A fiber-modified adenoviral vector interacts with immunoevasion molecules of the B7 family at the surface of murine leukemia cells derived from dormant tumors

Tumor cells can escape the immune system by overexpressing molecules of the B7 family, e.g. B7-H1 (PD-L1 or CD86), which suppresses the anti-tumor T-cell responses through binding to the PD-1 receptor, and similarly for B7.1 (CD80), through binding to CTLA-4. Moreover, direct interactions between B7-H1 and B7.1 molecules are also likely to participate in the immunoevasion mechanism. In this study, we used a mouse model of tumor dormancy, DA1-3b leukemia cells. We previously showed that a minor population of DA1-3b cells persists in equilibrium with the immune system for long periods of time, and that the levels of surface expression of B7-H1 and B7.1 molecules correlates with the dormancy time. We found that leukemia cells DA1-3b/d365 cells, which derived from long-term dormant tumors and overexpressed B7-H1 and B7.1 molecules, were highly permissive to Ad5FB4, a human adenovirus serotype 5 (Ad5) vector pseudotyped with chimeric human-bovine fibers. Both B7-H1 and B7.1 were required for Ad5FB4-cell binding and entry, since (i) siRNA silencing of one or the other B7 gene transcript resulted in a net decrease in the cell binding and Ad5FB4-mediated transduction of DA1-3b/d365; and (ii) plasmid-directed expression of B7.1 and B7-H1 proteins conferred to Ad5FB4-refractory human cells a full permissiveness to this vector. Binding data and flow cytometry analysis suggested that B7.1 and B7-H1 molecules played different roles in Ad5FB4-mediated transduction of DA1-3b/d365, with B7.1 involved in cell attachment of Ad5FB4, and B7-H1 in Ad5FB4 internalization. BRET analysis showed that B7.1 and B7-H1 formed heterodimeric complexes at the cell surface, and that Ad5FB4 penton, the viral capsomere carrying the fiber projection, could negatively interfere with the formation of B7.1/B7-H1 heterodimers, or modify their conformation. As interactors of B7-H1/B7.1 molecules, Ad5FB4 particles and/or their penton capsomeres represent potential therapeutic agents targeting cancer cells that had developed immunoevasion mechanisms

High Throughput Methods to Transfer DNA in Cells and Perspectives

Genome sequencing led to thousands of genes to study and their molecular cloning to provide ORF collection plasmids. The main approach to study their function involves analysis of the biological consequences of their expression or knockdown, in a cellular context. Given that, the starting point of such experiments is the delivery of the exogenous material, including plasmid DNA in cells. During the last decades, efforts were made to develop efficient methods and protocols to achieve this goal. The present chapter will first give a rapid overview of the main DNA transfer methods described so far: physical, chemical, and biological. Secondly, it will focus on the different methods having reached high-throughput nowadays. Finally, it will discuss the perspectives of this field in terms of future enhancements

Pre-return to work consultation and therapeutic part-time work: Cross-sectional study on level of knowledge and use by general practitioners in France

International audienceBackground In France, general practitioners (GPs) may use two tools specifically designed to help employees who experience difficulties in returning to work after sick leave: the pre-return-to-work (PRW) medical consultation and therapeutic part-time (TPT) work. Objectives The objective was to investigate the level of knowledge and use of these two tools by GPs in Maine-et-Loire, France.Methods This cross-sectional study was performed using a telephone questionnaire to evaluate the level of knowledge of GPs and the use of these two tools in patients having difficulties returning to work.Results: Among the 200 randomly selected GPs, 122 responded (response rate: 61%). More than half of the interviewed GPs declared they 'often' (46%) or 'always' (14%) contacted the occupational physician in these situations. Moreover, 62.2% and 32.7% believed that they had a 'vague' or 'very good' level of knowledge, and 41% and 51% declared either 'frequent' or 'regular' level of use of the PRW medical consultation, respectively. Regarding TPT work, 47% and 53% reported a 'very good' or 'vague' level of knowledge, and 41% and 51% a 'frequent' or 'regular' level of use, respectively. GPs who had a better level of knowledge of this tool reported a higher level of use (p < 0.001).Conclusion This study shows that while the level of knowledge and use of the PRW medical consultation and TPT work is good, it is not optimal. This could be improved by organising training courses for GPs. Obstacles to their wider use could be investigated further in a qualitative study

Improved donor/acceptor BRET couples for monitoring ß-arrestin recruitment to G protein-coupled receptors

