Biology subjects

Gras, D.

Publications and source records attributed to Gras, D..

3 recordsLinked to original sources

bioRxiv2024

Cell culture differentiation and proliferation conditions influence the in vitro regeneration of the human airway epithelium

The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an Air-Liquid Interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist between media used for ALI-cultured human airway epithelial cells, our study aimed to evaluate the impact of several media (BEGMTM, PneumaCultTM, "Half&Half" and "Clancy") on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling and epithelial morphology. We found higher proportions of multiciliated cells in PneumaCultTM-ALI and Half&Half, stronger EGF signaling from basal cells in BEGMTM-ALI, differential expression of the SARS-CoV-2 entry factor ACE2, and distinct secretome transcripts depending on media used. We also established that proliferation in PneumaCultTM-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will help to choose the most relevant medium according to the processes to be investigated such as cilia, mucus biology or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology.

cell biology↗

bioRxiv2022

A recurrent de novo splice site variant involving DNM1 alternative exon 10a causes developmental and epileptic encephalopathy through a dominant-negative mechanism

Heterozygous pathogenic variants in DNM1 cause developmental and epileptic encephalopathy (DEE) due to a dominant-negative mechanism impeding vesicular fission. Thus far, pathogenic variants in DNM1 have been studied using a canonical transcript that includes the alternatively spliced exon 10b. However, after performing RNA sequencing in thirty-nine pediatric brain samples, we find the primary transcript expressed in the brain includes the downstream exon 10a instead. Using this information, we evaluated genotype-phenotype correlations of variants affecting exon 10a and identified a cohort of eleven previously unreported individuals. Eight individuals harbor a recurrent de novo splice site variant, NG_029726.1(NM_001288739.1):c.1197-8G>A, which affects exon 10a and leads to DEE consistent with the classical DNM1 phenotype. We find this splice site variant leads to disease through an unexpected dominant-negative mechanism. Functional testing reveals an in-frame upstream splice acceptor causing insertion of two amino acids predicted to impair oligomerization-dependent activity. This is supported by neuropathological samples showing accumulation of synaptic vesicles adherent to the plasma membrane consistent with impaired vesicular fission. Two additional individuals with missense variants affecting exon 10a, p.(Arg399Trp) and p.(Gly401Asp), had a similar DEE phenotype. In contrast, a single individual with a missense variant affecting exon 10b, p.(Pro405Leu), which is less expressed in the brain, had a correspondingly less severe presentation. Thus, we implicate variants affecting exon 10a as causing the severe DEE typically associated with DNM1-related disorders. We highlight the importance of considering relevant isoforms for disease-causing variants, as well as the possibility of splice site variants acting through a dominant-negative mechanism.

genetics↗

bioRxiv2019

Active mucus-cilia hydrodynamic coupling drives self-organisation of human bronchial epithelium

The respiratory tract is protected by mucus, a complex fluid transported along the epithelial surface by the coordinated beating of millions of microscopic cilia, hence the name of mucociliary clearance. Its impairment is a strong marker of severe chronic respiratory diseases. Yet, the relationship between ciliary density and the spatial scale of mucus transport, as well as the mechanisms that drive ciliary-beat orientations during ciliogenesis are much debated. Here, we show on polarized human bronchial epithelia that mucus swirls and circular orientational order of the underlying ciliary beats emerge and grow during ciliogenesis, until a macroscopic mucus transport is achieved for physiological ciliary densities. By establishing that the macroscopic ciliary-beat order is lost and recovered by removing and adding mucus respectively, we demonstrate that cilia/mucus hydrodynamic interactions govern the collective dynamics of ciliary-beat directions. We propose a two-dimensional model that predicts a phase diagram of mucus transport in accordance with the experiments. It paves the way to a predictive in-silico modeling of bronchial mucus transport in health and disease.

biophysics↗