[Knee pain in a child : watch out for the hip !] / Gonalgie chez l'enfant : vérifier la hanche !

This is a case report of a 13-year old patient presenting with knee pain after a bike accident. Multiple investigations and medical examinations focused on the knee. They did not show any abnormality. About one year later, the patient comes back with increased hip pain, limited internal rotation and shortening of the left limb. Hip X-Ray exams reveal a bilateral slipped capital femoral epiphysis requiring already an osteotomy. Slipped capital femoral epiphysis is a disorder of the proximal femoral epiphysis. The femoral neck is displaced anteriorly and rotates externally while the epiphysis remains in the acetabulum. This pathology is more common in preadolescent boys and can be associated with hormonal disorders. Patients present with an externally rotated gait, a limited internal rotation, associated hip and knee pain. Diagnosis might be missed when patients present with knee pain and can lead to delayed treatment. When facing knee pain, it is essential to perform a clinical examination of the hip. Radiography is the first medical imaging. Surgical treatment by in situ fixation is recommended and prevents further slip with a high rate of success. When delayed, a much more invasive approach is required. A delay in diagnosis adversely affects long-term outcomes.

Il s'agit d'un patient de 13 ans présentant des gonalgies comme principale plainte. Vu l'apparition des douleurs suite à une chute, les investigations se sont focalisées sur le genou. De multiples examens complémentaires ont été réalisés sans mise en évidence de lésion. Après un an d'évolution, le patient se présente en consultation d'orthopédie en chaise roulante avec une impotence fonctionnelle majeure et des douleurs importantes au niveau des membres inférieurs. On note une boiterie, une inégalité des membres inférieurs et une raideur des hanches. Une radiographie du bassin démontre une épiphysiolyse fémorale supérieure bilatérale. Il s'agit d'un glissement de l'épiphyse par rapport au col fémoral, retrouvé plus fréquemment chez le garçon entre 10 et 15 ans. Le tableau clinique peut être caractérisé par des gonalgies, entrainant des retards diagnostiques. En cas de diagnostic précoce, un vissage in situ, peu invasif, empêche la progression de la déformation. Si le diagnostic est retardé, un traitement plus lourd et plus invasif comportant plus de risques et de moins bons résultats à long terme doit être pratiqué. En cas de mauvaise évolution, une arthroplastie précoce doit être réalisée. Une prise en charge rapide permet d'éviter cette escalade thérapeutique.

Epigallocatechin 3-Gallate as an Inhibitor of Tau Phosphorylation and Aggregation: A Molecular and Structural Insight.

Polyphenols such as Epigallocatechin-3 gallate (EGCG) are currently bearer of hope to prevent or at least to slow down the deleterious effect of Tauopathies such as Alzheimer disease. One of the main effects of these neurodegenerative pathologies is the hyperphosphorylation and consequent aggregation of the Tau protein that leads to the irremediable neuronal cells death. In the present paper, we show how EGCG can play a crucial role to prevent Tau aggregation: (i) in binding Tau in its phosphorylation region with an affinity of the same order of magnitude than kinases (0.5 mM), hindering their access to the protein and (ii) in modifying the 3D-structure of Tau whose preferential conformation changes in the presence of EGCG. For this purpose, two peptides were synthesized, one of 20 residues long issued from the first Proline-rich region of Tau (171Ile-190Lys), the second of 50 residues long (171Ile-220Thr) corresponding to more than 50% of the Tau Proline rich domaine. The total attribution of all the 1H, 13C and 15N resonances of the two peptides has been achieved thanks to a "divide and conquer" strategy leading to their 3D structure preference and their affinity towards EGCG.

Structure-analysis of the HIV-1 integrase Y143C/R raltegravir resistance mutation in association with the secondary mutation T97A.

