Biology subjects

Gabel, F.

Publications and source records attributed to Gabel, F..

11 recordsLinked to original sources

bioRxiv2026

X-CODE: a dual RNA barcoding system for multi-platform clonal tracking and spatial phenotyping

Experimental dissection of clonal dynamics in complex tissues requires barcoding systems that are scalable, compatible with different analytical platforms, providing phenotypic and spatial resolution. Here we introduce X-CODE, a dual-expressed RNA barcoding system designed to enable high-complexity clonal tracking across sequencing-based, cytometric, and spatial imaging modalities within a unified experimental framework. X-CODE combines a combinatorial, probe-detectable long RNA barcode with a matched short sequencing barcode, enabling seamless integration of probe-based readouts with sequencing and barcode-guided clonal retrieval. We demonstrate robust X-CODE detection by mass cytometry and imaging-based platforms, including spatial RNA barcode readout using via a repurposed Akoya PhenoCycler-Fusion protocol. In addition, we show compatibility with MALDI mass spectrometry imaging for co-registration of clonal and metabolic information. We further demonstrate the feasibility of X-CODE detection within probe-based spatial transcriptomics using the 10x Genomics Xenium platform. Applied to an in vivo model of androgen deprivation in prostate cancer, X-CODE reveals clonal architecture, selection and clone-specific phenotypic and metabolic plasticity underlying castration resistance. Together, X-CODE provides a flexible and broadly accessible platform for integrated clonal analysis across spatial, phenotypic, and molecular dimensions.

cancer biology↗

bioRxiv2025

ProMPt: A modular preclinical platform for functional modelling of prostate cancer heterogeneity and therapeutic vulnerabilities

Prostate cancer progression is driven by heterogenous genetic, phenotypic, and microenvironmental programs that remain challenging to model experimentally. Existing systems such as genetically engineered mouse models, xenografts, and patient-derived organoids have each advanced mechanistic insight but are limited by genetic scope, scalability, or lack of immune context. To overcome these constraints we developed ProMPt, a genetically-defined syngeneic mouse modelling platform that captures combinations of the most recurrent clinical prostate cancer genomic alterations to enable scalable in vitro and in vivo interrogation of prostate cancer evolution. Tumours derived from ProMPt organoids recapitulate the histologic and molecular diversity of human disease. Cross - species transcriptomic integration and multivariate single-cell analysis under defined culture permutations revealed conserved phenoscapes, highlighting a central role for MYC in disease progression and therapy resistance. Guided by these insights, preclinical intervention studies demonstrated that combined MAPK inhibition and blockade of protein translation synergistically suppressed tumour growth in castration-resistant models. This combination not only suppressed proliferation but also remodelled the tumour immune landscape, underscoring its dual epithelial and microenvironmental effects. Together, these findings establish ProMPt as a versatile framework for linking genotype, lineage plasticity, and therapeutic vulnerability in prostate cancer.

cancer biology↗

bioRxiv2023

Sex differences in neurotransmitter levels in different brain regions after acute and chronic morphine treatment in mice

Background and PurposePain management is a major health burden. Pain results from the integration of the nociceptive message and neuronal communication relying on neurotransmitters such as glutamate, {gamma}-aminobutyric acid (GABA), dopamine, noradrenaline and serotonin in brain regions including the periaqueductal gray (PAG), the nucleus accumbens (Nac), the caudate-putamen (Cpu) and the amygdala. Morphine remains the gold standard painkiller for severe pain via the activation of the mu opioid receptors. However, among side effects, morphine chronic treatment lead to antinociceptive tolerance. As antinociceptive tolerance might be linked to neurotransmission dysregulation, we have compared various neurotransmitter concentrations in acute vs chronic morphine conditions in the amygdala, PAG, Cpu, and the Nac of male and female mice. Experimental approachSex differences in morphine antinociception and tolerance were assessed using the tail-immersion test. The behavioural effects of acute and chronic morphine treatments, as well as sex differences in the levels of dopamine, serotonin, noradrenaline, glutamate and GABA in the amygdala, PAG, Cpu, and the Nac were determined by an absolute quantification LC-MS/MS approach using the isotopic dilution method. Key resultsThis study indicates, as previously reported, that female mice are less sensitive to morphine and develop morphine antinociceptive tolerance earlier than males (ED50 of 5.5{+/-}0.24 days vs 1.54{+/-}0.11 days, respectively). However, the rate at which the tolerance developed did not differ between both sex. We have found major differences in dopamine, serotonin, noradrenaline, glutamate and GABA levels between female and male mice in the amygdala, PAG, Cpu, and the Nac. Finally, no major effect of anti-nociceptive tolerance induced by chronic morphine was observed compared to acute administration of morphine. ConclusionNeurotransmitter differences are attributable mainly to sex differences in pain-related CNS regions. However, the impacts of morphine anti-nociceptive tolerance on dopamine, serotonin, noradrenaline, glutamate and GABA contents appeared to be limited.

