Development of a [68Ga]-ghrelin analogue for PET imaging of the ghrelin receptor (GHS-R1a).

The ghrelin receptor is a member of the growth hormone secretagogue receptor (GHS-R) family and is present at low concentrations in tissues such as the brain, kidney, cardiovascular system, and prostate. The ghrelin receptor plays an important role in cellular proliferation, apoptosis, invasion, and migration associated with the progression of many cancers, including prostate, breast, ovarian, testicular, and intestinal carcinomas. Ghrelin, the endogenous ligand, is a 28 amino acid peptide (IC50 = 3.1 nM) known to have poor in vivo stability. Herein, we report the synthesis and evaluation of [Dpr3(octanoyl),Lys19(Ga-DOTA)]ghrelin(1-19). This new ghrelin analogue has a binding affinity (IC50 = 5.9 nM) comparable to that of natural ghrelin. Preliminary in vivo evaluation shows higher uptake of [Dpr3(octanoyl),Lys19(68Ga-DOTA)]ghrelin(1-19) in HT1080/GHSR-1a xenografts than the non-transfected HT1080 xenografts in NOD-SCID mice, although considerable uptake is observed in the kidneys. This is the first example of ghrelin receptor PET imaging in a xenograft model using a peptide derived directly from the endogenous ligand and serves as motivation for developing more effective ghrelin-based radiopeptides.

Optimization of the method of the content-containing interaction evaluation for cosmetic products by gas chromatography-mass spectrometry.

In July 2013, the European Regulation (EC) No 1223/2009 came into effect in order to secure cosmetic products. The content-containing interaction between the packaging and the product must be considered for the safety assessment. Indeed, some compounds are able to migrate from the packaging to the product and may be harmful to the consumer health. This is why a first test was established by EXPERTOX laboratory in 2012 to deal with this new regulation. A new analytical method was developed and validated for the quantification of 23 substances able to migrate from the packaging to the product. It was applied on a plastic packaging with the five simulants of migration. To evaluate the content-containing interaction, a gas chromatography-mass spectrometry method was developed and validated. Liquid-liquid extraction was used to extract contaminants (thirteen phthalates and ten substances of very high concern) from migration simulants. Calibration curves showed good linearity regression from 2 to 50 µg mL-1 for nineteen molecules and from 5 to 45 µg mL-1 for the others. The limits of quantification were respectively 2 and 5 µg mL-1 . The accuracy, precision, repeatability of the analytical method and extraction yields were acceptable. No molecule was found in simulants of migration, so the potential contaminants present in the packaging did not migrate. A gas chromatography-mass spectrometry method and liquid-liquid extraction were validated for 23 molecules and can be used for the evaluation of the content-containing interaction of cosmetic products. Both quantification and extraction procedures are more robust and faster than previous method.

Molecular imaging probes derived from natural peptides.

Covering: up to the end of 2015.Peptides are naturally occurring compounds that play an important role in all living systems and are responsible for a range of essential functions. Peptide receptors have been implicated in disease states such as oncology, metabolic disorders and cardiovascular disease. Therefore, natural peptides have been exploited as diagnostic and therapeutic agents due to the unique target specificity for their endogenous receptors. This review discusses a variety of natural peptides highlighting their discovery, endogenous receptors, as well as their derivatization to create molecular imaging agents, with an emphasis on the design of radiolabelled peptides. This review also highlights methods for discovering new and novel peptides when knowledge of specific targets and endogenous ligands are not available.

[Staphylococcal toxic shock syndrome after breast surgery]. / Syndrome du choc toxique staphylococcique après chirurgie du sein.

The surgical site infection occurs within 30 days after surgery. It is the most common complication of surgery, with a rate of 1 to 5% without antibiotic prophylaxis and less than 1% with antibiotic prophylaxis. The toxic shock syndrome (TSS) is a dramatic complication. We report the case 39-year-old woman who presented a life-threatening TSS acquired after breast surgery. We describe the signs and symptoms of this condition as well as treatment principles.

Evolution of plant eukaryotic initiation factor 4E (eIF4E) and potyvirus genome-linked protein (VPg): a game of mirrors impacting resistance spectrum and durability.

