Fabrice Fleury

Quantum dot surface chemistry and functionalization for cell targeting and imaging.

Quantum dots (QDs) are highly fluorescent nanoscale crystals with size-dependent emission spectra. Due to their excellent photophysical properties, QDs are a promising alternative to organic fluorescent dyes and fluorescent proteins for cell targeting, imaging, and drug delivery. For biomedical applications, QDs should be chemically modified to be stable in aqueous solutions and tagged with the recognition molecules or drugs. Here, we review surface modification approaches to, and strategies for, conjugation of bioactive molecules with QDs. There are a variety of methods of QD surface modification and QD incorporation into larger delivery systems that yield fluorescent nanocarriers from 10 nm to several micrometers. Conjugates of QDs with peptides, proteins, antibodies, oligonucleotides, and small molecules have been used for fluorescent targeting, tracking, and imaging both in vitro and in vivo. Due to an extremely high stability to photobleaching, QDs were used for long-term visualization. QD applications pave the way for new generations of ultrasensitive detection, diagnostic systems, as well as drug delivery approaches, combining accurate targeting, delivery, and imaging in a single assay.

Interactions of lactone, carboxylate and self-aggregated forms of camptothecin with human and bovine serum albumins

Pronounced differences in the interactions of monomeric (lactone and carboxylate) and the J‐type self‐aggregated form of camptothecin (CPT), an inhibitor of DNA topoisomerase (topo) I, with human (HSA) and bovine (BSA) serum albumins were observed by using circular dichroism (CD) spectroscopy. HSA binding changes the geometry of the covalent structure of CPT due to hydrophobic contacts of the chromophore within the protein interior. The carbonyl group of the ring D of CPT ( Fig. 1 A) interacts with the positively charged amino acid residues of HSA. Interaction with HSA induces disaggregation of the J‐type self‐aggregates of CPT. On the other hand, neither heat‐denatured HSA nor native BSA participated in binding of the lactone or carboxylate or self‐aggregate forms of CPT. Analysis of HSA and BSA homology within the IIA and IIIA principle ligand‐binding structural domains suggests that the binding site for the CPT chromophore is located in subdomain IIA. Hydrophobic contacts with Leu‐203, Phe‐211, and Ala‐215 and electrostatic interactions with Lys‐199 and/or Arg‐222 of HSA may play a key role in formation of the drug‐HSA complex.

Inhibition of filament formation of human Rad51 protein by a small peptide derived from the BRC-motif of the BRCA2 protein

Human Rad51 is a key element of recombinational DNA repair and is related to the resistance of cancer cells to chemo‐ and radiotherapies. The protein is thus a potential target of anti‐cancer treatment. The crystallographic analysis shows that the BRC‐motif of the BRCA2 tumor suppressor is in contact with the subunit–subunit interface of Rad51 and could thus prevent filament formation of Rad51. However, biochemical analysis indicates that a BRC‐motif peptide of 69 amino acids preferentially binds to the N‐terminal part of Rad51. We show experimentally that a short peptide of 28 amino acids derived from the BRC4 motif binds to the subunit–subunit interface and dissociates its filament, both in the presence and absence of DNA, certainly by binding to dissociated monomers. The inhibition is efficient and specific for Rad51: the peptide does not even interact with Rad51 homologs or prevent their interaction with DNA. Neither the N‐terminal nor the C‐terminal half of the peptide interacts with human Rad51, indicating that both parts are involved in the interaction, as expected from the crystal structure. These results suggest the possibility of developing inhibitors of human Rad51 based on this peptide.

Targeting RAD51 phosphotyrosine-315 to prevent unfaithful recombination repair in BCR-ABL1 leukemia

Chronic myeloid leukemia chronic phase (CML-CP) CD34(+) cells contain numerous DNA double-strand breaks whose unfaithful repair may contribute to chromosomal instability and disease progression to blast phase (CML-BP). These phenomena are often associated with the appearance of imatinib-resistant BCR-ABL1 kinase mutants (eg, T315I) and overexpression of BCR-ABL1. Here we show that BCR-ABL1 (nonmutated and T315I mutant) promoted RAD51 recombinase-mediated unfaithful homeologous recombination repair (HomeoRR) in a dosage-dependent manner. BCR-ABL1 SH3 domain interacts with RAD51 proline-rich regions, resulting in direct phosphorylation of RAD51 on Y315 (pY315). RAD51(pY315) facilitates dissociation from the complex with BCR-ABL1 kinase, migrates to the nucleus, and enhances formation of the nuclear foci indicative of recombination sites. HomeoRR and RAD51 nuclear foci were strongly reduced by RAD51(Y315F) phosphorylation-less mutant. In addition, peptide aptamer mimicking RAD51(pY315) fragment, but not that with Y315F phosphorylation-less substitution, diminished RAD51 foci formation and inhibited HomeoRR in leukemia cells. In conclusion, we postulate that BCR-ABL1 kinase-mediated RAD51(pY315) promotes unfaithful HomeoRR in leukemia cells, which may contribute to accumulation of secondary chromosomal aberrations responsible for CML relapse and progression.

