The structure of RNA molecules and their complexes are crucial for understanding biology. Notorious examples of large RNAs include the genomes of RNA viruses (Influenza, HIV, Chikungunya, SARS-CoV2...), whose lengths exceed the current capabilities of predictive computational methods, as well as high-res experimental structural techniques. In the INSSANE project, we will develop integrated experimental protocols, together with efficient computational methods for the structural modeling of large RNAs. We will accurately probe and predict the genomic RNA architectures of, bio-medically relevant, viruses. The scope of applicability of our methodologies in bioinformatics will extend beyond viruses, and could be used to model the structure of other large RNAs (lncRNAS, Introns). Towards that goal, we will introduce a novel protocol, named SHAPE-Cut, to streamline the probing of large RNAs. SHAPE-Cut will measure position-specific solvent accessibility by combining novel chemistry and long-read sequencing. In comparison to existing protocols, we expect SHAPE-Cut to avoid typical biases, be easier to implement, and provide increased accuracy, when coupled with specific data analyses and computational methods. We will combine the complementary data of crosslinking and probing experiments: the former reveals long-range interactions, while the latter, through accessibility profiles, has been shown to greatly improve the prediction of local structures. We will implement a recent crosslinking protocol and use its data in index-based genome-wide search of thermodynamically stable RNA-RNA interactions. Then, we devise an integrative structure prediction method that combines SHAPE reactivity, long-range interactions, homology, and thermodynamic stability. Finally, a novel visualization tool will represent genome-scale RNAs and streamline the interdisciplinary dialogue. Algorithmic hurdles will be overcome to improve the processing of sequencing data produced by RNA structure-targeting experiments. All modern RNA probing protocols are based on sequencing technologies, and reveal structural information indirectly, through an alteration that is observable at the RNA sequence level (mutations, stops/cut). However, the crucial mapping of primary sequencing data has received relatively scarce attention in the context of probing techniques, despite specific challenges (chimeric reads, informative errors/stops) having been identified at the root of biases and technical artifacts. We will tailor mapping to our protocols, and develop data structures and indexing techniques to fully exploit sequencing data to its fullest extent. We will also inform mapping by predicted accessibility, e.g. to disambiguate the mapping of erroneous (but probably informative) reads. Beyond increasing mappability, we will deconvolute isoforms/subgenomes, which are known to occur in viral genomes. Our final integrative structure modeling method will consider evolutionary information, and will be formulated as a Maximum-Independent-Set (MIS) graph problem for a conflict graph including both alternative local structure and long-range interactions. We will implement a Fixed Parameter Tractable algorithm based on the treewidth to produce a model with maximal support and thermodynamic stability. By including experts in bioinformatics of RNA structure, sequence analysis, biochemistry, and organic chemistry, our consortium is uniquely positioned to address the timely challenges tackled in the project. Its implementation requires a combination of expertise from traditionally distinct areas of bioinformatics, namely combinatorial structure prediction and high-throughput sequencing analysis. Its synergies will build on existing pairwise collaborations and will streamline the communication between partners representing complementary perspectives on RNA as an object of study.

During the last decades, extensive work focused on the design of molecular systems displaying circularly polarised luminescence (CPL). A chiral light emitter shows different right- and left-handed circularly polarised emission which gives birth to CPL. CPL is key for the enhancement of CP-OLED screen brightness and was pointed as promising for bio-imaging. Therefore, designing systems displaying strong CPL emission and harnessing the produced CPL in devices applications are challenges that we aim to overcome. SupraCPL is a multidisciplinary project aiming at inducing or enhancing the CPL response of 3d complexes by a supramolecular approach. We will develop an innovative strategy, combining coordination complex emitters with chiral supramolecular environment to (i) induce CPL from an achiral chromophore (Cu(I), Cr(III)), or (ii) enhance CPL of chromium(III) chiral complexes showing record CPL for non-lanthanides. The originality of our approach comes from the chiral environment that will be provided by self-assembled capsules or cages structures enabling large effects and dynamic responses. First we plan to use emitting complexes as guests in chiral resorcin[4]arene or pyrogallol[4]arene hexameric self-assembled capsules. Then more complexity will be implemented using Cr(III) complexes as vertices of tetrahedral M4L6 cages. The latter cages are interesting platforms to be combined with CPL outcome for probing or controlling encapsulation by lock/unlock redox input. Therefore, we will finally implement NIR-CPL outcome in versatile applications inherent to cages architectures. To summarise, SupraCPL is an ambitious but realistic project that aim at generating or enhancing CPL in achiral or promising chiral emitters owing to supramolecular environment provided by capsules and cages. The strong CPL arising from this strategy will be valorised imputing CPL as new output in the molecular cages application fields like probing, substrate delivery or multicomponent dynamic systems.

