Plasma desorption mass spectrometry, an analytical tool in protein engineering: characterization of modified insulins

Californium-252 plasma desorption mass spectrometry (PDMS) hasbeen employed for the characterization of a series of humaninsulin derivatives in order to evaluate the performance ofthis technique as an analytical tool in protein engineering.Several of the characterized modifications result in a 1 a.m.u.mass change. The precision in mass determination obtainableby PDMS analysis is not sufficient for unambiguous verificationof such modifications based on the molecular weight alone. Itis, however, possible to carry out in situ enzymatic digestionof the sample. Subsequent PDMS analysis will in most cases revealif the modification has been introduced as intended.     

Quasi-spherical focal spot in two-photon scanning microscopy by three-ring apodization

We present a beam-shaping technique for two-photon excitation (TPE) fluorescence microscopy. We show that by inserting a properly designed three-ring pupil filter in the illumination beam of the microscope, the effective optical sectioning capacity of such a system improves so that the point spread function gets a quasi-spherical shape. Such an improvement, which allows the acquisition of 3D images with isotropic quality, is obtained at the expense of only a small increase of the overall energy in the axial sidelobes. The performance of this technique is illustrated with a scanning TPE microscopy experiment in which the image of small beads is obtained. We demonstrate an effective narrowing of 12.5% in the axial extent of the point spread function, while keeping the 82% of the spot-fluorescence efficiency.     

Spatio-temporal modeling of perimetric test data

This work describes the application of a spatio-temporal modeling to the study of glaucoma, a very serious ocular illness. The aim of this modeling is to solve various significant medical problems, namely the forecasting of future observations, the classification of observations as normal or defective, and the simulation of new longitudinal data sets. In order to ascertain whether a patient suffers from glaucoma, a perimetry is performed. The output of a perimetry is called a visual field and consists of a map with 52 numerical values plotted on a regular grid. In this work, a data set of healthy patients' visual fields is used. The work begins with an exploratory spatial data analysis. A semi-parametric approach is used to model the mean, and the variogram is fitted using a Matérn function. Once the spatial structure has been analysed, the spatial mean is subtracted from all the observations in the data set and the spatio-temporal correlation of the residuals is explored. All this information is used to build a space-time model, the parameters of which are estimated by maximum likelihood. Different methods are used to check the goodness of fit.     

Eicosapentaenoic acid (20∶5n-3) from the marine microalgaPhaeodactylum tricornutum

Eicosapentaenoic acid (EPA, 20∶5n-3) was obtained from the marine microalgaePhaeodactylum tricornutum by a three-step process: fatty acid extraction by direct saponification of biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds, and EPA isolation by semipreparative high-performance liquid chromatography (HPLC). Alternatively, EPA was obtained by a similar two-step process without the PUFA concentration step by the urea method. Direct saponification of biomass was carried out with two solvents that contained KOH for lipid saponification. An increase in yield was obtained because the problems associated with emulsion formation were avoided by separating the biomass from the soap solution before adding hexane for extraction of insaponifiables. The most efficient solvent, ethanol (96%) at 60°C for 1 h, extracted 98.3% of EPA. PUFA were concentrated by the urea method with a urea/fatty acid ratio of 4∶1 at a crystallization temperature of 28°C and by using methanol and ethanol as urea solvents. An EPA concentration ratio of 1.73 (55.2∶31.9) and a recover yield of 78.6% were obtained with methanol as the urea solvent. This PUFA concentrate was used to obtain 93.4% pure EPA by semipreparative HPLC with a reverse-phase, C₁₈, 10 mm i.d.×25-cm column and methanol/water (1% acetic acid), 80∶20 w/w, as the mobile phase. Eighty-five percent of EPA loaded was recovered, and 65.7% of EPA present inP. tricornutum biomass was recovered in highly pure form by this three-step downstream process. Alternatively, 93.6% pure EPA was isolated from the fatty acid extract (without the PUFA concentration step) with 100% EPA recovery yield. This two-step process increases the overall EPA yield to 98.3%, but it is only possible to obtain 20% as much EPA as that obtained by three-step downstream processing.     

