Catégorie d'actualités: significant publications

In Cellulo and In Vivo Comparison of 3 helper lipids for Lipid Nanoparticle Formulation of mRNA

LNPs are a leading class of mRNA delivery systems. LNPs are made of an ionizable lipid, a polyethyleneglycol (PEG)-lipid conjugate and helper lipids. The success of LNPs is due to proprietary ionizable lipids and appropriate helper lipids.

Using a benchmark lipid (D-Lin-MC3) researchers compared the ability of three helper lipids to transfect dendritic cells in cellulo and in vivo. Studies revealed that the choice of helper lipid does not influence the transfection efficiency of immortalized cells but, LNPs prepared with DOPE (dioleylphosphatidylethanolamine) and β-sitosterol were more efficient for mRNA transfection in murine dendritic cells than LNPs containing DSPC (distearoylphosphatidylcholine).

This higher potency of DOPE and β-sitosterol LNPs for mRNA expression was also evident in vivo but only at low mRNA doses.

References of the article published in Nanomaterials:
Ayoub Medjmedj, Albert Ngalle-Loth, Rudy Clemnçon, Josef Hamacek, Chantal Pichon and Federico Perche
In Cellulo and In Vivo Comparison of Cholesterol, Beta-Sitosterol and Dioleylphosphatidylethanolamine for Lipid Nanoparticle Formulation of mRNA
Nanomaterials 2022, 12(14), 2446; https://doi.org/10.3390/nano12142446

A unique biosensor able to detect as low as 10 nM bioavailable copper based on whole-cell eukaryotic Saccharomyces cerevisiae

Cooper in a micronutrient essential to life, its lack induces neurological and blood disorders. It is extensively used in industry, in particular in the batteries of electric cars, but also as a fertilizer and fungicide. However, it is toxic at high concentrations, and is considered as a critical emerging pollutant. Copper detection in water constitutes a major societal and environmental issue.

Currently, copper concentrations are monitored by sophisticated analytical methods requiring time, expansive equipments and deep expertises. Moreover, these technics quantify total copper present in a sample and not copper interacting with living organisms.

Researchers of CBM developed a new and atypical device able to detect copper in a very sensitive and easy way. Their biosensor is based on whole-cell eukaryotic living Saccharomyces cerevisiae, allowing detection of bioavailable copper. It is a ratiometric biosensor, measuring the ratio between (i) a constitutively expressed fluorescent protein and (ii) another fluorescent protein whose expression is directly correlated with copper concentrations as its expression is in under the control of CUP1 promoter, a well-known promoter in Saccharomyces.

By genetic engineering, researchers created many different variants to optimize the response of our biosensor. Their best biosensor is able to detect as low as 10 nM of copper in a linear range from 10-3 to 10-8 M, much better features compared to other currently reported whole cell copper biosensors. This biosensor was also validated on “real” samples: detected concentrations are totally in agreement with manufacturers’ values.

Reference of the article :
Bojan Zunar, Christine Mosrin, Hélène Bénédetti, Béatrice Vallée
Re-engineering of CUP1 promoter and Cup2/Ace1 transactivator to convert Saccharomyces cerevisiae into a whole-cell eukaryotic biosensor capable of detecting 10 nM of bioavailable copper
Biosensors and Bioelectronics 214 (2022) 114502

The article was reported by the CNRS Institute of Chemistry on its website and in its letter "En direct des labos".

 

A new mechanism of antiobiotic resistance

The bacterial Rho factor is a molecular motor that induces genome-wide transcription termination. Rho is essential in many species, including in Mycobacterium tuberculosis where inactivation of the rho gene leads to rapid death. Nevertheless, the Rho factor of M. tuberculosis [MtbRho] displays idiosyncrasies, including resistance to the antibiotic bicyclomycin [BCM], which remain unexplained. To identify the molecular origin of these idiosyncrasies, we solved the structure of MtbRho by cryo-EM at 3.3 Å. This atomic structure notably reveals a leucine → methionine substitution that creates steric hindrance in the binding pockets of BCM, close to the ATPase sites, thereby conferring resistance to BCM at the expanse of molecular motor efficiency. Our work contributes to explain the unusual properties of MtbRho and provides groundwork for the development of new antibiotics.

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J.-M. Bonmatin co-authored an article linking neonicotinoid insecticides to chronic kidney disease of undetermined etiology (CKDu)

Chronic Kidney Diseases (CKD) are a growing scourge worldwide, particularly in less developed countries with intensive agriculture. Several risk factors have been identified, but an undetermined etiology (CKDu) remains which may be linked to pesticides (Floris et al., 2021).

J.-M. Bonmatin participated in a study in Sri Lanka published in Scientific Reports late 2021 (Taira et al., 2021). Although the size of the study remains statistically modest, the authors (members of the Task Force on Systemic Pesticide) showed that the concentrations of several neonicotinoids measured in urine were related to the biomarkers Cystatin-C and L-FABP as well as to the neurophysiological symptoms observed. The authors conclude that the urinary concentrations of these neonicotinoids are a risk factor for tubular disorders of the kidney. This is another element that adds to our previous publications (e.g., Ichikawa et al., 2019 and Bonmatin et al., 2021) on the human health effects of these insecticides.

Neonicotinoids, known as "bee killers" and which are the source of major impacts on biodiversity (invertebrates and vertebrates) are still widely used for rice cultivation in Asia (Prihandiani et al., 2021).  These insecticides have been banned in France since 2018 (except for sugar beet). They are in the process of being banned in Sri Lanka.