Our goal is to apply for to the MSCA Doctoral Networks in 2023 to train highly skilled doctoral candidates, stimulate their creativity, enhance their innovation capacities, and boost their employability in the emerging field of Epigenetics and Epitranscriptomics. Our consortium called “Chemical analysis and manipulation of epitranscriptomic and epigenetic patterns” (CAME-EPICS) gathers 8 academic partners (6 beneficiaries and 2 partners) and 6 non-academic partners (6 beneficiaries) with complementarity knowledge/expertise, whose role and expertise are crucial for the application-driven research and training activities towards solving societal challenges, entrepreneurship and health care issues. The partners from the non-academic sector will actively participate in all aspects of the training program. The expertise brought by the different partners covers nucleosides and nucleic acids chemistry, analytical chemistry, biochemistry and cellular biology, which will ensure the high quality of an interdisciplinary training of the PhD students in the field of nucleic acids chemistry. Noteworthy, in academia or in industry, there is a growing and urgent need of scientists able to bridge nucleic acids chemistry and biology to fully explore epigenetic and epitranscriptomic processes. Indeed, the recent success of mRNA vaccines has clearly pushed many companies to re-investigate the broad field of nucleic acid chemical modifications in two directions: the use of modified nucleic acids for treatments and vaccins or the search for new drugs (small molecule entities or synthetic biomolecules) that can alter the epigenetic or epitranscriptomic signature of a pathology. In both cases, chemists trained in this interdisciplinary field are very rare and the current supply on the employment market does not satisfy the considerable demand for this type of profile. Our consortium will fill the gap in this field, within the MSCA DN, and will bring the chemical skills and experience necessary to explore and understand at the molecular level the epigenetic and epitranscriptomic fields.

Bacterial resistance to antibiotics is a growing threat to public health and endangers the effective treatment of infections. In Europe and other industrialized countries, the highest proportion of resistant bacteria is found in healthcare facilities. Today, about 70% of bacteria that cause nosocomial infections are resistant to at least one of the drugs most commonly used to treat them. About 25,000 patients die every year in E.U. because of bacterial resistance. It adds about 1.5 billion € of healthcare-associated costs. Although the situation is alarming, pharmaceutical companies have for a long time lost interest in the discovery and development of novel antibiotics. Public research is thus crucial in the field of antimicrobial therapy to identify novel and innovative approaches. In Gram-negative bacteria, the major mechanism of resistance to ß-lactam antibiotics (penicillins, cephalosporins, carbapenems), the most widely used antimicrobials, is the production of one or several ß-lactamase(s). One major approach to overcome this issue consists of combination therapy in which a ß-lactam drug is given along with a ß-lactamase inhibitor, which protects the former from inactivation. Several ß-lactamase inhibitors (clavulanic acid,…) are currently marketed, but they only target serine-ß-lactamases (SBL) and are not active on metallo-ß-lactamases (MBL). There is still much work to accomplish to find clinically useful inhibitors of the increasingly important Ambler class B MBL. MBLs contain one or two Zn cation(s) in their active site, and are structurally and mechanistically unrelated to SBLs. MBLs are classified into three subclasses, B1, B2, and B3 on the basis of structural features. Subclass B1 includes most of the acquired enzymes, encoded by mobile genetic elements. Clinically-relevant B1 MBLs, including IMP-, VIM- and NDM-type enzymes, are currently disseminating in important opportunistic Gram-negative pathogens such as Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumanii. Compared to most SBLs, MBLs exhibit an exceedingly broad substrate profile as they constantly inactivate carbapenems, antibiotics of last resort at the hospital. Moreover, MBL-producing clinical isolates very often show multidrug- and even pandrug-resistance phenotypes, thus representing an increasingly important medical threat that could be successfully addressed by the development of potent, safe and selective MBL inhibitors. In this project, we propose to develop a new and recently identified class of MBL inhibitors, which possess an original Zn-coordinating group. The synthesis of a first generation of compounds incorporating this group has been achieved and some inhibitors already showed efficient inhibition of selected enzymes of the three sub-classes, thus supporting the rationale that broad-spectrum