The HIV-1 integrase (IN) mutations Y143C/R are known as raltegravir (RAL) primary resistance mutations. In a previous study (S. Reigadas et al., PLoS One 5:e10311, 2010), we investigated the genetic pathway and the dynamics of emergence of the Y143C/R mutations in three patients failing RAL-containing regimens. In these patients, the Y143C/R mutation was associated with the T97A mutation. The aim of the present biochemical and molecular studies in vitro was to evaluate whether the secondary mutation, T97A, associated with the Y143C/R mutation could increase the level of resistance to RAL and impact IN activities. Site-directed mutagenesis experiments were performed with expression vectors harboring the region of the pol gene coding for IN. With a 3'-end processing assay, the 50% inhibitory concentrations (IC(50)) were 1.2 µM, 1.2 µM, 2.4 µM (fold change [FC], 2), and 20 µM (FC, 16.7) for IN wild type (WT), the IN T97A mutation, the IN Y143C/T97A mutation, and the IN Y143R/T97A mutation, respectively. FCs of 18 and 100 were observed with the strand transfer assay for IN Y143C/T97A and Y143R/T97A mutations, with IC(50) of 0.625 µM and 2.5 µM, respectively. In the strand transfer assay, the IN Y143C or R mutation combined with the secondary mutation T97A severely impaired susceptibility to RAL compared to results with the IN Y143C or R mutation alone. Assays without RAL suggested that the T97A mutation could rescue the catalytic activity which was impaired by the presence of the Y143C/R mutation. The combination of the T97A mutation with the primary RAL resistance mutations Y143C/R strongly reduces the susceptibility to RAL and rescues the catalytic defect due to the Y143C/R mutation. This result indicates that the emergence of the Y143C/R/T97A double-mutation pattern in patients is a signature of a high resistance level.

Protein kinases, from B to C.

The PKB (protein kinase B) and PKC (protein kinase C) families display highly related catalytic domains that require a largely conserved series of phosphorylations for the expression of their optimum activities. However, in cells, the dynamics of these modifications are quite distinct. Based on experimental evidence, it is argued that the underlying mechanisms determining these divergent behaviours relate to the very different manner in which their variant regulatory domains interact with their respective catalytic domains. It is concluded that the distinct behaviours of PKB and PKC proteins are defined by the typical ground states of these proteins.

Evaluation of the ability of antioxidants to counteract lipid oxidation: existing methods, new trends and challenges.

Oxidative degradation of lipids, especially that induced by reactive oxygen species (ROS), leads to quality deterioration of foods and cosmetics and could have harmful effects on health. Currently, a very promising way to overcome this is to use vegetable antioxidants for nutritional, therapeutic or food quality preservation purposes. A major challenge is to develop tools to assess the antioxidant capacity and real efficacy of these molecules. Many rapid in vitro tests are now available, but they are often performed in dissimilar conditions and different properties are thus frequently measured. The so-called 'direct' methods, which use oxidizable substrates, seem to be the only ones capable of measuring real antioxidant power. Some oxidizable substrates correspond to molecules or natural extracts exhibiting biological activity, such as lipids, proteins or nucleic acids, while others are model substrates that are not encountered in biological systems or foods. Only lipid oxidation and direct methods using lipid-like substrates will be discussed in this review. The main mechanisms of autoxidation and antioxidation are recapitulated, then the four components of a standard test (oxidizable substrate, medium, oxidation conditions and antioxidant) applied to a single antioxidant or complex mixtures are dealt with successively. The study is focused particularly on model lipids, but also on dietary and biological lipids isolated from their natural environment, including lipoproteins and phospholipidic membranes. Then the advantages and drawbacks of existing methods and new approaches are compared according to the context. Finally, recent trends based on the chemometric strategy are introduced as a highly promising prospect for harmonizing in vitro methods.

Homology model of the rainbow trout estrogen receptor (rtERalpha) and docking of endocrine disrupting chemicals (EDCs).

A model for rainbow trout (Oncorhynchus mykiss) estrogen receptor (rtERa) was built by homology with the human estrogen receptor (hERa). A high level of sequence conservation between the two receptors was found with 64% and 80% of identity and similarity, respectively. Selected endocrine disrupting chemicals were docked into the ligand binding domain (LBD) of rtERa and the corresponding free binding energies Delta(DeltaG(bind)) values were calculated. A Quantitative Structure-Activity Relationship (QSAR) model between the relative binding affinity data and the Delta(DeltaG(bind)) values was derived in order to predict which further organic pollutants are likely to bind to rtERa.

Stabilization of phospholipid multilayers at the air-water interface by compression beyond the collapse: a BAM, PM-IRRAS, and molecular dynamics study.

Compression beyond the collapse of phospholipid monolayers on a modified Langmuir trough has revealed the formation of stable multilayers at the air-water interface. Those systems are relevant new models for studying the properties of biological membranes and for understanding the nature of interactions between membranes and peptides or proteins. The collapse of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-di[cis-9-octadecenoyl]-sn-glycero-3-[phospho-l-serine] (DOPS), 1,2-di[cis-9-octadecenoyl]-sn-glycero-3-phosphocholine (DOPC), and 1,2-di[cis-9-octadecenoyl]-sn-glycero-3-[phospho-1-rac-glycerol] (DOPG) monolayers has been investigated by isotherm measurements, Brewster angle microscopy (BAM), and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). In the cases of DMPC and DOPS, the collapse of the monolayers revealed the formation of bilayer and trilayer structures, respectively. The DMPC bilayer stability has been analyzed also by a molecular dynamics study. The collapse of the DOPC and DOPG systems shows a different behavior, and the Brewster angle microscopy reveals the formation of luminous bundles, which can be interpreted as diving multilayers in the subphase.

