Stabilising cysteinyl thiol oxidation and nitrosation for proteomic analysis

Oxidation and S-nitrosylation of cysteinyl thiols (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H), sulfonic acids (Cys-SO3H), disulphides and S-nitrosothiols are suggested as important post-translational modifications that can activate or deactivate the function of many proteins. Non-enzymatic post-translational modifications to cysteinyl thiols have been implicated in a wide variety of physiological and pathophysiological states but have been difficult to monitor in a physiological setting because of a lack of experimental tools. The purpose of this review is to bring together the approaches that have been developed for stably trapping cysteine either in its reduced or oxidised forms for enrichment and or subsequent mass spectrometric analysis. These tools are providing insight into potential targets for post-translational modifications to cysteine modification in vivo. This article is part of a Special Issue entitled: Special Issue: Posttranslational Protein modifications in biology and Medicine. © 2013.

Post-translational modifications and mass spectrometry detection

In this review, we provide a comprehensive bibliographic overview of the role of mass spectrometry and the recent technical developments in the detection of post-translational modifications (PTMs). We briefly describe the principles of mass spectrometry for detecting PTMs and the protein and peptide enrichment strategies for PTM analysis, including phosphorylation, acetylation and oxidation. This review presents a bibliographic overview of the scientific achievements and the recent technical development in the detection of PTMs is provided. In order to ascertain the state of the art in mass spectrometry and proteomics methodologies for the study of PTMs, we analyzed all the PTM data introduced in the Universal Protein Resource (UniProt) and the literature published in the last three years. The evolution of curated data in UniProt for proteins annotated as being post-translationally modified is also analyzed. Additionally, we have undertaken a careful analysis of the research articles published in the years 2010 to 2012 reporting the detection of PTMs in biological samples by mass spectrometry. © 2013 Elsevier Inc.

Development of novel mass spectrometry methodology to detect post-translational modifications in oxidative stress and disease

This paper presented at the European Meeting of the Society-for-Free-Radical-Research-Europe 2007, discusses the development of novel mass spectrometry methodology to detect post-translational modifications in oxidative stress and disease.

Structural and chemical modifications of typical South African biomasses during torrefaction

Torrefaction experiments were carried out for three typical South African biomass samples (softwood chips, hardwood chips and sweet sorghum bagasse) to a weight loss of 30wt.%. During torrefaction, moisture, non-structural carbohydrates and hemicelluloses were reduced, resulting in a structurally modified torrefaction product. There was a reduction in the average crystalline diameter (La) (XRD), an increase in the aromatic fraction and a reduction in aliphatics (substituted and unsubstituted) (CPMAS 13C NMR). The decrease in the aliphatic components of the lignocellulosic material under the torrefaction conditions also resulted in a slight ordering of the carbon lattice. The degradation of hemicelluloses and non-structural carbohydrates increased the inclusive surface area of sweet sorghum bagasse, while it did not change significantly for the woody biomasses.

On some Modifications of the Nekrassov Method for Numerical Solution of Linear Systems of Equations

A modification of the Nekrassov method for finding a solution of a linear system of algebraic equations is given and a numerical example is shown.

PSD modifications of FHRV due to interpolation and CTG storage rate

Cardiotocographic data provide physicians information about foetal development and permit to assess conditions such as foetal distress. An incorrect evaluation of the foetal status can be of course very dangerous. To improve interpretation of cardiotocographic recordings, great interest has been dedicated to foetal heart rate variability spectral analysis. It is worth reminding, however, that foetal heart rate is intrinsically an uneven series, so in order to produce an evenly sampled series a zero-order, linear or cubic spline interpolation can be employed. This is not suitable for frequency analyses because interpolation introduces alterations in the foetal heart rate power spectrum. In particular, interpolation process can produce alterations of the power spectral density that, for example, affects the estimation of the sympatho-vagal balance (computed as low-frequency/high-frequency ratio), which represents an important clinical parameter. In order to estimate the frequency spectrum alterations of the foetal heart rate variability signal due to interpolation and cardiotocographic storage rates, in this work, we simulated uneven foetal heart rate series with set characteristics, their evenly spaced versions (with different orders of interpolation and storage rates) and computed the sympatho-vagal balance values by power spectral density. For power spectral density estimation, we chose the Lomb method, as suggested by other authors to study the uneven heart rate series in adults. Summarising, the obtained results show that the evaluation of SVB values on the evenly spaced FHR series provides its overestimation due to the interpolation process and to the storage rate. However, cubic spline interpolation produces more robust and accurate results. © 2010 Elsevier Ltd. All rights reserved.

