UltimSMM project proposes original synthetic pathways to generate ideal lanthanide-based sandwich metallocene complexes towards ground-breaking insights into the field of lanthanide Single Molecule Magnets (SMMs). Bulky cyclopentadienyl-based trivalent dysprosium complexes have recently led to impressive progress in the field of lanthanide SMMs, however, so far the perfect geometry has not been obtained in these sandwich complexes (Cp-Dy-Cp angle of 180°). This project aims to synthesize such unprecedented linear trivalent complexes based on pentaarylcyclopentadienyl ligands starting from zero-valent lanthanides and exploring straightforward C-FG bond cleavage (FG = P, Si, halides) routes driven by prospective quantum chemical calculations. The reactions will be performed in solution or, for the first time, under solvent-free ball-milling techniques. Variation of the aryl groups, introduction of heteroelements in the cyclopentadienyl ring and employing lanthanide metals beyond dysprosium are among the target modifications, providing complexes designed to lead the race in the field of high-temperature nanomagnets.

The combination of Bone Marrow Mesenchymal Stromal Cells (BM-MSC) with active injectable carriers brings about innovative solutions to current issues in the field of tissue engineering. In particular, repair of adult articular cartilage lesions remains a clinical challenge because of the limited self-healing capacity of cartilage. We demonstrated previously that the open porosity of homemade collagen microspheres allows for the entrapment and progressive release of TGF-β3, which efficiently triggered the chondrogenic differentiation of BM-MSC in vitro and in vivo, and the production of neo-cartilage tissue. However, one major hurdle in MSC-based therapies for cartilage repair is their late hypertrophic differentiation and subsequent tissue calcification, characterized by the secretion of specific markers such as type X collagen, alkaline phosphatase, osteocalcin and metalloprotease 13 (MMP13). To tackle this challenge, we identified Runx2, which plays a central role in chondrocyte hypertrophy, as the main molecular target to be repressed. Indeed, Runx2 has been widely described to up-regulate the expression of hypertrophic markers. We previously demonstrated that the transient down-regulation of this factor can be achieved with a specific siRNA targeting Runx2. Hence, the strategy of the Spacecart project is to use the transient down-regulation of Runx2 with siRNA (siRunx2) to prevent calcification and ultimately bone formation. We intend to deliver siRunx2 from collagen microspheres also used as an injectable support for BM-MSC and as a TGF-β3 reservoir. However, efficient down-regulation of Runx2 with siRNA requires transfection vectors to bring the nucleic acid to its nuclear target within the cells. Also, to maintain efficient chondrogenic differentiation of BM-MSC, it is important that Runx2 be repressed only after induction of the chondrocyte phenotype, i.e. approximatively day 14, thus calling for delayed delivery of the siRNA. For this project we have designed modified DOTAP-DOPE lipoplexes as the nucleic acid vector. These vectors will be loaded into collagen microspheres and anchored to the matrix via MMP13-sensitive peptides. The action of MMP13 secreted by MSCs will therefore trigger the local delivery of siRNA to the cells at the early stage of hypertrophic differentiation commitment. This cell-elicited spatio-temporal control of siRNA release is expected to help maintain the phenotype of mature chondrocytes in the long term and achieve fully functional hyaline cartilage regeneration. Our work plan includes three experimental tasks dedicated to 1) the design and synthesis of an optimal MMP13 peptide substrate and its use as cleavable linker between the collagen microspheres and siRNA vectors, 2) the investigation of the down-regulation of Runx2 in BM-MSC and its outcome on in vitro chondrogenesis and hypertrophy inhibition and 3) the study of neocartilage production in vivo in the absence of ossification in the long-term. Our consortium gathers complementary expertise in the fields of biomaterial elaboration and functionalization, peptide design and synthesis, and MSC-based therapy of cartilage pathologies. The main originality of our research project is to use the secretion of MMP13 by BM-MSC undergoing hypertrophic differentiation, to locally trigger the delivery of an anti-hypertrophic siRNA. We believe that our integrative approach is original and has a strong innovative potential. In addition, such highly specific self-induced retro-control of the cell behavior can potentially be transposed to other therapeutic indications by adapting the peptides to the enzymatic secretion profile of the specific cells. Therefore, we expect that Spacecart will have an impact on the broad community in the field of biomaterials and tissue engineering.