Référence :  Taira K, Kawakami T, Weragoda SK, Herath HMAS, Ikenaka Y, Fujioka K, Hemachandra M, Pallewatta N, Aoyama Y, Ishizuka M, Bonmatin JM & Komori M (2021) Scientific Reports, 11, 22484.

https://doi-org.inc.bib.cnrs.fr/10.1038/s41598-021-01732-2

The first molecules which really prefer manganese(II) over zinc(II)

Manganese(II) complexes meet increasing interest in biomedical applications, in particular as potential Magnetic Resonance Imaging contrast agents with a better biocompatibility and safety profile than the currently used gadolinium(III) chelates. However, we severely lack chelating ligands that enable high MnII complex stability and especially good selectivity for manganese(II) versus zinc(II), the most relevant biological competitor. In an article published in Angewandte Chemie Int. Ed., the “Metal complexes and MRI” group in collaboration with the group of Peter Comba at the University of Heidelberg, has identified novel cage molecules which, for the first time, satisfy these criteria. The bispidine derivative ligands provide rigid and large coordination cavities that perfectly match the slightly larger size of MnII, yielding eight-coordinate MnII complexes with record stabilities. In contrast, the smaller ZnII ion cannot accommodate all ligand donors, resulting in highly strained and less stable six-coordinate complexes.

Combined theoretical and experimental data demonstrate unprecedented selectivity, with up to 10 orders of magnitude higher stability for the manganese(II) than for the zinc(II) analogues, in sharp contrast to usual coordination chemistry rules.

 

https://onlinelibrary.wiley.com/doi/full/10.1002/anie.202115580

A new luminescence for the direct follow-up of drug delivery

Liposomes are nanocapsules widely used for the in vivo transport and delivery of therapeutic or diagnostic agents, or both. The therapeutic agent will only become active upon its release, thus sparing healthy tissues. The follow up of such release process is crucial to understand and control the drug action.

The previously reported approaches are based on optical imaging, using exclusively organic fluorophores or inorganic nanoparticles, co-encapsulated with the drug in the liposome.

The use of luminescent lanthanide complexes is an alternative which offers a number of advantages, including the capacity of those complexes to emit in the near infrared (NIR) region, enabling their detection in biological media.

The CBM research teams have developed an original nanocapsule : a liposome comprising an Ytterbium complex encapsulated with doxorubicin (anticancer agent). The NIR emission of the lanthanide complex is only observed when the drug is encapsulated. This NIR luminescence signal can therefore enable the direct follow-up, and in real time, of the integrity of the liposome, and can thus be used to detect the drug release.

An in vivo proof of concept was performed and the lanthanide luminescent signal could be detected in a mouse model of breast cancer.

Références de l'article :

Doxorubicin-sensitized Luminescence of NIR-emitting Ytterbium Liposomes: Towards Direct Monitoring of Drug Release,

Sara Lacerda, Anthony Delalande, Svetlana V. Eliseeva, Agnès Pallier, Célia S. Bonnet, Frédéric Szeremeta, Sandra Même, Chantal Pichon, Stéphane Petoud, Eva Toth

Angewandte Chemie Int. Ed. 13 août 2021  https://doi.org/10.1002/anie.202109408

 

See the news on CNRS Chemistry Institute site

 

 




New methodological breakthrough in total protein synthesis

Since the first synthesis of an amino acid dimer in the late 19th century, synthetic proteins represented a fascinating goal for generations of chemists. Invented in the 1960s, the solid phase peptide synthesis (SPPS) technology is since used routinely for peptides made up of a few dozen amino acids. The discovery of "chemical ligation" reactions in the 1990s paved the way to the synthesis of proteins of more than one hundred amino acids: extremely selective chemical reactions are used under aqueous conditions to couple peptide segments - themselves synthesized by SPPS -, devoted of side chain protective groups usually required in organic synthesis. Thanks to these methodological breackthroughs, the chemical approach to protein synthesis today advantageously complements biotechnological methods and enable the generation of native or modified proteins, tailor-made tools for deciphering biological processes at atomic resolution.

However, the synthesis of proteins of several hundred amino acids requires numerous successive chemical ligations, and therefore particularly delicate stages of purification of the reaction intermediates. One solution to overcome these steps is to assemble the proteins onto a solid support. Although very attractive, this approach has been limited to proofs of concept: one of the main reasons is the difficulty of grafting on a suitable solid support the first peptide segment through a linker that can be easily cleaved once the ligations carried out. Indeed, the conditions required for cleaving the linkers developed so far are incompatible with many proteins.

To overcome this problem, CBM scientists, in collaboration with colleagues from IC2MP in Poitiers, explored linkers programmed to be cleaved under very mild conditions by an enzymatic reaction. Remarkably, the size of the enzyme directly correlates with the rate at which the arm is cleaved, and therefore with the efficiency of the release of the synthesized protein. The method was applied to the synthesis of a peptide of 160 amino acids, which is to date the longest sequence ever synthesized by solid phase chemical ligations.

Reference of the article : S. A. Abboud, M. Amoura, J.-B. Madinier, B. Renoux, S. Papot, V. Piller, V. Aucagne. Enzyme-cleavable linkers for protein chemical synthesis through solid-phase ligations, Angew. Chem. Int. Ed., 2021, accepted article. https://doi.org/10.1002/anie.202103768

See the news on the website of the CNRS institute of chemistry