International audienceWe report highly sensitive Bioluminescence Resonance Energy Transfer (BRET) assays with optimized donor/acceptor couples. We combined the energy donors Renilla luciferase (Rluc) and the Rluc8 variant with the energy acceptors yellow fluorescent protein (YFP), the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well-established assays measuring ligand-induced ß-arrestin (ßARR) intramolecular rearrangements and recruitment to G protein-coupled receptors (GPCRs). We show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing ßARR assays and offer new options for the development of future BRET assays

Screening for Protein-Protein Interaction Inhibitors Using a Bioluminescence Resonance Energy Transfer (BRET)-Based Assay in Yeast

International audienceThe bioluminescence resonance energy transfer (BRET) technology is a widely used live cell-based method for monitoring protein-protein interactions as well as conformational changes within proteins or molecular complexes. Considering the emergence of protein-protein interactions as a new promising class of therapeutic targets, we have adapted the BRET method in budding yeast. In this technical note, we describe the advantages of using this simple eukaryotic model rather than mammalian cells to perform high-throughput screening of chemical compound collections: genetic tractability, tolerance to solvent, rapidity, and no need of expensive robotic systems. Here, the HDM2/p53 interaction, related to cancer, is used to highlight the interest of this technology in yeast. Sharing the protocol of this BRET-based assay with the scientific community will extend its application to other protein-protein interactions, even though it is toxic for mammalian cells, in order to discover promising therapeutic candidates

Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer.

Several G protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living cells. However, the link between ligand-induced receptor activation and its oligomerization state as well as the proportion of the total receptor population that can engage in oligomeric complexes remain open questions. Here, the closely related human MT1 and MT2 melatonin receptors (MT1R, MT2R) were used to address these issues. Bioluminescence resonance energy transfer (BRET) experiments in living HEK 293 cells revealed that these receptors form homo- and hetero-oligomers. Constitutive energy transfer was observed for all receptor combinations at physiological expression levels and could be detected in single cell BRET experiments. Inhibition of the energy transfer by dilution of the BRET partners identified MT1R and MT2R dimers as the predominant receptor species, and this oligomerization state did not change upon agonist and antagonist binding. Agonists, neutral antagonists, and inverse agonists all promoted increases in BRET values for MT2R but not for MT1R homodimers in living cells and isolated plasma membranes. This indicates that no correlation could be inferred between the receptor activation state and the dimerization state of the receptor. This also suggests that ligand-promoted BRET increases represent specific ligand-induced conformational changes of pre-existing dimers rather then increased dimerization. The observation that ligands favored the energy transfer within the hetero-oligomer from MT1R to MT2R but not in the reverse orientation, from MT2R to MT1R, supports this view

A reporter cell line for the automated quantification of SARS-CoV-2 infection in living cells

International audienceThe SARS-CoV-2 pandemic and the urgent need for massive antiviral testing highlighted the lack of a good cell-based assay that allowed for a fast, automated screening of antivirals in high-throughput content with minimal handling requirements in a BSL-3 environment. The present paper describes the construction of a green fluorescent substrate that, upon cleavage by the SARS-CoV-2 main protease, re-localizes from the cytoplasm in non-infected cells to the nucleus in infected cells. The construction was stably expressed, together with a red fluorescent nuclear marker, in a highly susceptible clone derived from Vero-81 cells. With this fluorescent reporter cell line, named F1G-red, SARS-CoV-2 infection can be scored automatically in living cells by comparing the patterns of green and red fluorescence signals acquired by automated confocal microscopy in a 384-well plate format. We show the F1G-red system is sensitive to several SARS-CoV-2 variants of concern and that it can be used to assess antiviral activities of compounds in dose–response experiments. This high-throughput system will provide a reliable tool for antiviral screening against SARS-CoV-2

Molecular Design in Practice: A Review of Selected Projects in a French Research Institute That Illustrates the Link between Chemical Biology and Medicinal Chemistry

Chemical biology and drug discovery are two scientific activities that pursue different goals but complement each other. The former is an interventional science that aims at understanding living systems through the modulation of its molecular components with compounds designed for this purpose. The latter is the art of designing drug candidates, i.e., molecules that act on selected molecular components of human beings and display, as a candidate treatment, the best reachable risk benefit ratio. In chemical biology, the compound is the means to understand biology, whereas in drug discovery, the compound is the goal. The toolbox they share includes biological and chemical analytic technologies, cell and whole-body imaging, and exploring the chemical space through state-of-the-art design and synthesis tools. In this article, we examine several tools shared by drug discovery and chemical biology through selected examples taken from research projects conducted in our institute in the last decade. These examples illustrate the design of chemical probes and tools to identify and validate new targets, to quantify target engagement in vitro and in vivo, to discover hits and to optimize pharmacokinetic properties with the control of compound concentration both spatially and temporally in the various biophases of a biological system