The pneumovirus family contains several viruses that infect the respiratory tract and induce severe or fatal pneumonia and bronchiolitis in humans and animals. Pneumoviruses include human respiratory syncytial virus (RSV), bovine RSV, human metapneumovirus, avian metapneumovirus, and the recently discovered swine orthopneumovirus (SOV). There are no effective vaccines against these viruses and antivirals are restricted to Ribavirin, a toxic and nonspecific nucleoside analogue. Developing new strategies against these viruses requires improving our knowledge of the molecular mechanisms of viral replication. The genome of Pneumoviruses is composed of an non segmented, single-stranded RNA with a negative polarity of around 15 kb and which encodes 9-11 genes. This RNA genome is always enwrapped by the viral nucleoprotein which prevents any detection of RNA by the cellular innate immunity sensors and protects it against cellular attacks. Pneumoviruses are enveloped viruses that replicate in the cytoplasm of the cells. Once the viral and cellular membranes are fused together, the virus is released into the cytoplasm in the form of a holo-nucleocapsid including the viral polymerase L, capable on the one hand of synthesizing viral mRNAs that will be translated by the cellular machinery, and on the other hand to replicate its genome to create new viral particles that will bud at the plasma membrane thanks to the neo-synthesized glycoproteins. In this project we want to elucidate the mechanisms of viral RNAs synthesis as well as the mechanisms of encapsidation of genomes by nucleoproteins. The viral machinery synthesizing the viral RNAs is autonomous and specific and formed by a multi-protein complex (no cellular equivalent). This complex consists of 3 central proteins: the large polymerase L (250 kDa), the phosphoprotein P, the nucleoprotein N which surrounds the genomic RNA. Pneumoviruses also have transcription (M2-1) or replication (M2-2) co-factors. To synthesize the mRNAs or replicate the genome, the polymerase must temporarily access the viral RNA by "opening" the nucleocapsid. Mirroring, after synthesis of the new genomes, they will be encapsidated by neosynthesized N proteins. In this project, we want to elucidate the molecular mechanisms and regulation of this complex process by associating 4 teams with complementary skills and expertise: (1) an INRA team with the molecular tools to synthesize and purify N, P, M2-1 and L proteins in recombinant form as well as genomic RNAs. (2) an IBS team able to solve structures by electron microscopy and cryo-microscopy. (3) an IBMM team (CNRS, Montpellier University, ENSCM) specialized in the synthesis of modified and varied RNAs at the level of their sequence (methylation at different nucleoside sites) or at the 5 'end by adding various caps. (4) a CNRS-U Aix-Marseille team with the tools and know-how to measure enzymatic activities carried by L proteins. Our main specific objectives will be: To determine the mechanisms controlling the specificity (sequence, chemical nature) of encapsidation of the RNAs by the N proteins To characterize the atomic structure of the N-RNA protein complexes To characterize the epitranscriptomic modifications of the RNAs mediated by the L protein (capping and methylations). Ultimately, we want to develop an autonomous system of in vitro synthesis of RNAs that will permit studying the functioning of this complex and testing antiviral compounds targeting the replication / transcription steps in vitro.

