1D NMR WaterLOGSY as an efficient method for fragment-based lead discovery

WaterLOGSY is a sensitive ligand-observed NMR experiment for detection of interaction between a ligand and a protein and is now well-established as a screening technique for fragment-based lead discovery. Here we develop and assess a protocol to derive ligand epitope mapping from WaterLOGSY data and demonstrate its general applicability in studies of fragment-sized ligands binding to six different proteins (glycogen phosphorylase, protein peroxiredoxin 5, Bcl-xL, Mcl-1, HSP90, and human serum albumin). We compare the WaterLOGSY results to those obtained from the more widely used saturation transfer difference experiments and to the 3D structures of the complexes when available. In addition, we evaluate the impact of ligand labile protons on the WaterLOGSY data. Our results demonstrate that the WaterLOGSY experiment can be used as an additional confirmation of the binding mode of a ligand to a protein

Characterization of Macrophages and Osteoclasts in the Osteosarcoma Tumor Microenvironment at Diagnosis: New Perspective for Osteosarcoma Treatment?

Biological and histopathological techniques identified osteoclasts and macrophages as targets of zoledronic acid (ZA), a therapeutic agent that was detrimental for patients in the French OS2006 trial. Conventional and multiplex immunohistochemistry of microenvironmental and OS cells were performed on biopsies of 124 OS2006 patients and 17 surgical (“OSNew”) biopsies respectively. CSF-1R (common osteoclast/macrophage progenitor) and TRAP (osteoclast activity) levels in serum of 108 patients were correlated to response to chemotherapy and to prognosis. TRAP levels at surgery and at the end of the protocol were significantly lower in ZA+ than ZA− patients (padj = 0.0011; 0.0132). For ZA+-patients, an increase in the CSF-1R level between diagnosis and surgery and a high TRAP level in the serum at biopsy were associated with a better response to chemotherapy (p = 0.0091; p = 0.0251). At diagnosis, high CD163+ was associated with good prognosis, while low TRAP activity was associated with better overall survival in ZA− patients only. Multiplex immunohistochemistry demonstrated remarkable bipotent CD68+/CD163+ macrophages, homogeneously distributed throughout OS regions, aside osteoclasts (CD68+/CD163−) mostly residing in osteolytic territories and osteoid-matrix-associated CD68−/CD163+ macrophages. We demonstrate that ZA not only acts on harmful osteoclasts but also on protective macrophages, and hypothesize that the bipotent CD68+/CD163+ macrophages might present novel therapeutic targets

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PARIS-BIUP (751062107) / SudocSudocFranceF

The Dynamics of Copper Uptake in Human Cu,Zn-Superoxide Dismutase: a Solid-State NMR Study

http://thcgerm.free.fr/spip.php?article123National audienceLes expériences de saturation transfer difference (STD) et de Water–ligand observed via gradient spectroscopy (WaterLOGSY) sont des techniques simples et rapides de criblage, largement utilisées en RMN (interactions protéine-ligand, criblage de fragments). Adaptées à la détection de ligands faible affnité (Kd ~ mM-uM), ces méthodes présentent l’avantage d’être exclusivement centrées sur l’analyse des signaux 1D 1H des ligands et ne nécessitent pas de posséder des informations sur la cible protéique elle-même. Nous montrons ici l’intérêt particulier de ces deux expériences pour étudier l’orientation du ligand, renseignement crucial pour la validation et la sélection de ligands de protéine, notamment dans le cadre du criblage de fragments

Influence du nourrissement exogène sur la composition analytique de la gelée royale.

communication par afficheNational audienc

Physicochemical characterisation of French royal jelly: Comparison with commercial royal jellies and royal jellies produced through artificial bee-feeding.

International audienceA reliable database is established from the analysis of 500 French royal jellies (RJ). Diversity of geographical origins, harvesting seasons, and forage sources were taken into account. Authenticity intervals for numerous physicochemical parameters have been established from French RJs, obtained without bee-feeding with artificial sugars or proteins. RJs from Italy and non-European countries, derived from feeding experiments, were analysed and compared with the database. Differences in 10-HDA, d13C and sugars (sucrose, erlose, maltose, maltotriose) contents were observed. Sucrose and erlose contents in French RJs were less than 1.8 and 0.4%, respectively, whereas they attained 3.9 and 2.0% in some commercial samples and 7.7 and 1.7% in RJs produced by sugarcane feeding. Maltose and maltotriose contents were less than 1.0 and 0.2% in French RJs, and they can attain levels of 2.6 and 0.4% in commercial samples; they can be as high as 5.5 and 1.7% in RJs obtained by bee-feeding with starch hydrolysate syrups. RJs obtained with and without feeding exhibit d13C values ranging from 26.45 to 23.73%, and from 26.05 to 14.65%, respectively. This study could be used to discriminate different production methods and provide a useful tool for identifying unknown commercial RJs. Highlights A total of 750 royal jellies (RJs) from different origins were studied. Origins included domestic, imported, obtained by bee-feeding. French RJ samples were chemically characterised. Authenticity intervals were calculated for authentic RJs. Sucrose, erlose, maltose, maltotriose and δ13C contents are bee-feeding markers

