Analysis of protein carbonylation - pitfalls and promise in commonly used methods

Abstract Oxidation of proteins has received a lot of attention in the last decades due to the fact that they have been shown to accumulate and to be implicated in the progression and the patho-physiology of several diseases such as Alzheimer, coronary heart diseases, etc. This has also resulted in the fact that research scientist became more eager to be able to measure accurately the level of oxidized protein in biological materials, and to determine the precise site of the oxidative attack on the protein, in order to get insights into the molecular mechanisms involved in the progression of diseases. Several methods for measuring protein carbonylation have been implemented in different laboratories around the world. However, to date no methods prevail as the most accurate, reliable and robust. The present paper aims at giving an overview of the common methods used to determine protein carbonylation in biological material as well as to highlight the limitations and the potential. The ultimate goal is to give quick tips for a rapid decision making when a method has to be selected and taking into consideration the advantage and drawback of the methods.

ANALYSIS OF HYPORHEIC FLOW INDUCED BY A BAR IN A GRAVEL STREAM: AN EXPERIMENTAL STUDY

Pipelines are one of the safest means to transport crude oil, but are not spill-free. This is of concern in North America, due to the large volumes of crude oil shipped by Canadian producers and the lengthy network of pipelines. Each pipeline crosses many rivers, supporting a wide variety of human activities, and rich aquatic life. However, there is a knowledge gap on the risks of contamination of river beds due to oil spills. This thesis addresses this knowledge gap by focussing on mechanisms that transport water (and contaminants) from the free surface flow to the bed sediments, and vice-versa. The work focuses on gravel rivers, in which bed sediments are sufficiently permeable that pressure gradients caused by the interactions of flow with topographic elements (gravel bars), or changes in direction induce exchanges of water between the free surface flow and the bed, known as hyporheic flows. The objectives of the thesis are: to present a new method to visualize and quantify hyporheic flows in laboratory experiments; to conduct a novel series of experiments on hyporheic flow induced by a gravel bar under different free surface flows. The new method to quantify hyporheic flows rests on injections of a solution of dye and water. The method yielded accurate flow lines, and reasonable estimates of the hyporheic flow velocities. The present series of experiments was carried out in a 11 m long, 0.39 m wide, and 0.41 m deep tilting flume. The gravel had a mean particle size of 7.7 mm. Different free surface flows were imposed by changing the flume slope and flow depth. Measured hyporheic flows were turbulent. Smaller free surface flow depths resulted in stronger hyporheic flows (higher velocities, and deeper dye penetration into the sediment). A significant finding is that different free surface flows (different velocities, Reynolds number, etc.) produce similar hyporheic flows as long as the downstream hydraulic gradients are similar. This suggests, that for a specified bar geometry, the characteristics of the hyporheic flows depend on the downstream hydraulic gradients, and not or only minimally on the internal dynamics of the free surface flow.

Measuring the Diversity and the Integration of Film Production in Mercosur: A Social Network Analysis

Based on an original and comprehensive database of all feature fiction films produced in Mercosur between 2004 and 2012, the paper analyses whether the Mercosur film industry has evolved towards an integrated and culturally more diverse market. It provides a summary of policy opportunities in terms of integration and diversity, emphasizing the limiter role played by regional policies. It then shows that although the Mercosur film industry remains rather disintegrated, it tends to become more integrated and culturally more diverse. From a methodological point of view, the combination of Social Network Analysis and the Stirling Model opens up interesting research tracks to analyse creative industries in terms of their market integration and their cultural diversity.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Statistical Analysis of the heavy neutral Atoms measured by IBEX

We investigate the directional distribution of heavy neutral atoms in the heliosphere by using heavy neutral maps generated with the IBEX-Lo instrument over three years from 2009 to 2011. The interstellar neutral (ISN) O&Ne gas flow was found in the first-year heavy neutral map at 601 keV and its flow direction and temperature were studied. However, due to the low counting statistics, researchers have not treated the full sky maps in detail. The main goal of this study is to evaluate the statistical significance of each pixel in the heavy neutral maps to get a better understanding of the directional distribution of heavy neutral atoms in the heliosphere. Here, we examine three statistical analysis methods: the signal-to-noise filter, the confidence limit method, and the cluster analysis method. These methods allow us to exclude background from areas where the heavy neutral signal is statistically significant. These methods also allow the consistent detection of heavy neutral atom structures. The main emission feature expands toward lower longitude and higher latitude from the observational peak of the ISN O&Ne gas flow. We call this emission the extended tail. It may be an imprint of the secondary oxygen atoms generated by charge exchange between ISN hydrogen atoms and oxygen ions in the outer heliosheath.