Polymorphism in the plant eukaryotic translation initiation factor 4E (eIF4E) and potyvirus genome-linked protein (VPg) determine, in many cases, the outcome of the confrontation between these two organisms: compatibility (i.e. infection of the plant by the virus) or incompatibility (i.e. resistance of the plant to the virus). The two interacting proteins eIF4E and VPg show strikingly similar evolution patterns. Most codon positions in their coding sequences are highly constrained for nonsynonymous substitutions but a small number shows evidence for positive selection. Several of these latter positions were shown to be functionally important, conferring resistance to the host or pathogenicity to the virus. Determining the mutational pathways involved in pepper eIF4E diversification revealed a link between an increase of the pepper resistance spectrum towards a panel of potyvirus species and an increase of durability of the resistance towards Potato virus Y. This relationship questions the interest of using more generally the spectrum of action of a plant resistance gene as a predictor of its durability potential.

A novel macrolide/fluoroketolide, solithromycin (CEM-101), reverses corticosteroid insensitivity via phosphoinositide 3-kinase pathway inhibition.

BACKGROUND AND PURPOSE: Corticosteroid insensitivity is a major therapeutic problem for some inflammatory diseases including chronic obstructive pulmonary disease (COPD), and it is known to be induced by reduced histone deacetylase (HDAC)-2 activities via activation of the phosphoinositide 3-kinase (PI3K) pathway. The aim of this study is to evaluate effects of a novel macrolide/fluoroketolide, solithromycin (SOL, CEM-101), on corticosteroid sensitivity induced by oxidative stress. EXPERIMENTAL APPROACH: Corticosteroid sensitivity was determined by IC50/EC50 of dexamethasone (Dex) on TNF-α-induced CXCL8 production in U937 monocytic cell line and peripheral blood mononuclear cells (PBMC) from COPD patients. Activities of HDAC and protein phosphatase 2A (PP2A) were measured by fluorescence-based assay in cells exposed to hydrogen peroxide (H2O2). We also investigated steroid insensitive airway neutrophilia in cigarette smoke exposed mice in vivo. KEY RESULTS: SOL (10 µM) restored Dex sensitivity in PBMC from COPD patients, H2O2-treated U937 cells and phorbol 12-myristate 13-acetate-differentiated U937 cells. In addition, SOL restored HDAC activity with concomitant inhibition of Akt phosphorylation as surrogate marker of PI3K activation. The inhibition of Akt phosphorylation by SOL was due to increased PP2A phosphatase activity, which was reduced in COPD and oxidative stress model. Other known macrolides, such as eryhthromycin, clarithromycin and azithromycin, were significantly less effective in these responses. In cigarette smoke-exposed mice, SOL (100 mg kg(-1), po) showed significant but weak inhibition of neutrophilia, whereas Dex (10 mg kg(-1), p.o.) showed no such effect. However, a combination of SOL and Dex inhibited neutrophilia by over 50%. CONCLUSIONS AND IMPLICATIONS: SOL has potential as novel therapy for corticosteroid-insensitive diseases such as COPD.

Right ventricular failure in acute lung injury and acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a clinical entity involving not only alveolar lesions but also capillary lesions, both of which have deleterious effects on the pulmonary circulation, leading to constant pulmonary hypertension and to acute cor pulmonale (ACP) in 20-25% of patients ventilated with a limited plateau pressure (Pplat). Considering the poor prognosis of patients suffering from such acute right ventricular (RV) dysfunction, RV protection by appropriate ventilatory settings has become a crucial issue in ARDS management. The goal of this review is to emphasize the importance of analyzing RV function in ARDS, using echocardiography, in order to limit RV afterload. Any observed acute RV dysfunction should lead physicians to consider a strategy for RV protection, including strict limitation of Pplat, diminution of positive end-expiratory pressure (PEEP) and control of hypercapnia, all goals achieved by prone positioning.

Effective mechanical properties of diaphyseal cortical bone in the canine femur.

