Agnès Paquet

SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: Reproducibility and predictivity results from an inter-laboratory study.

The SENS-IS test protocol for the in vitro detection of sensitizers is based on a reconstructed human skin model (Episkin) as the test system and on the analysis of the expression of a large panel of genes. Its excellent performance was initially demonstrated with a limited set of test chemicals. Further studies (described here) were organized to confirm these preliminary results and to obtain a detailed statistical analysis of the predictive capacity of the assay. A ring-study was thus organized and performed within three laboratories, using a test set of 19 blind coded chemicals. Data analysis indicated that the assay is robust, easily transferable and offers high predictivity and excellent within- and between-laboratories reproducibility. To further evaluate the predictivity of the test protocol according to Cooper statistics a comprehensive test set of 150 chemicals was then analyzed. Again, data analysis confirmed the excellent capacity of the SENS-IS assay for predicting both hazard and potency characteristics, confirming that this assay should be considered as a serious alternative to the available in vivo sensitization tests.

RiboProfiling: a Bioconductor package for standard Ribo-seq pipeline processing.

The ribosome profiling technique (Ribo-seq) allows the selective sequencing of translated RNA regions. Recently, the analysis of genomic sequences associated to Ribo-seq reads has been widely employed to assess their coding potential. These analyses led to the identification of differentially translated transcripts under different experimental conditions, and/or ribosome pausing on codon motifs. In the context of the ever-growing need for tools analyzing Ribo-seq reads, we have developed ‘RiboProfiling’, a new Bioconductor open-source package. ‘RiboProfiling’ provides a full pipeline to cover all key steps for the analysis of ribosome footprints. This pipeline has been implemented in a single R workflow. The package takes an alignment (BAM) file as input and performs ribosome footprint quantification at a transcript level. It also identifies footprint accumulation on particular amino acids or multi amino-acids motifs. Report summary graphs and data quantification are generated automatically. The package facilitates quality assessment and quantification of Ribo-seq experiments. Its implementation in Bioconductor enables the modeling and statistical analysis of its output through the vast choice of packages available in R. This article illustrates how to identify codon-motifs accumulating ribosome footprints, based on data from Escherichia coli.

SigmaR1 regulates membrane electrical activity in response to extracellular matrix stimulation to drive cancer cell invasiveness

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K(+) channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition.

Characterizing isomiR variants within the microRNA‐34/449 family

miR‐34/449 microRNAs are conserved regulators of multiciliated cell differentiation. Here, we evidence and characterize expression of two isomiR variant sequences from the miR‐34/449 family in human airway epithelial cells. These isomiRs differ from their canonical counterparts miR‐34b and miR‐449c by one supplemental uridine at their 5′‐end, leading to a one‐base shift in their seed region. Overexpression of canonical miR‐34/449 or 5′‐isomiR‐34/449 induces distinct gene expression profiles and biological effects. However, some target transcripts and functional activities are shared by both canonical microRNAs and isomiRs. Indeed, both repress important targets that result in cell cycle blockage and Notch pathway inhibition. Our findings suggest that 5′‐isomiR‐34/449 may represent additional mechanisms by which miR‐34/449 family finely controls several pathways to drive multiciliogenesis.

Inflammatory and Repair Pathways Induced in Human Bronchoalveolar Lavage Cells with Ozone Inhalation

Background Inhalation of ambient levels of ozone causes airway inflammation and epithelial injury. Methods To examine the responses of airway cells to ozone-induced oxidative injury, 19 subjects (7 with asthma) were exposed to clean air (0ppb), medium (100ppb), and high (200ppb) ambient levels of ozone for 4h on three separate occasions in a climate-controlled chamber followed by bronchoscopy with bronchoalveolar lavage (BAL) 24h later. BAL cell mRNA expression was examined using Affymetrix GeneChip Microarray. The role of a differentially expressed gene (DEG) in epithelial injury was evaluated in an in vitro model of injury [16HBE14o- cell line scratch assay]. Results Ozone exposure caused a dose-dependent up-regulation of several biologic pathways involved in inflammation and repair including chemokine and cytokine secretion, activity, and receptor binding; metalloproteinase and endopeptidase activity; adhesion, locomotion, and migration; and cell growth and tumorigenesis regulation. Asthmatic subjects had 1.7- to 3.8-fold higher expression of many DEGs suggestive of increased proinflammatory and matrix degradation and remodeling signals. The most highly up-regulated gene was osteopontin, the protein level of which in BAL fluid increased in a dose-dependent manner after ozone exposure. Asthmatic subjects had a disproportionate increase in non-polymerized osteopontin with increasing exposure to ozone. Treatment with polymeric, but not monomeric, osteopontin enhanced the migration of epithelial cells and wound closure in an α9β1 integrin-dependent manner. Conclusions Expression profiling of BAL cells after ozone exposure reveals potential regulatory genes and pathways activated by oxidative stress. One DEG, osteopontin, promotes epithelial wound healing in an in vitro model of injury.