Methods and tools for analysis and management of risks and regulatory compliance in the healthcare sector: the hospital at home – HaH

Changing or creating an organisation means creating a new process. Each process involves many risks that need to be identified and managed. The main risks considered here are procedural and legal risks. The former are related to the risks of errors that may occur during processes, while the latter are related to the compliance of processes with regulations. Managing the risks implies proposing changes to the processes that allow the desired result: an optimised process. In order to manage a company and optimise it in the best possible way, not only should the organisational aspect, risk management and legal compliance be taken into account, but it is important that they are all analysed simultaneously with the aim of finding the right balance that satisfies them all. This is the aim of this thesis, to provide methods and tools to balance these three characteristics, and to enable this type of optimisation, ICT support is used. This work isn’t a thesis in computer science or law, but rather an interdisciplinary thesis. Most of the work done so far is vertical and in a specific domain. The particularity and aim of this thesis is not to carry out an in-depth analysis of a particular aspect, but rather to combine several important aspects, normally analysed separately, which however have an impact and influence each other. In order to carry out this kind of interdisciplinary analysis, the knowledge base of both areas was involved and the combination and collaboration of different experts in the various fields was necessary. Although the methodology described is generic and can be applied to all sectors, the case study considered is a new type of healthcare service that allows patients in acute disease to be hospitalised to their home. This provide the possibility to perform experiments using real hospital database.

Critical applications of SW 846 US EPA methods to evaluation of marine samples quality

Technical evaluation of analytical data is of extreme relevance considering it can be used for comparisons with environmental quality standards and decision-making as related to the management of disposal of dredged sediments and the evaluation of salt and brackish water quality in accordance with CONAMA 357/05 Resolution. It is, therefore, essential that the project manager discusses the environmental agency's technical requirements with the laboratory contracted for the follow-up of the analysis underway and even with a view to possible re-analysis when anomalous data are identified. The main technical requirements are: (1) method quantitation limits (QLs) should fall below environmental standards; (2) analyses should be carried out in laboratories whose analytical scope is accredited by the National Institute of Metrology (INMETRO) or qualified or accepted by a licensing agency; (3) chain of custody should be provided in order to ensure sample traceability; (4) control charts should be provided to prove method performance; (5) certified reference material analysis or, if that is not available, matrix spike analysis, should be undertaken and (6) chromatograms should be included in the analytical report. Within this context and with a view to helping environmental managers in analytical report evaluation, this work has as objectives the discussion of the limitations of the application of SW 846 US EPA methods to marine samples, the consequences of having data based on method detection limits (MDL) and not sample quantitation limits (SQL), and present possible modifications of the principal method applied by laboratories in order to comply with environmental quality standards.

Evaluation of a simple hyphenated system for flow injection solid-phase pre-concentration and capillary electrophoresis

In this work, the development and evaluation of a hyphenated flow injection-capillary electrophoresis system with on-line pre-concentration is described. Preliminary tests were performed to investigate the influence of flow rates over the analytical signals. Results revealed losses in terms of sensitivity of the FIA-CE system when compared to the conventional CE system. To overcome signal decrease and to make the system more efficient, a lower flow rate was set and an anionic resin column was added to the flow manifold in order to pre-concentrate the analyte. The pre-concentration FIA-CE system presented a sensitivity improvement of about 660% and there was only a small increase of 8% in total peak dispersion. These results have confirmed the great potential of the proposed system for many analytical tasks especially for low concentration samples.

Determination of paracetamol at a graphite-polyurethane composite electrode as an amperometric flow detector

A bare graphite-polyurethane composite was evaluated as an amperometric flow injection detector in the determination of paracetamol (APAP) in pharmaceutical formulations. A linear analytical curve was observed in the 5.00 x 10-5 to 5.00 x 10-3 mol L-1 range with a minimum detectable net concentration of 18.9 µmol L-1 and 180 determinations h-1, after optimization of parameters such as the detection potential, sample loop volume, and carrier solution flow rate. Interference of ascorbic acid was observed, however, it was possible overcome the interference, reaching results that agreed with HPLC within 95% confidence level. These results showed that the graphite-polyurethane composite can be used as an amperometric detector for flow analysis in the determination of APAP.

VLT-FLAMES analysis of 8 giants in the bulge metal-poor globular cluster NGC 6522: oldest cluster in the Galaxy? Analysis of 8 giants in NGC 6522