Pseudomonas aeruginosa (PA) is one of the most common pathogen identified in respiratory track infections. This bacterium is particularly able to develop biofilm structure giving a selective advantage to the pathogen toward antibiotics therapy. Moreover, resistant strains are emerging. As a consequence, the development of new antibacterial agents able to escape the mechanisms of resistance or using new modes of action had become imperative and represents a major research challenge. PA takes advantage of a large arsenal of molecular tools and weapons to adapt and proliferate in human airways. The ability to inhibit such virulent factors offers the perspective for a new approach for antibacterial therapies in which the bacterial functions contributing to the infection are targeted. Inhibiting the adhesion of PA to the secreted mucins as well as the cell membrane of bronchial epithelium is an attractive alternative to antibiotic treatment and a promising approach. This adhesion process is mainly mediated through the interactions of flagellum (FliC, FliD), pili (type IV, CupB) or soluble lectins (PA-IL, PA-IIL) with mucin exposed oligosaccharides, cell exposed asialo-ganglioside or the glycocalyx surrounding the host’s cells. Several reports in the literature have described the synthesis of PA lectins/adhesins inhibitors based on the glycoside cluster effect. However, their selectivity and their mechanisms of action in-vitro and in-vivo remain to be assessed. The aim of this project is thus to design, synthesize and screen molecular mimetics inhibiting these carbohydrate-bacterial lectin interactions and to assess their selectivity and mechanism of action. The present project is willing to discover high affinity multivalent ligands towards seven identified PA lectins (PA-IL, PA-IIL, FliC, FliD, PilA, PilY1 and CupB6) in order to test these molecules from first model affinity experiments to in vivo assays. Another challenging aspect of the work will be to isolate, identify and characterize new PA lectins to further define a complete library of glycomimetics, as inhibitors of PA adhesion. In order to design high affinity ligands, we have developed a fast and versatile chemistry based on a “Lego-like” chemistry for the synthesis of glycomimetics. Glycoside building blocks will be assembled on a phosphorylated scaffold using a combination of DNA solid phase synthesis and microwave assisted click chemistry. Thanks to glycoarray technology and to nanotechnology tools (AFM adhesive force measurements), the glycosides building blocks and the glycomimetics will be studied at the nanomole scale. The results from this study and molecular simulations will be used to assess a structure-binding relationship. The synthesis of the best hits will be scaled up for their study in-vitro, in-vivo and in murin model as anti-biofilm and anti-adhesive molecules. In the second aspect of this project, new lectins will be identified by two approaches: 1) lectin fishing with glycomimetic modified magnetic colloids and 2) competition assays between purified human bronchial mucins and their free exposed oligosaccharides. Our project combines original synthetic and screening strategies as well as complementary know-how of four interdisciplinary laboratories specialized in the synthesis of oligonucleotides and their conjugates, in glycosides synthesis, in glycoarrays and nanotechnology and in glycobiology, molecular simulation and microbiology.

The SURHYMI project aims at developing a sustainable and integrated approach for the synthesis of hybrid mesoporous films and membranes densely and homogeneously functionalized by polymers, designed as platform materials for the elaboration of micropollutant removal devices. The control of the textural and chemical properties of the supported films and membranes (pore diameter and topology, and functions (acid, basic, cyclodextrin) in the mesopores), will allow to evaluate their performances in the reversible sorption of anionic, cationic and hydrophobic micropollutants based on electrostatic interactions or host-guest complexes. Novel polyion complex micelles (PIC) as well as host-guest inclusion complex (InC) micelles will be evaluated for the first time for their ability to controllably form a variety of ordered mesostructures by the sol-gel route, first as powders by macroscopic precipitation and then as films and membranes by deposition-evaporation. PIC micelles will be formed by electrostatic complexation between double-hydrophilic block copolymers (DHBC) and oppositely charged polyions, auxiliaries of micellisation. Poly(acrylic acid) and poly(aminoethylacrylamide) based DHBCs will be synthesized by RAFT in acidic media in order to protect the chain-transfer agent. Then, they will be used as platform polymers for the preparation by amidation reactions of a range of new DHBCs with beta-cyclodextrin (CD). Polymers with beta-CD functionalities will enable the formation of inclusion complex micelles (InC) with ditopic/multitopic guest species, which will be studied as silica structuring agents. PIC and InC assemblies will be evaluated for the first time as structuring, functionalizing and porogenic agents of functional mesoporous supported films and membranes. The new methodology developed should allow the preparation of materials whose mesopores will be intrinsically functionalized in a homogeneous and dense way by the targeted and previously prepared functions. The films will be prepared by evaporation induced assembly (EISA process). Depositions will be performed first on dense substrates, then on porous substrates. The disassembly of PIC and InC will allow the elution of the micellisation auxiliaries and will reveal the intrinsic functionalization of the layers with the three types of functions, acid, basic, and CD. The porous textures, thicknesses, density profiles and permeability of the functional films will be characterized. The influence of these properties will be evaluated for the sorption of model micropollutants as a function of physicochemical parameters (pH, concentration, ionic strength). Four organic micropollutants (hydrophilic cationic and anionic, as well as hydrophobic) were selected to demonstrate the specific and reversible character of the sorption mechanism in hybrid mesoporous silica-based platform materials. The kinetics, reversibility and repeatability of the sorption process will be studied as well as the chemical and structural recyclability and aging of the adsorbent materials. These results will be transferred to supported membranes to evaluate their filtration performance, chemical and structural stability and the mechanical properties of the prepared membrane materials.