The effective elastic modulus, yield strength, yield strain, ultimate strength, ultimate strain, strain energy density at yield and strain energy density at ultimate failure of femoral diaphyseal cortical bone were investigated on canine femurs. Four femurs representative of the canine population were selected from four statistically-determined clusters based on increasing size and weight comprising the Toy poodle (5 kg), Poodle (12 kg), German shorthaired pointer (25 kg) and Doberman (50 kg). The zones of interest were the lateral, medial, cranial, and caudal quadrants of the mid-diaphysis. Effective mechanical properties were measured using quasi-static three-point bending tests on strips. The averages ± SD were 15.6 ± 2.6 GPa for effective elastic modulus, 174.3 ± 32.1 MPa for yield strength, 0.012 ± 0.003 for yield strain, 251.0 ± 49.1 MPa for ultimate strength, 0.021 ± 0.005 for ultimate strain, 10.7 ± 4.0J m(-3) × 10(5) for strain energy density at Yield and 33.0 ± 14.1 Jm(-3)× 10(5) for strain energy density at ultimate failure. Significant differences were found between dogs and the effective elastic modulus increased with breed weight and size (13.9 GPa for the Toy poodle to 17.2 GPa for the Doberman). The ultimate strength σ(u) and strain energy density at ultimate failure U(u) were significantly lower in the Toy poodle than in the Poodle and German shorthaired pointer indicating that the cortical bone material in the Toy poodle differed from that of the other dogs. Examination of the zones of interest revealed that the cranial quadrant showed the greatest stiffness, whereas strength was highest at the medial site. The caudal cortex was less stiff and strong than the cranial cortex.

Reversal of refractory septic shock with drotrecogin alpha (activated).

OBJECTIVE: We previously reported that early continuous veno-venous hemodiafiltration (CVVHDF) enables rapid identification of a subgroup of patients with "refractory" septic shock and a 100% risk of death. The objective of this study was to investigate whether early administration of drotrecogin alpha (activated) (DrotAA) to this selected subgroup of septic patients at extremely high risk of death would significantly improve prognosis. METHOD: Prospective observational study in a medical intensive-care unit of a University Hospital. Twenty-three patients with refractory septic shock were included. "Refractory" shock was defined as persistent circulatory failure despite adequate circulatory support, associated with persisting lactic acidosis despite early CVVHDF. Response to CVVDHF was assessed after 6 h of this continuous procedure. Patients selected by this strategy received DrotAA infusion for four days. RESULTS: The 28-day mortality rate of the 23 patients was 39%. No difference was observed at inclusion between survivors and nonsurvivors. In patients who finally survived, 12 h of DrotAA infusion was associated with a significant decrease in lactic acidosis and in norepinephrine dose. CONCLUSION: DrotAA therapy was associated with unexpectedly high 28-day survival in patients with "refractory" septic shock.

Crystal quality and differential crystal-growth behaviour of three proteins crystallized in gel at high hydrostatic pressure.

Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75-100 MPa (lysozymes) and 250 MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with approximately 20% less ordered water molecules in crystals grown at 150 MPa when compared with those grown at atmospheric pressure (0.1 MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered.

The archaeal sRNA binding protein L7Ae has a 3D structure very similar to that of its eukaryal counterpart while having a broader RNA-binding specificity.

The ribosomal L7Ae protein of archaea has the peculiarity to be a component of the C/D and H/ACA snRNPs, that guide rRNA post-transcriptional modifications. Its yeast (Snu13p) and human (15.5kDa protein) homologs are only found in C/D snoRNPs and the (U4/U6, U5) spliceosomal tri-snRNP. By using a large variety of RNAs, we compared the RNA-binding specificities of the recombinant Pyrococcus abyssi L7Ae and Saccharomyces cerevisiae Snu13 proteins. Unlike Snu13p, protein L7Ae binds terminal loops closed by two A:G and G:A pairs and canonical K-turn structures with similar efficiencies, provided that the terminal loop contains at least 5nt. In contrast to Snu13p, binding of protein L7Ae to canonical K-turn structures is not dependent on the identity of the residue at position 2 in the bulge. The peculiar KT-15 motif of P. abyssi 23S rRNA, that is recognized by L7Ae, does not associate with Snu13p. To get more information on the P. abyssi L7Ae protein, we solved its X-ray structure at 1.9A resolution. In spite of their sequence divergence, the free P. abyssi and bound H. marismortui proteins were found to have highly similar structures. Only a limited number of side-chain conformational changes occur at the protein-RNA interface upon RNA binding. In particular, one ion pair that is formed by residues Glu43 and Lys46 in the free protein is disrupted in the ribosomal 50S subunit, so that, residue Glu43 can interact with the RNA residue G264. The Glu43-Lys46 ion pair of protein L7Ae belongs to a complex network of ion pairs that may participate to protein thermostability.

From conventional crystallization to better crystals from space: a review on pilot crystallogenesis studies with aspartyl-tRNA synthetases.