Context. NGC 6522 has been the first metal-poor globular cluster identified in the bulge by Baade. Despite its importance, very few high-resolution abundance analyses of stars in this cluster are available. The bulge metal-poor clusters may be important tracers of the early chemical enrichment of the Galaxy. Aims. The main purpose of this study is to determine metallicity and elemental ratios in individual stars of NGC 6522. Methods. High-resolution spectra of 8 giants of the bulge's globular cluster NGC 6522 were obtained at the 8m VLT UT2-Kueyen telescope with the FLAMES+GIRAFFE spectrograph. Multiband V, I, J, K(s) photometry was used to derive effective temperatures as reference values. Spectroscopic parameters were derived from Fe I and Fe II lines, and adopted for the derivation of abundance ratios. Results. The present analysis provides a metallicity [Fe/H] = -1.0 +/- 0.2. The alpha-elements oxygen, magnesium and silicon show [O/Fe] = +0.4 +/- 0.3, [Mg/Fe] = [Si/Fe] = +0.25 +/- 0.15, whereas calcium and titanium show shallower ratios of [Ca/Fe] = [Ti/Fe] = +0.15 +/- 0.15. The neutron-capture r-process element europium appears to be overabundant by [Eu/Fe] = +0.4 +/- 0.4. The neutron-capture s-elements lanthanum and barium are enhanced by [La/Fe] = +0.35 +/- 0.2 and [Ba/Fe] = +0.5 +/- 0.5. The large internal errors, indicating the large star-to-star variation in the barium and europium abundances, are also discussed. Conclusions. The moderate metallicity combined to a blue horizontal branch (BHB), are characteristics similar to those of HP 1 and NGC 6558, pointing to a population of very old globular clusters in the Galactic bulge. Also, the abundance ratios in NGC 6522 resemble those in HP 1 and NGC 6558. The ultimate conclusion is that the bulge is old, and went through an early prompt chemical enrichment.

Functional microarray analysis suggests repressed cell-cell signaling and cell survival-related modules inhibit progression of head and neck squamous cell carcinoma

Background: Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+) and recurrent head and neck squamous cell carcinoma (HNSCC) may increase our understanding of the complex biology of this disease. Methods: Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative) or tumor recurrence (recurrent or non-recurrent tumor) after treatment (surgery with neck dissection followed by radiotherapy). Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. Results: The most frequent alterations were the repression of modules in negative lymph node (N0) and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. Conclusions: The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway, specially apoptosis and interactions between tumor cells and the stroma.

A flow injection procedure based on solenoid micro-pumps for spectrophotometric determination of free glycerol in biodiesel

A flow system designed with solenoid micro-pumps is proposed for fast and greener spectrophotometric determination of free glycerol in biodiesel. Glycerol was extracted from samples without using organic solvents. The determination involves glycerol oxidation by periodate, yielding formaldehyde followed by formation of the colored (3,5-diacetil-1,4-dihidrolutidine) product upon reaction with acetylacetone. The coefficient of variation, sampling rate and detection limit were estimated as 1.5% (20.0 mg L(-1) glycerol, n =10), 34 h(-1), and 1.0 mg L(-1) (99.7% confidence level), respectively. A linear response was observed from 5 to 50 mg L(-1), with reagent consumption estimated as 345 mu g of KIO(4) and 15 mg of acetylacetone per determination. The procedure was successfully applied to the analysis of biodiesel samples and the results agreed with the batch reference method at the 95% confidence level. (C) 2010 Elsevier B.V. All rights reserved.

An environmentally friendly flow system for high-sensitivity spectrophotometric determination of free chlorine in natural waters

A green and highly sensitive analytical procedure was developed for the determination of free chlorine in natural waters, based on the reaction with N,N-diethyl-p-phenylenediamine (DPD). The flow system was designed with solenoid micro-pumps in order to improve mixing conditions by pulsed flows and to minimize reagent consumption as well as waste generation. A 100-cm optical path flow cell based on a liquid core waveguide was employed to increase sensitivity. A linear response was observed within the range 10.0 to 100.0 mu g L(-1), with the detection limit, coefficient of variation and sampling rate estimated as 6.8 mu g (99.7% confidence level), 0.9% (n = 20) and 60 determinations per hour, respectively. The consumption of the most toxic reagent (DPD) was reduced 20,000-fold and 30-fold in comparison to the batch method and flow injection with continuous reagent addition, respectively. The results for natural and tap water samples agreed with those obtained by the reference batch spectrophotometric procedure at the 95% confidence level. (C) 2010 Elsevier By. All rights reserved.

Exploitation of a single interface flow system for on-line aqueous biphasic extraction

The exploitation of aqueous biphasic extraction is proposed for the first time in flow analysis This extraction strategy stands out for being environmentally attractive since it is based in the utilization of two immiscible phases that are intrinsically aqueous The organic solvents of the traditional liquid-liquid extractions ale no longer used, being replaced by non-toxic, non-flammable and non-volatile ones. A single interface flow analysis (SIFA) system was implemented to carry out the extraction process due to its favourable operational characteristics that include the high automation level and simplicity of operation, the establishment of a dynamic interface where the mass transfer occurred between the two immiscible aqueous phases, and the versatile control over the extraction process namely the extraction time The application selected to demonstrate the feasibility of SIFA to perform this aqueous biphasic extraction was the pre-concentration of lead. After extraction, lead reacted with 8-hydroxyquinoline-5-sulfonic acid and the resulting product was determined by a fluorimetric detector included in the flow manifold. Therefore, the SIFA single interface was used both as extraction (enrichment) and reaction interface. (C) 2010 Elsevier B.V All rights reserved.