inhibition of MBLs could be achieved. Our objective is to optimize, on the basis of our current knowledge, this completely novel and still unexploited series of MBL inhibitors toward lead compounds that could potentially be amenable to clinical development. The iterative optimization of the inhibitors will be supported by an innovative combination of in silico methods using quantum chemistry and polarizable molecular dynamics approaches, which are the most reliable methods for metallo-enzymes, and X-ray crystallography. The inhibition of clinically-relevant B1 enzymes is the main target, but we will also explore the feasibility of optimizing inhibitors with a broader spectrum of activity. This might be important considering the recent emergence of several acquired subclass B3 enzymes in relevant opportunistic pathogens (AIM-1, SMB-1). If successful, this project is expected to have an important impact in the field of antimicrobial research, as it could represent a viable starting point to develop therapies to treat infections caused by MBL-producing MDR/XDR Gram-negative pathogens, which represent an extremely urgent clinical need.

Human and wildlife animals are exposed to multiple sources of environmental stressors including chemicals such as persistent organic pollutants (POPs) and endocrine disrupting compounds (EDCs). In addition to the important public health issues related to such exposures, EDCs are suspected to elicit ecosystems toxicity with an impact on the food chain and biodiversity and a significant economic burden linked to the increase of metabolic and neurodevelopmental disorders. In this complex and multifactorial context, new and innovative approaches are warranted to address potential linkages between such environmental exposure and health outcomes. Whereas exposure models in toxicology and ecotoxicology traditionally link a given external exposure source with a target organism, the vision of CREATIvE is to consider the organism as both an internal exposure source and a target. Specifically, its ambition is to assess potential health consequences from the release of POP mixtures from an internal storage site (the source) by understanding their complex biological modes of action (MoAs) on the target tissues of the same organism. It is well known that POPs bio-accumulate in living organisms and are stored in specific tissues e.g. adipose tissue (AT) brain, and liver, for long periods of time. Therefore, these tissues represent internal chronic sources of pollutants possibly leading to various disorders including metabolic and neurodegenerative diseases. Such “internal” exposures are not satisfactorily captured by current methods based on investigating different types of external POPs exposure via gavage, injection or acute inhalation. The proposed protocol will not replace the existing ones but will be complementary, taking into account for the first time internal sources of exposure. The aim of CREATIvE is to develop a novel strategy exploring the effects of an internal exposure from grafted contaminated AT. The kinetics and consequences from a redistribution of POPs and their metabolites from grafted contaminated AT on several tissues and organs, e.g. liver, brain and host AT will be studied. The proposed integrated approach is a combination of experimental studies (chemical quantitative measurements in tissues, metabolomics, transcriptomics) and computational modeling (PBPK and systems biology approaches). The advantage of developing such integrated approaches is the possibility to identify the systemic effects of internal mixture exposure at different biological levels, by mimicking the reality of human and animal exposure. As results, new biomarkers will be characterized, and novel complementary models will be proposed which will help at increasing Agregated Exposure Pathways (AEPs) information. To our knowledge such a strategy is clearly innovative and different from existing studies. In a recent preliminary study, an allograft model was developed at Paris Descartes, consisting in a mouse graft of contaminated AT to a non-contaminated mouse. We demonstrated that four weeks after transplantation, the grafts are vascularized and functional. In those initial studies, donor AT was contaminated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and we showed that this contaminant was indeed redistributed to different tissues with different kinetics. Based on this acquired proof of concept, CREATIvE will explore the kinetics of a low dose POP mixture release from an internal source of exposure, and most importantly will assess the toxic effects of such mixtures on other tissues and organs. After improvement of the experimental model, a mixture of twelve environmentally relevant POPs will be studied at low doses with the aim to better understand the consequences of POP mixture release from a unique internal source of exposure.