neuroscience↗

bioRxiv2022

Cotranscriptional folding of the lncRNA Xist A-repeats indicates a modular structure

LncRNAs are emerging to play crucial roles in the regulation of many essential cellular processes and have been linked to human disease, but a detailed understanding of their structure and how this relates to underlying molecular mechanisms is still limited. The structure that a lncRNA adopts can interconvert between multiple conformations. However, characterizing the structure and dynamics is challenging given their large size. Here, we present an integrated approach, combining biochemical and biophysical techniques to investigate the core structural elements and conformational dynamics of the A-repeats of the lncRNA Xist. We combine chemical RNA structure probing, SAXS, NMR-spectroscopy and cryo-EM to comprehensively describe the conformational landscape of the Xist A-repeats. We show that under native-like conditions, the A-repeats are modular, comprising building blocks made from stable AUCG tetraloop hairpins and inter-repeat dimers separated by flexible uracil-rich regions. The structural core of the A-repeats involves dimerization of sequential repeats to form two subdomains, comprising repeats 1-4 and 5-8. The overall topology of the A-repeats is dynamic, with structural variability linked to the uracil-rich linker regions. Our results rationalize context and buffer-dependent structural variations of the Xist lncRNA. The integrative approach presented here establishes a general pipeline for investigating lncRNA structure and dynamics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/501616v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1761cbeorg.highwire.dtl.DTLVardef@15905e0org.highwire.dtl.DTLVardef@190bf0corg.highwire.dtl.DTLVardef@1eeca5a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Binding stoichiometry and structural model of the HIV-1 Rev/Importin beta complex

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein Importin {beta} (Imp{beta}), but how Rev associates with Imp{beta} is poorly understood. Here we report biochemical, biophysical and structural studies of the Imp{beta}/Rev complex. Gel shift, native mass spectrometry and isothermal titration calorimetry data reveal that Imp{beta} binds two Rev monomers through independent binding sites. Small-angle X-ray scattering (SAXS) data suggest that the HEAT repeats of Imp{beta} retain an extended conformation upon binding Rev, which according to NMR data is primarily recognized through its helical hairpin domain. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix 2 within Revs Arginine-Rich Motif (ARM) as a primary Imp{beta} binding epitope. Crosslinking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Imp{beta} with Rev helix 2 roughly parallel to the HEAT-repeat superhelical axis while the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Imp{beta} and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular Imp{beta} cargos.

biochemistry↗

bioRxiv2021

Mechanistic Insight from Full Quantum Mechanical Modeling: Laccase as a Detoxifier of Aflatoxins

We demonstrate a path towards full Quantum Mechanics (QM) characterization of enzymatic activity. As a case-study, we investigate the detoxification of aflatoxin, a carcinogenic food contaminant, by laccase, a versatile oxidase capable of--but not efficient for--degrading aflatoxin. We use a combination of quantitative experimentation and QM modeling to show that low enzymatic steric affinity for aflatoxin is the main bottleneck, rather that the oxidative activity of laccase. To identify the structural elements responsible for low reaction rates, we perform a density functional theory (DFT) based modeling of both the substrate and the enzyme in a full QM simulation of more than 7,000 atoms. Thanks to our approach we point to amino acid residues that determine the affinity of laccase for aflatoxin. We show that these residues are substrate-dependent, making a full QM approach necessary for enzyme optimization. Altogether, we establish a roadmap for rational enzyme engineering applicable beyond our case study.

