Biology subjects

Ricci, V.

Publications and source records attributed to Ricci, V..

5 recordsLinked to original sources

bioRxiv2026

Anisomycin-induced ribosome collisions follow a dose-dependent positional rule, distinct from the UV-induced collision landscape

Ribosome collisions trigger stress pathways such as the ribotoxic stress response (RSR), yet how different ribosome-impairing stimuli shape where collisions form on mRNAs remains unclear. Here, we combine monosome- and disome-resolved ribosome profiling with proteomics of collided-ribosome fractions to compare anisomycin- and UV-induced ribosome impairment in mouse embryonic stem cells. Low-dose anisomycin, a widely used collision-inducing paradigm, does not generate collisions dispersed along transcripts but a post-initiation collision zone, with stalled monosomes and disomes accumulating within the first ~70 nucleotides of coding sequences across most translated mRNAs. High-dose anisomycin, commonly considered collision-poor, produces the same pattern shifted closer to the start codon and reduced in amplitude. These observations support a single dose-dependent positional rule, in which drug occupancy determines how far ribosomes progress before arrest and ongoing initiation supplies trailing ribosomes. In contrast, 30 min after UV irradiation, collisions are detectable biochemically but appear more dispersed, with few recurrent hotspots and preferential accumulation on highly translated mRNAs. Collided-ribosome fractions share enrichment of the collision sensor EDF1 across conditions, whereas recruitment of ZNF598 and initiation factors is most evident after UV. Our results refine the interpretation of anisomycin-based collision paradigms and show that collision landscapes depend on the nature of the stimulus.

molecular biology↗

bioRxiv2025

Double-stranded RNA responses, neoantigen presentation and suppression of hepatocellular carcinoma through NMD inhibition via endonuclease SMG6

Nonsense-mediated mRNA decay (NMD) eliminates aberrant transcripts to maintain transcriptome integrity, yet its broader physiological roles remain incompletely defined. Here, using a liver-specific, inducible mouse model, we selectively inactivate the endonuclease SMG6 - the terminal effector of NMD - in a genetic model of hepatocellular carcinoma (HCC). SMG6 loss completely prevents tumour development and triggers robust innate and adaptive immune responses. Mechanistically, SMG6 inactivation causes the cytoplasmic accumulation of endogenous double-stranded RNAs (dsRNAs), leading to type I interferon induction via the dsRNA sensor MDA5 and revealing an unexpected physiological role for NMD in maintaining dsRNA homeostasis. In parallel, stabilisation and translation of normally degraded transcripts generate non-canonical MHC-I-presented peptides that elicit potent CD8+ T-cell responses. These findings identify SMG6-dependent NMD as a central gatekeeper that restrains dsRNA-driven antiviral signalling and suppresses immunogenic transcript expression. Our work establishes selective SMG6 inhibition as a promising strategy to enhance tumour immunogenicity and suggests that targeting terminal NMD activity could represent a conceptually distinct avenue for cancer immunotherapy.

cancer biology↗

bioRxiv2025

Mutation experiments with AcrB and MexB D408A mutants * indicate the efflux liability of antibiotics

BackgroundWe showed that exposure of an AcrB D408A mutant to efflux inhibitors applied evolutionary pressure to select bacteria with the wild type acrB sequence. This suggested that reversion to wild type can differentiate between efflux inhibitors. Thus, we hypothesized that this experiment could identify inhibitors of the primary RND pump, AcrB or its homologues in other species. ObjectivesTo construct three mutants, Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A and expose the mutants to substrates and non-substrates of AcrB and/or MexB and determine the rate of reversion to wildtype acrB/mexB sequence. MethodsMutant Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A were constructed with site-directed mutagenesis of the relevant nucleotide in the acrB/mexB gene. Mutants were exposed on agar to substrates and the mutation frequency and mutation rate determined. The MIC of antibiotics and the presence/absence of the D408A substitution was determined for mutants. ResultsExposure to the AcrB substrates chlorpromazine and minocycline reverted the D408A genotypes to wild type in a species-dependent manner. Exposure to a non-AcrB substrate, spectinomycin, did not select wild type acrB. Chlorpromazine selected for wild type acrB K. pneumoniae as it had for S. Typhimurium, whereas minocycline selected for wild type E. coli acrB. None of the antibiotics selected wild type mexB, including the tested MexB substrates. ConclusionsEvolutionary paths depend upon the genetic background of the species and availability of alternative routes/genetic pathways that can confer resistance/decreased susceptibility to antibiotics.

microbiology↗

bioRxiv2020

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

Resistance-nodulation-division (RND) efflux pumps play a key role in inherent and evolved multidrug-resistance (MDR) in bacteria. AcrB is the prototypical member of the RND family and acts to recognise and export a wide range of chemically distinct molecules out of bacteria, conferring resistance to a variety of antibiotics. Although high resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown, preventing a complete mechanistic understanding of efflux and inhibition. Here, we determine these structural dynamics in the presence of AcrB substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular modelling, drug binding and bacterial susceptibility studies. We show that the well-studied efflux pump inhibitor phenylalanine-arginine-{beta}-naphthylamide (PA{beta}N) potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within an MDR clinical isolate, AcrBG288D, modifies the plasticity of the transport pathway, which could explain its altered substrate specificity. Our results provide molecular insight into drug export and inhibition of a major MDR-conferring efflux pump and the important directive role of its dynamics.

biochemistry↗

bioRxiv2019

Two simple criteria to estimate an objective’s performance when imaging in non design tissue clearing solutions

Tissue clearing techniques are undergoing a renaissance motivated by the need to image fluorescence deep in biological samples without physical sectioning. Optical transparency is achieved by equilibrating tissues with high refractive index (RI) solutions, which require expensive optimized objectives to avoid aberrations. One may thus need to assess whether an available objective is suitable for a specific clearing solution, or the impact on imaging of small mismatches between cleared sample and objective design RIs. We derived closed form approximations for image quality degradation versus RI mismatch and other parameters available to the microscopist. We validated them with computed (and experimentally confirmed) aberrated point spread functions, and by imaging fluorescent neurons in high RI solutions. Crucially, we propose two simple numerical criteria to establish: (i) the degradation in image quality (brightness and resolution) from optimal conditions of any clearing solution/objective combination; (ii) which objective, among several, achieves the highest resolution in a given immersion medium. These criteria apply directly to the widefield fluorescent microscope but are also closely relevant to more advanced microscopes.

neuroscience↗