Molecular dynamics simulations of the insertion of two ideally amphipathic lytic peptides LK(15) and LK(9) in a 1,2-dimyristoylphosphatidylcholine monolayer.

We present here the results of 1-ns molecular dynamics (MD) simulations of two ideally amphipathic lytic peptides, namely LK(15) and LK(9), in a 1,2-dimyristoylphosphatidylcholine monolayer with two different cross-sectional areas per lipid of 80 A(2) (loose film) and 63 A(2) (tight standard film). These peptides are lytic, ideally amphipathic with a minimalist composition L(i)K(j) and the following sequences: H(2)N-KLLKLLLKLLLKLLK-CO-Ph for LK(15) and H(2)N-KLKLKLKLK-CO-Ph for LK(9). From experimental data, LK(15) exhibits an alpha-helical secondary structure, whereas LK(9) was found to form antiparallel beta-sheets at the interface of a DMPC monolayer. Whatever the specific lipid surface is, the two peptides exhibit very different behavior: the alpha-helix inserts deeply into the monolayer whereas the beta-sheeted peptide stays at the surface within the upper polar part of the monolayer. In all cases, a loose monolayer (80 A(2)) results in noticeable artifacts whereas a monolayer with standard specific surface leads to very reliable behavior well in accordance with experimental data. Despite their different insertion depth, the two peptides exhibit identical lytic efficiency. This is very likely a direct consequence of the very strong Van der Waals interactions between the fatty alkyl chains of the lipids and the highly lipophilic lower part of the peptide, resulting in an identical thinning of the two monolayers.

Modelling drugs and receptors using potentials: examples in the GPCRs' domain.

Shape complementarity, electrostatic and hydrophobic matching, were used to model drugs and receptors. From known experimental data on alpha1A/alpha2A-adrenergic ligands and alpha1A/alpha2A-adrenoceptors, a model for the ligand binding sites, based on the structure of bacteriorhodopsin as a template, was proposed and built. Agonists and antagonists have overlapping but different binding sites. Emphasis was given on the role of the disulphide bridge and on the role of the sodium site. The model was extended to other G-protein coupled receptors.

Structure, orientation and affinity for interfaces and lipids of ideally amphipathic lytic LiKj(i=2j) peptides.

The behavior of lytic ideally amphipathic peptides of generic composition LiKj(i=2j) and named LKn, n=i+j, is investigated in situ by the monolayer technique combined with the recently developed polarization modulation IR spectroscopy (PMIRRAS). A change in the secondary structure occurs versus peptide length. Peptides longer than 12 residues fold into alpha-helices at interfaces as expected from their design, while enough shorter peptides, from 9 down to 5 residues, form intermolecular antiparallel beta-sheets. Analysis of experimental and calculated PMIRRAS spectra in the amide I and II regions show that peptides are flat oriented at the interfaces. Structures and orientation are preserved whatever the nature of the interface, air/water or DMPC monolayer, and the lateral pressure. Peptide partition constants, KaffPi, are estimated from isobar surface increases of DMPC monolayers. They strongly increase when Pi decreases from 30 mN/m to 8 mN/m and they vary with peptide length with an optimum for 12 residues. This non-monotonous dependence fits with data obtained in bilayers and follows the hemolytic activity of the peptides. Lipid perturbations due to peptide insertion essentially detected on the PO4- and CO bands indicate disorder of the lipid head groups. Lysis induced on membranes by such peptides is proposed to first result from their flat asymmetric insertion.

Incidence of Molecular Structure in Oxidation of Grape Seed Procyanidins.

