Biology subjects

Conte, V.

Publications and source records attributed to Conte, V..

8 recordsLinked to original sources

bioRxiv2026

Increased Nuclear Susceptibility to Mechanical Stress in Phospholamban R14del Cardiomyopathy: A New Perspective on Disease Heterogeneity

Background: Clinical presentation of phospholamban (PLN) R14del cardiomyopathy is highly variable, from asymptomatic carriers to rapidly progressing heart failure. which cannot be fully explained by its known disturbance of sarcoplasmic reticulum calcium handling. Nuclear calcium signalling, shaped by sarcoplasmic reticulum calcium release, plays a key role in regulating gene expression and the cellular response to mechanical stress. However, how the R14del variant affects nuclear signalling under mechanical loading remains unclear. Objective: To uncover the impact of the PLN R14del variation on mechanosensing, nuclear calcium handling and its link to the variable clinical representation of this cardiomyopathy. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the PLN R14del variant, an isogenic control, and wildtype control were subjected to mechanical stimulation by cyclic stretch or cultured on substrates with physiological (10-20 kPa) and pathological (65-85 kPa) stiffness. Nuclear morphology, cytoskeletal organisation and nuclear body formation were assessed via immunofluorescence. Traction force microscopy was used to assess mechanical stress and force generation. Cardiac tissues from PLN R14del cardiomyopathy patients, dilated cardiomyopathy patients, and healthy controls were analysed under mechanical loading (at left ventricular assist device implantation) and unloading (at heart transplantation). Living myocardial slices from a PLN R14del patient were maintained under overloading or physiological loading conditions. Results: PLN R14del hiPSC-CMs showed impaired nuclear calcium handling and, under mechanical stress, exhibited impaired mechanotransduction and elevated nuclear bodies. A strain-avoidance response was triggered but failed to prevent apoptosis. Nuclear abnormalities were confirmed in patient myocardial slices and cardiac biopsies which normalized upon relative mechanical unloading. Conclusion: PLN R14del increases nuclear susceptibility to mechanical stress, linking nuclear dysfunction to new disease insights and highlighting potential for mechanobiology-based therapeutic interventions.

cell biology↗

bioRxiv2024

New insight in cyclic monoterpenoids mechanism of action: an in silico approach

Clarifying the mechanism of action of natural terpenoids is challenging. Further, their efficacy is inspiring in developing new antimycotic agents. Among all, thymol, carvacrol and thymyl acetate are largely scrutinized, while the new brominate thymol, namely bromothymol (4-bromo-2-isopropyl-5-methylphenol), needs deeper investigation. Here its antimycotic efficacy was evaluated and, in parallel, a careful in silico investigation of the mechanism of action was proposed. In vivo experiments, on species of acclaimed resistance, demonstrated that bromothymol had a Minimum Inhibitory Concentration (MIC) equal [~]40 g/ml, 6 times more active than thymol. Partition coefficient (LogP) in heptane, determined through density functional theory (DFT), and Molecular Dynamics (MD) simulations, based on a Minimum Bias Approach, in the presence of neutral bilayers, indicated that bromothymol inserts into cellular membrane, such as thymol, carvacrol, and Thymyl acetate. Monoterpenoids bearing the hydroxyl group induces a shrinkage of the membrane thickness, while only thymol affected membrane density of the leaflets in which it inserted. Thymol, carvacrol, and bromothymol interacted with the polar head of the lipids causing an electrostatic imbalance into the membrane, justifying their biological activity. For the first time a detailed in silico characterization on the mechanism of these compounds is afforded, returning a coherent and clear picture of their mechanism of action.

bioinformatics↗

bioRxiv2024

De novo gene synthesis by an antiviral reverse transcriptase

Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands in DRT2-expressing cells revealed that reverse transcription generates concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this DNA product is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA. One-Sentence SummaryBacterial reverse transcriptases synthesize extrachromosomal genes via rolling-circle amplification to confer potent antiviral immunity.

molecular biology↗

bioRxiv2023

Belt and braces: two escape ways to maintain the cassette reservoir of large chromosomal integrons

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, it was found that the experimental inversion of the SCI of Vibrio cholerae led to a dramatic increase of the cassette excision rate associated to a fitness defect. Here, we question the evolutionary sustainability of this apparently counter selected genetic context through experimental evolution. We find that the integrase is rapidly inactivated and that the inverted SCI can recover its original orientation by homologous recombination between two insertion sequences (ISs) present in the array. These two outcomes of SCI inversion restore the normal growth and prevent the loss of cassettes, enabling SCIs to retain their roles as reservoirs of functions. These results illustrate an interesting interplay between gene orientation, genome rearrangement, bacterial fitness and demonstrate how integrons can benefit from their embedded ISs.