Triplet‐Triplet Annihilation Upconversion‐Based Photolysis: Applications in Photopharmacology

International audienceThe emerging field of photopharmacology is a promising chemobiological methodology for optical control of drug activities that could ultimately solve the off-target toxicity outside the disease location of many drugs for the treatment of a given pathology. The use of photolytic reactions looks very attractive for a light-activated drug release but requires to develop photolytic reactions sensitive to red or near-infrared light excitation for better tissue penetration. This review will present the concepts of triplet-triplet annihilation upconversion-based photolysis and their recent in vivo applications for light-induced drug delivery using photoactivatable nanoparticles

Optimization of peptide-based cancer vaccine compositions, by sequential screening, using versatile liposomal platform

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Bone Like Arterial Calcification in Femoral Atherosclerotic Lesions: Prevalence and Role of Osteoprotegerin and Pericytes

International audienceOBJECTIVE/BACKGROUND:Arterial calcification, a process that mimics bone formation, is an independent risk factor of cardiovascular morbidity and mortality, and has a significant impact on surgical and endovascular procedures and outcomes. Research efforts have focused mainly on the coronary arteries, while data regarding the femoral territory remain scarce.METHODS:Femoral endarterectomy specimens, clinical data, and plasma from a cohort of patients were collected prospectively. Histological analysis was performed to characterize the cellular populations present in the atherosclerotic lesions, and that were potentially involved in the formation of bone like arterial calcification known as osteoid metaplasia (OM). Enzyme linked immunosorbent assays and cell culture assays were conducted in order to understand the cellular and molecular mechanisms underlying the formation of OM in the lesions.RESULTS:Twenty-eight of the 43 femoral plaques (65%) displayed OM. OM included osteoblast and osteoclast like cells, but very few of the latter exhibited the functional ability to resorb mineral tissue. As in bone, osteoprotegerin (OPG) was significantly associated with the presence of OM (p = .04). Likewise, a high plasma OPG/receptor activator for the nuclear factor kappa B ligand (RANKL) ratio was significantly associated with the presence of OM (p = .03). At the cellular level, there was a greater presence of pericytes in OM+ compared with OM- lesions (5.59 ± 1.09 vs. 2.42 ± 0.58, percentage of area staining [region of interest]; p = .04); in vitro, pericytes were able to inhibit the osteoblastic differentiation of human mesenchymal stem cells, suggesting that they are involved in regulating arterial calcification.CONCLUSION:These results suggest that bone like arterial calcification (OM) is highly prevalent at femoral level. Pericyte cells and the OPG/RANK/RANKL triad seem to be critical to the formation of this ectopic osteoid tissue and represent interesting potential therapeutic targets to reduce the clinical impact of arterial calcification

Photoactivatable Liposomes for Blue to Deep Red Light-Activated Surface Drug Release: Application to Controlled Delivery of the Antitumoral Drug Melphalan

International audienceLiposome-based nanoparticles able to release, via a photolytic reaction, a payload anchored at the surface of the phospholipid bilayer were prepared. The liposome formulation strategy uses an original drug-conjugated blue light-sensitive photoactivatable coumarinyl linker. This is based on an efficient blue light-sensitive photolabile protecting group modified by a lipid anchor, which enables its incorporation into liposomes, leading to blue to green light-sensitive nanoparticles. In addition, the formulated liposomes were doped with triplet–triplet annihilation upconverting organic chromophores (red to blue light) in order to prepare red light sensitive liposomes able to release a payload, by upconversion-assisted photolysis. Those light-activatable liposomes were used to demonstrate that direct blue or green light photolysis or red light TTA-UC-assisted drug photolysis can effectively photorelease a drug payload (Melphalan) and kill tumor cells in vitro after photoactivation