biophysics↗

bioRxiv2020

Central metabolism as a potential origin of sex differences in morphine analgesia but not in the induction of analgesic tolerance in mice

In rodents, morphine analgesia is influenced by sex. However, conflicting results exist regarding the interaction between sex and morphine analgesic tolerance. Morphine is metabolized in the liver and brain into morphine-3-glucuronide (M3G). Sex differences in morphine metabolism and differential metabolic adaptations during tolerance development might explain the behavioral discrepancies. The present article investigates the differences in peripheral and central morphine metabolism after acute and chronic morphine treatment in male and female mice. The first experiment aimed to determine whether morphine analgesia and tolerance differ between male and female mice using the tail-immersion test. The second experiment evaluated morphine and M3G metabolic kinetics in the blood using LC-MS/MS. Morphine and M3G were also quantified in several central nervous system (CNS) regions after acute and chronic morphine treatment. Finally, the blood-brain barrier permeability of M3G was assessed in male and female mice. This study demonstrated that female mice showed weaker morphine analgesia. In addition, tolerance appeared earlier in females but the sex discrepancies observed seemed to be due to the initial differences in morphine analgesia rather than to sex-specific mechanisms involving metabolism. Additionally, compared to male mice, female mice showed higher levels of M3G in the blood and in several CNS regions, whereas lower levels of morphine were observed in these brain regions. These differences are attributable mainly to morphine central metabolism, which differed between males and females in pain-related brain regions, consistent with the weaker analgesic effect in females. However, the role of morphine metabolism in analgesic tolerance seems rather limited.

neuroscience↗

bioRxiv2020

Structure and dynamics of the quaternary hunchback mRNA translation repression complex

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. The suppression of the RNA is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins in the RNA suppression remains a long-standing open question. We characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex is flexible with the unoccupied nucleotides of the RNA functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, Brat and Pum with Nanos bind the RNA completely independently. In accordance with previous studies, showing that Brat can suppress hunchback mRNA independently and is distributed uniformly throughout the embryo, this suggests that hunchback mRNA suppression by Brat is functionally separate from the suppression by Pumilio and Nanos.

molecular biology↗

bioRxiv2020

RNA binding regulates TRIM25-mediated RIG-I ubiquitylation

TRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25's E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA binding have been described, neither the precise RNA binding site has been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. We also show that TRIM25 not only binds RNA in mammalian cells but that interfering with RNA binding has an effect on cellular RIG-I ubiquitination.

molecular biology↗

bioRxiv2018

Merging in-solution X-ray and neutron scattering data allows fine structural analysis of membrane-protein detergent complexes

AbstractIn-solution small angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labelled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. Firstly, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS which has been adapted to predict SANS data. Secondly, the use of MONSA which allows to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein-detergent complexes from unlabeled samples than SAXS or SANS alone.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC=\"FIGDIR/small/324103_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (67K):\norg.highwire.dtl.DTLVardef@f7e17corg.highwire.dtl.DTLVardef@1f4987dorg.highwire.dtl.DTLVardef@1475c79org.highwire.dtl.DTLVardef@839b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2017

Structural basis for terminal loop recognition and processing of pri-miRNA-18a by hnRNP A1

Post-transcriptional mechanisms play a predominant role in the control of microRNA (miRNA) production. Recognition of the terminal loop of precursor miRNAs by RNA-binding proteins (RBPs) influences their processing; however, the mechanistic and structural basis for how levels of individual or subsets of miRNAs are regulated is mostly unexplored. We previously described a role for hnRNP A1, an RBP implicated in many aspects of RNA processing, as an auxiliary factor that promotes the Microprocessor-mediated processing of pri-mir-18a. Here, we reveal the mechanistic basis for this stimulatory role of hnRNP A1 by combining integrative structural biology with biochemical and functional assays. We demonstrate that hnRNP A1 forms a 1:1 complex with pri-mir-18a that involves binding of both RNA recognition motifs (RRMs) to cognate RNA sequence motifs in the conserved terminal loop of pri-mir-18a. Terminal loop binding induces an allosteric destabilization of base-pairing in the pri-mir-18a stem that promotes its down-stream processing. Our results highlight terminal loop RNA recognition by RNA-binding proteins as a general principle of miRNA biogenesis and regulation.

molecular biology↗