Aspartyl-tRNA synthetases were the model proteins in pilot crystallogenesis experiments. They are homodimeric enzymes of Mr approximately 125 kDa that possess as substrates a transfer RNA, ATP and aspartate. They have been isolated from different sources and were crystallized either as free proteins or in association with their ligands. This review discusses their crystallisability with emphasis to crystal quality and structure determination. Crystallization in low diffusivity gelled media or in microgravity environments is highlighted. It has contributed to prepare high-resolution diffracting crystals with better internal order as reflected by their mosaicity. With AspRS from Thermus thermophilus, the better crystalline quality of the space-grown crystals within APCF is correlated with higher quality of the derived electron density maps. Usefulness for structural biology of targeted methods aimed to improve the intrinsic physical quality of protein crystals is highlighted.

Crystallization and preliminary X-ray diffraction data of the second and archaebacterial-type aspartyl-tRNA synthetase from Thermus thermophilus.

The archaebacterial-type aspartyl-tRNA synthetase (AspRS2) from the thermophilic eubacterium Thermus thermophilus was crystallized using the hanging-drop vapour-diffusion method. Crystals grew at pH 9.5 in the presence of PEG 8000 and NaCl. A native diffraction data set has been collected at 2.5 A resolution using synchrotron radiation and cryocooling. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 57.3, b = 121.9, c = 166.9 A and V(M) = 3.03 A(3) Da(-1). There is one dimer of M(r) 96 000 per asymmetric unit. A molecular-replacement analysis gave solutions for the rotation and translation functions.

1,6-anhydro-2, 3-di-O-benzyl-5C-[(R)-ethoxycarbonyl(hydroxy)methyl]-beta-L-altrofur ano se.

The crystal structure of the title compound, C(24)H(28)O(8), has been determined. The conformation of the furanose ring can be described as 58% ideal envelope (O)E conformer and 42% ideal twisted (O)T(1) conformer. The 1,3-dioxane ring adopts a chair conformation with the anhydro-O atom pointing upwards. Both phenyl rings are quasi-perpendicular to the mean plane of the furanose ring. The hydrogen bonding is intermolecular and consists of infinite chains parallel to the a axis.

The crystal structure of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus in the presence of NADP(+) at 2.1 A resolution.

The crystal structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaeon Methanothermus fervidus has been solved in the holo form at 2.1 A resolution by molecular replacement. Unlike bacterial and eukaryotic homologous enzymes which are strictly NAD(+)-dependent, GAPDH from this organism exhibits a dual-cofactor specificity, with a marked preference for NADP(+) over NAD(+). The present structure is the first archaeal GAPDH crystallized with NADP(+). GAPDH from M. fervidus adopts a homotetrameric quaternary structure which is topologically similar to that observed for its bacterial and eukaryotic counterparts. Within the cofactor-binding site, the positively charged side-chain of Lys33 decisively contributes to NADP(+) recognition through a tight electrostatic interaction with the adenosine 2'-phosphate group. Like other GAPDHs, GAPDH from archaeal sources binds the nicotinamide moiety of NADP(+) in a syn conformation with respect to the adjacent ribose and so belongs to the B-stereospecific class of oxidoreductases. Stabilization of the syn conformation is principally achieved through hydrogen bonding of the carboxamide group with the side-chain of Asp171, a structural feature clearly different from what is observed in all presently known GAPDHs from bacteria and eukaryotes. Within the catalytic site, the reported crystal structure definitively confirms the essential role previously assigned to Cys140 by site-directed mutagenesis studies. In conjunction with new mutation results reported in this paper, inspection of the crystal structure gives reliable evidence for the direct implication of the side-chain of His219 in the catalytic mechanism. M. fervidus grows optimally at 84 degrees C with a maximal growth temperature of 97 degrees C. The paper includes a detailed comparison of the present structure with four other homologous enzymes extracted from mesophilic as well as thermophilic organisms. Among the various phenomena related to protein thermostabilization, reinforcement of electrostatic and hydrophobic interactions as well as a more efficient molecular packing appear to be essentially promoted by the occurrence of two additional alpha-helices in the archaeal GAPDHs. The first one, named alpha4, is located in the catalytic domain and participates in the enzyme architecture at the quaternary structural level. The second one, named alphaJ, occurs at the C terminus and contributes to the molecular packing within each monomer by filling a peripherical pocket in the tetrameric assembly.

