Multi-laboratory validation of SkinEthic HCE test method for testing serious eye damage/eye irritation using liquid chemicals.

A prospective multicentric study of the reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted to evaluate its usefulness to identify chemicals as either not classified for serious eye damage/eye irritation (No Cat.) or as classified (Cat. 1/Cat. 2) within UN GHS. The aim of this study was to demonstrate the transferability and reproducibility of the SkinEthic™ HCE EITL protocol for liquids and define its predictive capacity. Briefly, 60 chemicals were three times tested (double blinded) in 3 laboratories and 45 additional chemicals were tested three times in one laboratory. Good within laboratory reproducibility was achieved of at least 88.3% (53/60) and 92.4% (97/105) for the extended data set. Furthermore, the overall concordance between the laboratories was 93.3% (56/60). The accuracy of the SkinEthic™ HCE EITL for the extended dataset, based on bootstrap resampling, was 84.4% (95% CI: 81.9% to 87.6%) with a sensitivity of 99.0% (95% CI: 96.4% to 100%) and specificity of 68.5% (95% CI: 64.0% to 74.0%), thereby meeting all acceptance criteria for predictive capacity. This efficient transferable and reproducible assay is a promising tool to be integrated within a battery of assays to perform an eye irritation risk assessment.

In-source fragmentation of peptide aldehydes and acetals: influence of peptide length and charge state.

The use of in-source collision-induced dissociation (CID) was evaluated to generate structural information on peptide aldehydes, which represent an important class of compounds as inhibitors for serine and cysteine proteases and as key intermediates for protein engineering. By studying five peptide aldehydes of different lengths, and their peptide acetal counterparts, mass to charge (m/z) dependency of in-source fragmentation was established for peptides that differ only by their C-terminal functionalization. In-source fragmentation of peptide aldehydes and acetals leads to the same final ion, probably via a similar mechanism. Moreover, the gas-phase information obtained here reflects the equilibrium occurring in solution between the peptide aldehyde and its hydrated form, which was retained during the ionization process. The equilibrium constant was determined to be close to unity. Disturbance of this equilibrium should enable the stability of covalent hydration of a given series of aldehydes to be compared.

An innovative experimental setup for the measurement of sputtering yield induced by keV energy ions.

An innovative experimental equipment allowing to study the sputtering induced by ion beam irradiation is presented. The sputtered particles are collected on a catcher which is analyzed in situ by Auger electron spectroscopy without breaking the ultra high vacuum (less than 10(-9) mbar), avoiding thus any problem linked to possible contamination. This method allows to measure the angular distribution of sputtering yield. It is now possible to study the sputtering of many elements such as carbon based materials. Preliminary results are presented in the case of highly oriented pyrolytic graphite and tungsten irradiated by an Ar(+) beam at 2.8 keV and 7 keV, respectively.

Online in situ x-ray diffraction setup for structural modification studies during swift heavy ion irradiation.

The high energy density of electronic excitations due to the impact of swift heavy ions can induce structural modifications in materials. We present an x-ray diffractometer called ALIX ("Analyse en Ligne sur IRRSUD par diffraction de rayons X"), which has been set up at the low-energy beamline (IRRadiation SUD - IRRSUD) of the Grand Accélérateur National d'Ions Lourds facility, to allow the study of structural modification kinetics as a function of the ion fluence. The x-ray setup has been modified and optimized to enable irradiation by swift heavy ions simultaneously to x-ray pattern recording. We present the capability of ALIX to perform simultaneous irradiation-diffraction by using energy discrimination between x-rays from diffraction and from ion-target interaction. To illustrate its potential, results of sequential or simultaneous irradiation-diffraction are presented in this article to show radiation effects on the structural properties of ceramics. Phase transition kinetics have been studied during xenon ion irradiation of polycrystalline MgO and SrTiO(3). We have observed that MgO oxide is radiation-resistant to high electronic excitations, contrary to the high sensitivity of SrTiO(3), which exhibits transition from the crystalline to the amorphous state during irradiation. By interpreting the amorphization kinetics of SrTiO(3), defect overlapping models are discussed as well as latent track characteristics. Together with a transmission electron microscopy study, we conclude that a single impact model describes the phase transition mechanism.

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side-chain and backbone anchoring strategies, and compatible protection for N-terminal cysteine.

Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C-terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N(alpha)-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N(alpha)-t-butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side-chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N-terminal cysteine residue have been developed: (i) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-(N-methyl-N-phenylcarbamoyl)sulfenylcysteine [Fmoc-Cys(Snm)-OH], allowing creation of an otherwise fully protected resin-bound intermediate with N-terminal free Cys; (ii) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-triphenylmethylcysteine [Fmoc-Cys(Trt)-OH], generating a stable Fmoc-Cys(H)-peptide upon acidolytic cleavage; and (iii) incorporation of N(alpha)-t-butyloxycarbonyl-S-fluorenylmethylcysteine [Boc-Cys(Fm)-OH], generating a stable H-Cys(Fm)-peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C-termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on-resin, as well as in solution after cleavage/purification.

Comparative interaction of alpha-helical and beta-sheet amphiphilic isopeptides with phospholipid monolayers.