The kinetics of decomposition of the following flavan-3-ol derivatives isolated from grape seeds under oxidative conditions by airing and using metal ion catalysis (iron and copper) are determined: (+)-catechin and (-)-epicatechin; seven natural procyanidin dimers, B1 [(-)-epicatechin-(4-->8)-(+)-catechin], B2 [(-)-epicatechin-(4-->8)-(-)-epicatechin], B3 [(+)-catechin-(4-->8)-(+)-catechin], B4 [(+)-catechin-(4-->8)-(-)-epicatechin], B6 [(+)-catechin-(4-->6)-(+)-catechin], B7 [(-)-epicatechin-(4-->6)-(+)-catechin], and B8 [(-)-epicatechin-(4-->6)-(-)-epicatechin]; trimers, C1 [(-)-epicatechin-(4-->8)-(-)-epicatechin-(4-->8)-(-)-epicatechin], (+)-catechin-(4-->8)-(+)-catechin-(4-->8)-(-)-epicatechin, and (+)-catechin-(4-->8)-(-)-epicatechin-(4-->6)-(+)-catechin, monogallate esters of B2 and B4 and digallate of B2, which were isolated from grape seeds. Kinetic decomposition comparisons were monitored by HPLC. The following order was found for oxidative decomposition for procyanidin dimers: B3 approximately B4 > B7 approximately B6 > B1 approximately B2 > B8. In the conditions of this study, the gallate ester of (-)-epicatechin is more unstable than (-)-epicatechin; inversely, kinetic decompositions of dimeric procyanidins B2 and B4 are much more important than those of their gallate esters. Molecular mechanics (MM2) and (1)H NMR studies of dimeric 3-O-gallate structures show a pi-pi stacking arrangement between the aromatic gallate and catechol rings, absent in analogous dimeric procyanidins, which reduces the total surface accessible to oxidizing agents.

A pharmacophore for high affinity PAF antagonists. II. Hydrophobicity study using the molecular lipophilicity potential.

Platelet-activating factor (PAF) is a powerful phospholipid-derived autacoid involved in many physiopathological mechanisms. Many PAF antagonists have been synthesized and evaluated as therapeutic candidates. In a previous report, we have described an electronic pharmacophore of PAF antagonists using the molecular electrostatic potential. In the present study, a molecular lipophilicity potential is used to compare the hydrophobic properties of 49 "heterocyclic sp2 nitrogen' highly potent PAF antagonists, belonging to six structurally different series (nine hetrazepines, five pyrrolo[1,2-c]thiazoles, 14 carboxamides, nine dihydropyridines, nine pyridinyl-thiazolidines and three imidazo[4,5-c]pyridines). Their common features consist of three hydrophilic (HYD2, HY14(3)B and HYD3) and two lipophilic zones (LIP3 and LIP4), defining the lipophilic pharmacophore of the antagonists. This pharmacophore is also characterized by several zone-to-zone distances: HYD3-HYD2 = 1.3 +/- 1.0 A, HY3B-HYD2 = 7.8 +/- 1.1, HYD3-HY3B = 5.1 +/- 1.1 A, LIP4-LIP3 = 5.4 +/- 1.1 A, LIP3-HYD2 = 11.3 +/- 1.6 A, LIP3-HY3B = 5.9 +/- 1.0 A, LIP3-HYD3 = 4.3 +/- 0.9 A, LIP4-HYD2 = 14.7 +/- 1.6 A, LIP4-HY3B = 8.1 +/- 1.2 A and LIP4-HYD3 = 3.9 +/- 1.1 A. These results represent a new step in the determination of a global pharmacophore for PAF antagonists.

A pharmacophore for high affinity PAF antagonists. I. Electronic model using molecular electrostatic potential.

PAF is a powerful phospholipid-derived autacoid involved in many physio-pathological mechanisms. Many PAF antagonists have been synthesized and assayed for therapeutic purposes. In this study, molecular electrostatic potential is used to compare the electronic properties of 48 'heterocyclic sp2 nitrogen' highly potent PAF antagonists, belonging to six series (nine hetrazepines, five pyrrolo[1,2-c]thiazoles, 14 carboxamides, nine dihydropyridines, nine pyridinylthiazolidines and two imidazo[4,5-c]pyridines). Their common features consist of three main electronegative zones (A, B1 and B2) describing the electronic pharmacophore of these ligands. The high affinity of these PAF antagonists seems to be related to this electronegative system A-B(x), which is characterized by three distances A-B1 (9.3 +/- 1.0 A), A-B2 (13.4 +/- 0.7 A) and B1-B2 (4.9 +/- 0.9 A). Moreover, B1 and B2 may surround a common anchorage point in the binding site of the receptor.

Molecular modeling of 5-HT3 receptor antagonists: geometrical, electronic and lipophilic features of the pharmacophore and 3D-QSAR study.

If the geometrical pharmacophore of 5-HT3 receptor antagonists has been proposed by different authors, the electronic and lipophilic features of the ligands had to be precised. A 3-D QSAR study has enlightened the importance of three parameters derived from molecular electrostatic and molecular lipophilicity potentials. A multiple linear regression equation has been established. Its predictive character (non specific binding of 3[H]-ICS 205-930) has been tested with success for three different new ligands.

2-amino-2-oxazolines, VII: Influence of structural parameters on the antidepressant activity of 5-(1-aryl-4-piperazino)methyl-2-amino-2-oxazolines.