Crystallization and preliminary X-ray diffraction studies of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus.

The homotetrameric holo-D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus has been crystallized in the presence of NADP+ using the hanging-drop vapour-diffusion method. Crystals grew from a solution containing 2-methyl-2,4-pentanediol and magnesium acetate. A native data set has been collected to 2.1 A using synchrotron radiation and cryocooling. Diffraction data have been processed in the orthorhombic system (space group P21212) with unit-cell dimensions a = 136.7, b = 153.3, c = 74.9 A and one tetramer per asymmetric unit.

A system and methodology for measuring volatile organic compounds produced by hydroponic lettuce in a controlled environment.

A system and methodology were developed for the nondestructive qualitative and quantitative analysis of volatile emissions from hydroponically grown 'Waldmann's Green' leaf lettuce (Lactuca sativa L.). Photosynthetic photon flux (PPF), photoperiod, and temperature were automatically controlled and monitored in a growth chamber modified for the collection of plant volatiles. The lipoxygenase pathway products (Z)-3-hexenal, (Z)-3-hexenol, and (Z)-3-hexenyl acetate were emitted by lettuce plants after the transition from the light period to the dark period. The volatile collection system developed in this study enabled measurements of volatiles emitted by intact plants, from planting to harvest, under controlled environmental conditions.

Photosynthetic photon flux, photoperiod, and temperature effects on emissions of (Z)-3-hexenal, (Z)-3-hexenol, and (Z)-3-hexenyl acetate from lettuce.

To investigate the effects of environment on plant volatile emissions, 'Waldmann's Green' leaf lettuce was cultivated under different levels of photosynthetic photon flux (PPF), photoperiod, and temperature. A modified growth chamber was used to sample plant volatile emissions nondestructively, over time, and under controlled conditions. Total volatile emission rates were significantly higher from lettuce cultivated under PPF of 360 or 200 micromoles m-2 s-1 compared to 105 micromoles m-2 s-1, and significantly higher under a 16-h photoperiod than an 8-h photoperiod. No differences were detected among emission rates from different temperature treatments. In controlled environments, emissions could be regulated by adjusting environmental conditions accordingly.

Quantitation of immunoglobulin classes and subclasses of autoantibodies bound to red cells in patients with and without hemolysis.

The concentration of IgG, IgA, and IgM, as well as IgG subclasses, was measured by an enzyme-linked immunosorbent assay in autoantibodies eluted from red cells (RBCs); the number of molecules of each isotype per RBC was calculated. Three groups were analyzed: Group 1 included 23 patients with autoimmune hemolytic anemia (AIHA) associated with warm autoantibodies of IgG class; Group 2 included 11 patients without anemia but with a positive direct antiglobulin test (DAT); Group 3 included 10 healthy DAT-negative subjects. The mean number of IgG molecules per RBC in Group 1 (920) was about three times that in Group 2 (306) and about 17 times that in Group 3 (54). The range of RBC-bound IgG showed an overlap between the two groups of patients. The mean number of IgM and IgA molecules per RBC was low in the three groups. IgG1 predominated in all groups except in two patients with AIHA, in whom IgG3 made up at least 50 percent of total IgG. The mean number of IgG1, IgG2, and IgG4 molecules per RBC in Group 1 was about three times that in Group 2, whereas the mean number of IgG3 molecules per RBC was 10 times as high (p < 0.001). It follows that IgG3 was more common in patients of Group 1, but it was also detected in patients of Group 2.

Treatment of myelodysplastic syndromes with daily oral idarubicin. A phase I-II study.

BACKGROUND: Idarubicin, a new anthracycline analogue, is available in an oral preparation, and responses have been observed using relatively aggressive therapy in patients with myelodysplastic syndromes (MDS). The authors studied whether a chronic low-dose schedule would be effective but less myelotoxic. METHODS: Forty-two patients with MDS received daily low-dose oral idarubicin in 5-week courses that included 3 weeks of treatment, followed by a 2-week rest period. Doses were escalated when possible after the second course, and each patient was to receive six courses. RESULTS: Only one partial response was observed. Although no patient had fatal bone marrow toxicity, only eight patients received the full six courses, primarily because of myelosuppression. CONCLUSIONS: This schedule of oral idarubicin is relatively safe but produces fewer responses than are reported with the high-dose pulse regimens.