Preliminary characterisation of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

Haslea ostrearia is a common marine tychopelagic diatom which has the particularity of synthesizing a blue-green hydrosoluble pigment called “marennine”. This pigment, when released into the external medium, is known to be responsible for the colour of oyster gills. Here we present results for main biophysical and biochemical characteristics of pure intra- and extracellular marennine. Tests for chemical determination show that the nature of the two forms of marennine cannot be distinguished and could be related to a polyphenolic compound. Nevertheless, based on spectral properties and the molecular weight, which is about 10751 ± 1 and 9893 ± 1 Da, for the intracellular and extracellular forms respectively, we assess that the pigment accumulated in the apex of the cell and the one released in the external medium have probably distinct molecular structures.

c-ABL tyrosine kinase stabilizes RAD51 chromatin association.

The assembly of RAD51 recombinase on DNA substrates at sites of breakage is essential for their repair by homologous recombination repair (HRR). The signaling pathway that triggers RAD51 assembly at damage sites to form subnuclear foci is unclear. Here, we provide evidence that c-ABL, a tyrosine kinase activated by DNA damage which phosphorylates RAD51 on Tyr-315, works at a previously unrecognized, proximal step to initiate RAD51 assembly. We first show that c-ABL associates with chromatin after DNA damage in a manner dependent on its kinase activity. Using RAD51 mutants that are unable to oligomerize to form a nucleoprotein filament, we separate RAD51 assembly on DNA to form foci into two steps: stable chromatin association followed by oligomerization. We show that phosphorylation on Tyr-315 by c-ABL is required for chromatin association of oligomerization-defective RAD51 mutants, but is insufficient to restore oligomerization. Our findings suggest a new model for the regulation of early steps of HRR.

Detection of c-Abl kinase-promoted phosphorylation of Rad51 by specific antibodies reveals that Y54 phosphorylation is dependent on that of Y315

MINT‐7034009: cABL (uniprotkb:P00519) physically interacts (MI:0218) with RAD51 (uniprotkb:Q06609) by pull down (MI:0096)

Protein Encapsulation into PLGA Nanoparticles by a Novel Phase Separation Method Using Non-Toxic Solvents

Nanoparticles of biocompatible and biodegradable polymers such as poly-lactic-co-glycolic acid (PLGA) are widely used as drug delivery systems for the administration of biomolecules like proteins. The purpose of this work is to validate a novel formulation method by a phase separation phenomenon using the non-toxic solvent glycofurol (GF) in order to encapsulate proteins into PLGA nanoparticles. Nanoprecipitates of a model protein (lysozyme) and a therapeutic protein (TGF-β1) were formed to ensure their stability upon subsequent encapsulation in PLGA nanoparticles. Good encapsulation efficiency was obtained with preservation of the structure integrity and protein bioactivity after encapsulation. PLGA nanoparticles were then characterized in terms of size, zeta potential and morphology. Moreover, residual solvent was quantified and in vitro release study of the encapsulated proteins was performed to demonstrate the efficacy of our encapsulation method in drug sustained release. Finally, cytocompatibility study of nanoparticles was performed. Thus, we developed an effective method based on the preliminary step of protein precipitation for the formulation of PLGA nanoparticles as protein carriers for biomedical applications.

Approach for the Preparation of Various Classes of Peroxides Based on the Reaction of Triketones with H2O2: First Examples of Ozonide Rearrangements

The reaction of β,δ-triketones with an ethereal solution of H2O2 catalyzed by heteropoly acids in the presence of a polar aprotic co-solvent proceeds via three pathways to form three classes of peroxides: tricyclic monoperoxides, bridged tetraoxanes, and a pair of stereoisomeric ozonides. The reaction is unusual in that produces bridged tetraoxanes and ozonides with one of the three carbonyl groups remaining intact. In the synthesis of bridged tetraoxanes, the peroxide ring is formed by the reaction of hydrogen peroxide with two carbonyl groups at the β positions. The synthesis of ozonides from ketones and hydrogen peroxide is a unique process in which the ozonide ring is formed with the participation of two carbonyl groups at the δ positions. Rearrangements of ozonides were found for the first time after more than one century of their active investigation. Ozonides are interconverted with each other and rearranged into tricyclic monoperoxides, whereas ozonides and tricyclic monoperoxides are transformed into bridged tetraoxanes. The individual reaction products were isolated by column chromatography and characterized by NMR spectroscopy, mass spectrometry, and elemental analysis. One representative of each class of peroxides was characterized by X-ray diffraction.

Assessment of new cationic porphyrin binding to plasma proteins by planar microarray and spectroscopic methods.

Porphyrins have a unique aromatic structure determining particular photochemical properties that make them promising photosensitizers for anticancer therapy. Previously, we synthesized a set of artificial porphyrins by modifying side-chain functional groups and introducing different metals into the core structure. Here, we have performed a comparative study of the binding properties of 29 cationic porphyrins with plasma proteins by using microarray and spectroscopic approaches. The porphyrins were noncovalently immobilized onto hydrogel-covered glass slides and probed to bio-conjugated human and bovine serum albumins, as well as to human hemoglobin. The signal detection was carried out at the near-infrared fluorescence wavelength (800 nm) that enabled the effect of intrinsic visible wavelength fluorescence emitted by the porphyrins tested to be discarded. Competition assays on porphyrin microarrays indicated that long-chain fatty acids (FAs) (palmitic and stearic acids) decrease porphyrin binding to both serum albumin and hemoglobin. The binding affinity of different types of cationic porphyrins for plasma proteins was quantitatively assessed in the absence and presence of FAs by fluorescent and absorption spectroscopy. Molecular docking analysis confirmed results that new porphyrins and long-chain FAs compete for the common binding site FA1 in human serum albumin and meso-substituted functional groups in porphyrins play major role in the modulation of conformational rearrangements of the protein.