Quantitative HIV-1 RNA as a marker of clinical stability and survival in a cohort of 302 patients with a mean CD4 cell count of 300 x 10(6)/l

OBJECTIVE: To analyse plasma HIV-1 RNA levels as a marker of clinical stability and survival in a cohort of HIV-infected patients whose time of seroconversion is unknown. DESIGN: Retrospective cohort study. SETTING: Retrovirology laboratory and AIDS Unit in a teaching hospital. PATIENTS: A total of 916 samples from 302 patients, most on antiretroviral therapy, were analysed. Mean initial CD4 cell counts and HIV-1 RNA were 299 x 10(6)/l (range: 0-1600) and 134,261 copies/ml (range: < 200-4,300,000), respectively. Sixty-six cases had been diagnosed previously with AIDS. METHODS: Analysis of progression to AIDS and survival, according to initial and longitudinal viral load (VL) and CD4 cell count measurements was performed by Kaplan-Meier test. Relative risks were calculated by Cox's proportional hazards model. RESULTS: During a mean follow-up of 444 +/- 309 days, 29 patients developed AIDS and 21 died. Relative risk (RR) of progression related to the group with VL < 35,000 was: 10.4 when CD4 > or = 250 x 10(6)/l and VL > or = 35,000 (P = 0.001); and 45.3 when CD4 < 250 x 10(6)/l and VL > or = 35,000 (P < 0.0001). Cumulative probability of progression was: 0%, 0% and 12.3%, at the first, second and third year respectively, for patients with all their sequential VL determinations < 60,000; and 13.3%, 34.7% and 79.3% for patients who did not maintain VL values always < 60,000 (RR = 23; P < 0.0001). The minimum value of VL that reached statistical significance for the survival analysis was 100,000 copies/ml (P < 0.0001). CONCLUSIONS: VL > or = or < 35,000 is a better discriminant for progression than a CD4 cell count > or = or < 250 x 10(6)/l. Sequential VL determinations < 60,000 are associated with a better prognosis.     

Friction stir welding of 25%SiC/2124Al composite with optimal mechanical properties and minimal tool wear

In this research, 8-mm-thick plates of 25%SiC/2124Al composite have been joined successfully by using an un-threaded WC–Co tool which showed minimal tool wear. Decreasing the rotational and advancing speeds is a key factor to obtain successful joints. Defect-free welds, and a joint efficiency (100UTS_joint/UTS_BaseMaterial) of up to 97%, can be achieved if a high forge force, F_z, (above some 15?kN) is reached. Proportionality between the torque, M, and F_z is observed. High F_z values, and corresponding M, lead to high values of the total energy per unit length of weld, H_tot, supplied by the tool to the workpiece. It is proposed that most of this energy is spent in plastic deformation of the high strength composite.     

Dehydrogenation of Light Alkanes Over Rhenium Catalysts on Conventional and Mesoporous MFI Supports

Recently, Re/HZSM-5 (Si/Al = 15) was shown to be an efficient catalyst for ethane dehydrogenation and aromatization at 823 K and atmospheric pressure. In this reaction, the major initial products were benzene, toluene and xylene (BTX), but increasing amounts of ethene were produced with time on stream due to deactivation of the catalyst. We show that by use of rhenium impregnated MFI supports with very few or no acidic sites (Si/Al > 500), highly selective ethane dehydrogenation catalysts are obtained with ethene selectivities of 98%. By use of mesoporous MFI supports (Si/Al >500) the lifetime of the catalyst appears to be slightly improved compared to conventional MFI crystals. The beneficial effect of a mesoporous MFI support is convincingly demonstrated in propane dehydrogenation, where both conversion and selectivities on the mesoporous MFI (Si/Al > 500) impregnated with Re are significantly higher than on Re supported on a comparable conventional MFI support.     

Metabolomics of Genetically Modified Crops

Metabolomic-based approaches are increasingly applied to analyse genetically modified organisms (GMOs) making it possible to obtain broader and deeper information on the composition of GMOs compared to that obtained from traditional analytical approaches. The combination in metabolomics of advanced analytical methods and bioinformatics tools provides wide chemical compositional data that contributes to corroborate (or not) the substantial equivalence and occurrence of unintended changes resulting from genetic transformation. This review provides insight into recent progress in metabolomics studies on transgenic crops focusing mainly in papers published in the last decade.