coli, which accumulated PFAS to a much lesser extent, substantially increased PFAS bioaccumulation when lacking TolC efflux pump indicating trans-membrane transport in PFAS bioaccumulation. Electron microscopy and cryogenic Focused Ion Beam-Secondary Ion Mass-spectrometry revealed distinct morphological changes and intracellular localisation of PFNA aggregates. Bioaccumulation of PFAS and transmembrane transport is also evident in proteomics, metabolomics, thermal proteome profiling, and mutations following adaptive laboratory evolution. In an in vivo context, mice colonized with human gut bacteria showed, compared to germ-free controls or those colonized with low-bioaccumulating bacteria, higher PFNA levels in excreted feces. As the gut microbiota is a critical interface between exposure and human body, our results have implications for understanding and utilizing microbial contribution to PFAS clearance.

microbiology↗

bioRxiv2024

A genome resource for the marine annelid Platynereis dumerilii

The marine annelid Platynereis dumerilii is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes of P. dumerilii (laboratory culture reference and a single individual assembly) and of the closely related P. massiliensis and P. megalops (single individual assembly) to facilitate comparative genomic approaches and help explore Platynereis biology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of [~]1.47 Gbp for P. dumerilii, of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 105 miRNA loci. We further explore the high genetic variation ([~]3% heterozygosity) within the Platynereis species complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development of Platynereis genomic resources.

genomics↗

bioRxiv2024

Comprehensive analysis of the microbial consortium in the culture of flagellate Monocercomonoides exilis

Monocercomonoides exilis is the only known amitochondriate eukaryote, making it an excellent model for studying the implications of mitochondrial reduction from a cellular and evolutionary point of view. Although M. exilis is an endobiotic heterotroph, it can grow in vitro, albeit with an uncharacterized and complex prokaryotic community. All attempts to grow M. exilis axenically have been unsuccessful. Here, we use metagenomic sequencing at different time points during culture growth to describe the composition and dynamics of this community. We assembled genomes of 24 from at least the 30 different bacterial species within. Based on DNA read abundances, M. exilis represents less than 1.5%, and the representation of dominant bacterial members changes over time. Genome-scale metabolic reconstruction, differential expression analysis and measurements of metabolites in the media showed that the community depends on organic carbon oxidation, fermentation, and hydrogen production without methanogenesis. This is consistent with the rapid decline of amino acids, nucleotides, glyceraldehyde, lactate, fatty acids, and alcohols in the media. The community depends on recycling the external supply of amino acids since it has a limited capacity to fix nitrogen gas and lacks ammonia oxidizers to close the nitrogen cycle. With the senescence of the culture, we observe changes in the expression of several metabolic pathways in M. exilis, particularly those adapting to starvation. We do not reveal any clear metabolic link to explain the dependence of M. exilis on prokaryotes.

microbiology↗

bioRxiv2023

Aging impairs muscle regeneration by desynchronizing matrix mechano-signaling and macrophage immunomodulation via fibro-adipogenic progenitors

Skeletal muscle regeneration depends on the function of fibro/adipogenic progenitors (FAPs). Here we show that aging impairs myogenic stem cells by disrupting the integration of extracellular matrix and immunomodulatory functions within the stem cell niche, thereby promoting fibro/fatty degeneration. We identify the FAP-secreted protein Periostin as a niche factor that is decreased in aged muscle and in circulation of aged humans with low-exercise lifestyle. Periostin controls FAP-expansion after injury and its depletion fate-regulates FAPs towards adipogenesis. This leads to delayed pro- to anti-inflammatory macrophage transition during regeneration. Transplantation of young FAPs with high Periostin secretion, but not Periostin-deficient FAPs, into aged muscle restores inflammation resolution and successful regeneration. Mechanistically, Periostin activates Focal adhesion kinase- and AKT-signaling in macrophages via integrins to promote an anti-inflammatory profile, which synchronizes matrix-derived mechanosensory signaling and immunomodulation. These results uncover a novel role of FAP-based regulation that orchestrates successful muscle regeneration and prevents fibro/fatty degeneration.

physiology↗