A series of 5-(1-aryl-4-piperazino)methyl-2-amino-2-oxazolines has been prepared and screened for antidepressant activity. Their lipophilic behaviour has been discussed in relation to the nature and the position of substituents on the aromatic ring. The influence of steric effects on the pharmacological activity has been investigated using experimental methods (X-ray diffraction, NMR) and theoretical calculations (semi-empirical quantum mechanics). The ortho-substitution on the phenyl ring or the C-alpha substitution on the piperazine ring by a methyl group results in the same effects i.e. an increase of the angle between the two rings up to 64 degrees (X-ray and calculation) and a loss of the antidepressant activity. Using NMR, only the influence of the ortho-substitution has been observed.

Erythrocytes as carriers for L-asparaginase. Methodological and mouse in-vivo studies.

L-Asparaginase has been encapsulated in Swiss mouse or human erythrocytes by hypotonic haemolysis followed by isotonic resealing and reannealing. The details of incorporation and properties of carrier erythrocytes are presented. When L-asparaginase loaded into 51Cr-labelled erythrocytes, was infused intravenously, the same half-life was found for asparaginase and 51Cr. In addition, L-asparaginase loaded into erythrocytes was much more effective in eliminating plasma asparagine compared with the same dose of free L-asparaginase injected in solution, during a sustained period (14 days).

Several aspects of red blood cell engineering: potential therapeutic applications.

Erythrocytes can be used to entrap drugs, enzymes or other molecules with active properties, with various encapsulation procedures. The method of internalization we are using includes an hypotonic dialysis step. Carrier erythrocytes survival depends on the dialysis process and the carried molecule. Research has led us to perform preclinical trials on animals for several drugs and enzyme therapies and for the improvement of oxyphoric capacity of erythrocytes. There exist many potential clinical applications for each kind of internalized molecules.

In vivo accelerated acetaldehyde metabolism using acetaldehyde dehydrogenase-loaded erythrocytes.

Human erythrocytes were loaded with homogeneous acetaldehyde dehydrogenase (AcDH) purified from Alcaligenes Eutrophus (an enzyme species with an apparent Km for acetaldehyde similar to the mitochondrial enzyme), using an encapsulation procedure based on hypotonic haemolysis, isotonic resealing and reannealing. The AcDH-overloaded erythrocytes contained 1.55 +/- 0.25 I.U. of AcDH activity per ml of packed erythrocytes, a value 12-15 times higher than that of corresponding unloaded or native red cells. The AcDH-loaded erythrocytes were found to metabolize 4 +/- 0.8 mumol of acetaldehyde/hr/ml of red blood cells, whereas the glycolytic activity was almost unmodified. Estimates of intracellular adenine nucleotides showed 50% ATP decay in the AcDH-loaded cells when incubated in the presence of acetaldehyde concentrations higher than 50 microM, whereas the [NAD+]/[NADH] ratio was strongly decreased but to the same extent as in control cells, suggesting that this was due to the acetaldehyde itself and not to the presence of encapsulated AcDH. Similar results were obtained using mouse erythrocytes. AcDH-overloaded mouse red blood cells from donor animals were also injected intraperitoneally into compatible recipients (Balb/C) and 80 to 85% of these were found to enter into circulation within 24 hr and to circulate with a half-life of 6-7.3 days (normal half-life 11 days). Following an acute dose of ethanol (2g/kg intraperitoneally), blood levels of acetaldehyde were significantly lower in mice receiving the AcDH-loaded erythrocytes than in controls. Blood levels of ethanol were also lower in the treated mice compared to controls. These results show that AcDH-overloaded erythrocytes can perform in vitro and in vivo as bioreactors improving alcohol and acetaldehyde metabolism, and suggest that administration of these cells to alcoholic patients could be of value in restoring to normal, or improving, alcohol and acetaldehyde metabolism.

Improved stability of 2,3-bisphosphoglycerate during storage of hexokinase-overloaded erythrocytes.

Human red blood cells were overloaded with homogeneous human hexokinase using a procedure of encapsulation based on hypotonic hemolysis and isotonic resealing and reannealing to achieve a final activity that was 15 times higher than that in control cells. Storage for 5 weeks at 4 degrees C of hexokinase-overloaded erythrocytes shows that these cells undergo small K+ leakage and mean cell volume increase compared with control cells. Furthermore, after these 5 weeks of storage the 2,3-bisphosphoglycerate content was normal while the ATP concentration was slightly reduced. These results and other properties suggest that encapsulation of key glycolytic enzymes in erythrocytes can provide a new way to maintain in vitro functionally active red blood cells for at least 5 weeks.