genetics↗

bioRxiv2023

Environmental stiffness restores mechanical homeostasis in vimentin-depleted cells

Recent experimental evidence indicates a role for the intermediate filament vimentin in regulating cellular mechanical homeostasis, but its precise contribution remains to be discovered. Mechanical homeostasis requires a balanced bi-directional interplay between the cells microenvironment and the cellular morphological and mechanical state - this balance being regulated via processes of mechanotransduction and mechanoresponse, commonly referred to as mechanoreciprocity. Here, we systematically analyze vimentin-expressing and vimentin-depleted cells in a swatch of in vitro cellular microenvironments varying in stiffness and/or ECM density. We find that vimentin-expressing cells maintain mechanical homeostasis by adapting cellular morphology and mechanics to micromechanical changes in the microenvironment. However, vimentin-depleted cells lose this mechanoresponse ability on short timescales, only to reacquire it on longer time scales. Indeed, we find that the morphology and mechanics of vimentin-depleted cell in stiffened microenvironmental conditions can get restored to the homeostatic levels of vimentin-expressing cells. Additionally, we observed vimentin-depleted cells increasing collagen matrix synthesis and its crosslinking, a phenomenon which is known to increase matrix stiffness, and which we now hypothesize to be a cellular compensation mechanism for the loss of vimentin. Taken together, our findings provide further insight in the regulating role of intermediate filament vimentin in mediating mechanoreciprocity and mechanical homeostasis.

biophysics↗

bioRxiv2023

CRB3 and ARP2/3 regulate cell biomechanical properties to set epithelial monolayers for collective movement

Several cellular processes during morphogenesis, tissue healing or cancer progression involve epithelial to mesenchymal plasticity that leads to collective motion (plasticity?). Even though a rich variety of EMP programs exist, a major hallmark unifying them is the initial breaking of symmetry that modifies the epithelial phenotype and axis of polarity. During this process, the actin cytoskeleton and cellular junctions are extensively remodelled correlating with the build-up of mechanical forces. As the collective migration proceeds, mechanical forces generated by the actin cytoskeleton align with the direction of migration ensuring an organized and efficient collective cell behaviour, but how forces are regulated during the breaking of symmetry at the onset of EMP remains an unaddressed question. It is known that the polarity complex CRB3/PALS1/PATJ, and in particular, CRB3 regulates the organization of the actin cytoskeleton associated to the apical domain thus pointing at a potential role of CRB3 in controlling mechanical forces. Whether and how CRB3 influences epithelial biomechanics during the epithelial-mesenchymal plasticity remains, however, largely unexplored. Here, we systematically combine mechanical and molecular analyses to show that CRB3 regulates the biomechanical properties of collective epithelial cells during the initial breaking of symmetry of the EMP. CRB3 interacts with ARP2/3 and controls the remodelling of actin throughout the monolayer via the modulation of the Rho-/Rac-GTPase balance. Taken together, our results identified CRB3, a polarity protein, as a regulator of epithelial monolayer mechanics during EMP.

cell biology↗

bioRxiv2022

Genetic drivers of chromosomal integron stability

Integrons are adaptive bacterial devices that rearrange promoter less gene cassettes into variable ordered arrays under stress conditions, to sample combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here we show a key role of promoter-containing toxin-antitoxin (TA) cassettes as abortive systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes. TeaserThe accumulation of cassette functions in integrons is ensured by toxin-antitoxin systems which kill the cell when the cassette excision rate is too high.

genetics↗

bioRxiv2021

Oncogenic RAS instructs morphological transformation of human epithelia via differential tissue mechanics.

The RAS proto-oncogene is a critical regulator of cell state, morphology and mechanics, and plays a key role in cancer progression. Here, by using a human epithelial model in vitro, we ask how morpho-mechanical changes driven by oncogenic RAS activation at the level of individual cells are collectively integrated to drive changes in tissue behaviour. We found that the uniform oncogenic expression of HRAS.V12 in confined epithelial monolayers causes reproducible changes in the structure and organization of the tissue, which acquires a transitory bilayered morphology. RAS-driven bilayering associates with reproducible layer-specific differences in cell-cell contractility and cell-matrix forces. These drive the initially flat tissues to form three-dimensional structures mimicking some of the behaviours seen in human cancers. Our findings establish a physical mechanism of cellular collectives through which uniform expression of RAS can be interpreted differently in different places of the same tissue to regulate its physiological and pathological morphology.

cancer biology↗