HITS-CLIP in various brain areas reveals new targets and new modalities of RNA binding by fragile X mental retardation protein

Abstract Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the functional deficiency of the fragile X mental retardation protein (FMRP), an RNA-binding protein involved in translational regulation of many messenger RNAs, playing key roles in synaptic morphology and plasticity. To date, no effective treatment for FXS is available. We searched for FMRP targets by HITS-CLIP during early development of multiple mouse brain regions (hippocampus, cortex and cerebellum) at a time of brain development when FMRP is most highly expressed and synaptogenesis reaches a peak. We identified the largest dataset of mRNA targets of FMRP available in brain and we defined their cellular origin. We confirmed the G-quadruplex containing structure as an enriched motif in FMRP RNA targets. In addition to four less represented motifs, our study points out that, in the brain, CTGKA is the prominent motif bound by FMRP, which recognizes it when not engaged in Watson–Crick pairing. All of these motifs negatively modulated the expression level of a reporter protein. While the repertoire of FMRP RNA targets in cerebellum is quite divergent, the ones of cortex and hippocampus are vastly overlapping. In these two brain regions, the Phosphodiesterase 2a (Pde2a) mRNA is a prominent target of FMRP, which modulates its translation and intracellular transport. This enzyme regulates the homeostasis of cAMP and cGMP and represents a novel and attractive therapeutic target to treat FXS.

A cost effective 5΄ selective single cell transcriptome profiling approach with improved UMI design

Abstract Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5΄ selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3΄ selective approaches which just provide internal sequences close to the 3΄ end. The only currently existing 5΄ selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5΄ selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.

CDC20B is required for deuterosome-mediated centriole production in multiciliated cells

Multiciliated cells (MCCs) harbour dozens to hundreds of motile cilia, which beat in a synchronized and directional manner, thus generating hydrodynamic forces important in animal physiology1, 2. In vertebrates, MCC differentiation critically depends on the synthesis and release of numerous centrioles by specialized structures called deuterosomes1-5. Little is known about the composition, organization and regulation of deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome-stage MCCs are characterized by the expression of many cell cycle-related genes. Among those, we further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene. We show that the CDC20B protein associates to the deuterosome, and is required for the production of centrioles and cilia in mouse and Xenopus MCCs. In Xenopus, centrioles and cilia were efficiently rescued in absence of CDC20B by over-expression of the protease Separase, linking CDC20B function to centriole release from deuterosomes, in analogy to centriole disengagement in mitotic cells. This work reveals the shaping of a new biological function, deuterosome-mediated centriole production in vertebrate MCCs, by adaptation between ancestral and recently evolved cell cycle-related molecules.

Targeting the DNM3OS / miR-199a~214 cluster for the treatment of fibroproliferative diseases

Given the paucity of effective treatments for fibrotic disorders, new insights into the deleterious mechanisms controlling fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. Here, we identified the long non-coding RNA DNM3OS as a critical downstream effector of TGF-β-induced myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to 3 distinct profibrotic mature miRNAs (i.e. miR-199a-5p/3p and miR-214-3p), which influence both SMAD and non-SMAD components of TGF-β signaling in a multifaceted way, through two modes of action consisting of either signal amplification or mediation. Finally, we provide preclinical evidence that interfering with DNM3OS function using distinct strategies not only prevents lung and kidney fibrosis but also improves established lung fibrosis, providing thus a novel paradigm for the treatment of refractory fibrotic diseases such as idiopathic pulmonary fibrosis. One Sentence Summary The DNM3OS lncRNA is a reservoir of fibromiRs with major functions in fibroblast response to TGF-β and represents a valuable therapeutic target for refractory fibrotic diseases such as idiopathic pulmonary fibrosis (IPF).

Single-cell RNA sequencing reveals novel cell differentiation dynamics during human airway epithelium regeneration

Background It is usually considered that the upper airway epithelium is composed of multiciliated, goblet, secretory and basal cells, which collectively constitute an efficient first line of defense against inhalation of noxious substances. Upon injury, regeneration of this epithelium through proliferation and differentiation can restore a proper mucociliary function. However, in chronic airway diseases, the injured epithelium frequently displays defective repair leading to tissue remodeling, characterized by a loss of multiciliated cells and mucus hyper-secretion. Delineating drivers of differentiation dynamics and cell fate in the human airway epithelium is important to preserve homeostasis. Results We have used single cell transcriptomics to characterize the sequence of cellular and molecular processes taking place during human airway epithelium regeneration. We have characterized airway subpopulations with high resolution and lineage inference algorithms have unraveled cell trajectories from basal to luminal cells, providing markers for specific cell populations, such as deuterosomal cells, i.e. precursors of multiciliated cells. We report that goblet cells, like secretory cells, can act as precursors of multiciliated cells. Our study provides a repertoire of molecules involved in key steps of the regeneration process, either keratins or components of the Notch, Wnt or BMP/TGFβ signaling pathways. Our findings were confirmed in independent experiments performed on fresh human and pig airway samples, and on mouse tracheal epithelial cells. Conclusions Our single-cell RNA-seq study provides novel insights about airway epithelium differentiation dynamics, clarifies cell trajectories between secretory, goblet and multiciliated cells, identifies novel cell subpopulations, and maps the activation and repression of key signaling pathways.