The two sequential amphiphilic peptide isomers, (Leu-Lys-Lys-Leu)4 and (Leu-Lys)8, were chosen as models for alpha-helical and beta-sheet peptides, respectively. In order to evaluate the contribution of the secondary structure of a peptide to its penetration into cellular membranes, interactions of these isopeptides with L-alpha-dimyristoyl phosphatidylcholine (DMPC) monolayers were studied. Both isopeptides penetrate into DMPC monolayers up to an exclusion pressure of approximately 27 mN/m, but a discontinuity is observed in the penetration profile of the alpha-helical (LKKL)4. The main parameters extracted from the compression isotherms of the mixed peptide/DMPC monolayers-namely, transition pressure, mean area, elasticity modulus, and energy of mixing-were analyzed. These analyses indicate that the alpha-helical isomer interacts strongly with DMPC by forming a 1:32 (LKKL)4-DMPC complex. When engaged in this complex, (LKKL)(4) behaves as an hydrophobic helix and has a tendency to become vertically oriented in the course of the compression process. The beta-sheet (LK)8 interacts more weakly with DMPC and no complex can be detected.

Band deconvolution analysis of the absorption and magnetic circular dichroism spectral data of a planar phthalocyanine dimer.

The electronic absorption and magnetic circular dichroism spectral data of a phthalocyanine dicopper complex that is deduced to be very planar and to share a common benzene ring have been studied by band deconvolution analysis. The results were compared with those of the molecular orbital (MO) calculations within the framework of the Pariser-Parr-Pople (PPP) approximation. The results of the band deconvolution analysis are in good agreement with those of the PPP calculations, allowing many bands to be reasonably assigned on the basis of the MO calculations. The validity of the PPP method for the MO calculation of large molecules is also emphasized.

Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometry.

Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (aminooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with liquid chromatography and tandem mass spectrometry has been used. In addition to the target tri-branched conjugate, two other conjugates were characterized allowing documentation of transoximation reactions in peptide chemistry. A fourth conjugate was identified as a side product appearing after the first ligation. Data obtained by low-energy collision-induced dissociation led to a rapid and reliable identification of impurities observed in the (aminooxy)acetyl peptide despite a previous high performance liquid chromatography (HPLC) purification. This highlights the great reactivity of the aminooxy group towards carbonyl-containing compounds.

Conformational study of sequential Lys and Leu based polymers and oligomers using vibrational and electronic CD spectra.

Vibrational CD (VCD) and electronic CD (ECD) spectra of some sequential Lys and Leu based oligo- and polypeptides were studied as a function of added salt and (for ECD) as a function of concentration in aqueous solution. For these samples, the VCD spectra can only be measured at relatively high concentrations under which the well-known salt-induced transition to a beta-sheet form can be observed for the KL based species, but only the end-state alpha-helical conformation is obvious for the LKKL based samples. ECD concentration dependence demonstrates that, at high concentration with no added or with added salt, LKKL based oligomers and polymers give alpha-helical spectra. These data provide evidence of aggregation induced secondary structure formation in an exceptionally simple peptide system. Similarly, the KL based oligomers and polymers give beta-sheet like spectra at high concentration or at high salt. These systems further provide model systems under "normal" aqueous conditions that yield VCD band shapes that correlate to the major secondary structural types of polypeptides. They are in substantial agreement with those spectra obtained on homopolypeptides and on proteins, confirming the relative independence of the VCD technique from side-chain and solvent effects.

Linear gramicidins at the air-water interface.

The behavior of four linear gramicidins, which differ by the nature of their 9, 11, 13, and 15 aromatic residues, together with a covalent "head to tail" retro GA-DAla-GA dimer, has been examined at the air-water interface. It is shown that all four "monomers" have almost the same molecular area, which is compatible with either a single-stranded or a double-stranded helical model, whereas it is suggested that retro GA-DAla-GA could adopt another conformation. The surface potential measurements agree with those of different groups of molecules characterized by their single-channel behaviors.

Structural properties of chimeric peptides containing a T-cell epitope linked to a fusion peptide and their importance for in vivo induction of cytotoxic T-cell responses.

We have previously shown that when administered to mice without adjuvant, a chimeric peptide consisting of the fusion peptide F from measles virus protein linked at the C-terminus of a cytotoxic T-cell epitope from the M2 protein of respiratory syncytial virus efficiently primes for an major histocompatibility complex (MHC) class-I restricted cytotoxic T lymphocyte (CTL) response. In this report, we demonstrated by microspectrofluorometry that the fusion-peptide moiety bound to the plasma membrane of living cells. When the fusion peptide was linked to the C-terminus of the CTL epitope, the chimeric peptide (M2-F) adopted a marked beta-sheet conformation. In contrast, when the fusion peptide was linked to the N-terminus of the T-cell epitope (F-M2), the chimeric peptide adopted an alpha-helical conformation in the presence of trifluoroethanol. The immunogenicity of the two chimeric peptides for class-I restricted CTL was also significantly different, the one adopting the alpha-helical conformation being more immunogenic. Probably due to its obvious conversion to an alpha-helical conformation, the F-M2 peptide could have a higher propensity to insert into membranes, as shown by microspectrofluorometry, with a resultant better immunogenicity than the M2-F peptide.

Tests for allergy to housedust.

In an attempt to find a satisfactory combination of method and antigen extract for tests of hypersensitivity to housedust, variations of skin test, RAST, histamine release and basophil degranulation tests have been used to test efficacy of five commonly used extracts of housedust or its components. The Lincoln Multitest for skin prick tests in its present form induced too much trauma for type-I hypersensitivity tests, though it has advantages for comparison of several extracts. Histamine release and two versions of basophil degranulation tests all gave good results but not consistently. RAST gave the best correlation between all extracts, but neither the in vivo nor in vitro tests gave perfect concordance with sensitivity in all patients and all extracts.

Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes. Evidence of affinity for negatively charged membranes.

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.