PSD modifications of FHRV due to CTG storage rate

Cardiotocographic data provide physicians information about foetal development and, through assessment of specific parameters (like accelerations, uterine contractions, ...), permit to assess conditions such as foetal distress. An incorrect evaluation of foetal status can be of course very dangerous. In the last decades, to improve interpretation of cardiotocographic recordings, great interest has been dedicated to FHRV spectral analysis. It is worth reminding that FHR is intrinsically an uneven series and that to obtain evenly sampled series, many commercial cardiotocographs use a zero-order interpolation (storage rate of CTG data equal to 4 Hz). This is not suitable for frequency analyses because interpolation introduces alterations in the FHR power spectrum. In particular, this interpolation process can produce artifacts and an attenuation of the high-frequency components of the PSD that, for example, affects the estimation of the sympatho-vagal balance (SVB - computed as low-frequency/high-frequency ratio), which represents an important clinical parameter. In order to estimate the frequency spectrum alterations due to zero-order interpolation and other CTG storage rates, in this work, we simulated uneven FHR series with set characteristics, their evenly spaced versions (with different storage rates) and computed SVB values by PSD. For PSD estimation, we chose the Lomb method, as suggested by other authors in application to uneven HR series. ©2009 IEEE.

Exploring oxidative modifications of tyrosine:an update on mechanisms of formation, advances in analysis and biological consequences

Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.

Simulation and Robust Modifications of Estimates in Branching Processes

This study is focused on the comparison and modification of different estimates arising in the branching processes. Simulations of models with or without migration are put through. Due to the complexity of the computations the algorithms are designed with the language of technical computing MATLAB. Using the simulations, estimates of the o spring mean of the generated processes are calculated. It is well known in the literature that under certain conditions the asymptotic distribution of the estimates is proved to be normal. Using the asymptotic normality a modified method of maximum likelihood is proposed. The aim is to obtain trimmed maximum likelihood estimates based on several sample paths with the same number of generations. Thus in a natural way the observations, inconsistent with the aprior information about the asymptotic normality are excluded from the model. The computation of the standard error allows the comparison of different types of estimates.

Mapping nitro-tyrosine modifications in fibrinogen by mass spectrometry as a biomarker for inflammatory disease

There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25mM and 1mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5nmol/mg protein in 0, 0.25mM and 1mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75%. The oxidised samples showed increases in oxidative modifications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identification of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions.

Brain activity modifications following spinal cord stimulation for chronic neuropathic pain:a systematic review

Background and objective: Spinal cord stimulation (SCS) is believed to exert supraspinal effects; however, these mechanisms are still far from fully elucidated. This systematic review aims to assess existing neurophysiological and functional neuroimaging literature to reveal current knowledge regarding the effects of SCS for chronic neuropathic pain on brain activity, to identify gaps in knowledge, and to suggest directions for future research. Databases and data treatment: Electronic databases and hand-search of reference lists were employed to identify publications investigating brain activity associated with SCS in patients with chronic neuropathic pain, using neurophysiological and functional neuroimaging techniques (fMRI, PET, MEG, EEG). Studies investigating patients with SCS for chronic neuropathic pain and studying brain activity related to SCS were included. Demographic data (age, gender), study factors (imaging modality, patient diagnoses, pain area, duration of SCS at recording, stimulus used) and brain areas activated were extracted from the included studies. Results: Twenty-four studies were included. Thirteen studies used neuroelectrical imaging techniques, eight studies used haemodynamic imaging techniques, two studies employed both neuroelectrical and haemodynamic techniques separately, and one study investigated cerebral neurobiology. Conclusions: The limited available evidence regarding supraspinal mechanisms of SCS does not allow us to develop any conclusive theories. However, the studies included appear to show an inhibitory effect of SCS on somatosensory evoked potentials, as well as identifying the thalamus and anterior cingulate cortex as potential mediators of the pain experience. The lack of substantial evidence in this area highlights the need for large-scale controlled studies of this kind.

Targeted mass spectrometry methods for detecting oxidative post-translational modifications

Oxidative post-translational modifications (oxPTMs) can alter the function of proteins, and are important in the redox regulation of cell behaviour. The most informative technique to detect and locate oxPTMs within proteins is mass spectrometry (MS). However, proteomic MS data are usually searched against theoretical databases using statistical search engines, and the occurrence of unspecified or multiple modifications, or other unexpected features, can lead to failure to detect the modifications and erroneous identifications of oxPTMs. We have developed a new approach for mining data from accurate mass instruments that allows multiple modifications to be examined. Accurate mass extracted ion chromatograms (XIC) for specific reporter ions from peptides containing oxPTMs were generated from standard LC-MSMS data acquired on a rapid-scanning high-resolution mass spectrometer (ABSciex 5600 Triple TOF). The method was tested using proteins from human plasma or isolated LDL. A variety of modifications including chlorotyrosine, nitrotyrosine, kynurenine, oxidation of lysine, and oxidized phospholipid adducts were detected. For example, the use of a reporter ion at 184.074 Da/e, corresponding to phosphocholine, was used to identify for the first time intact oxidized phosphatidylcholine adducts on LDL. In all cases the modifications were confirmed by manual sequencing. ApoB-100 containing oxidized lipid adducts was detected even in healthy human samples, as well as LDL from patients with chronic kidney disease. The accurate mass XIC method gave a lower false positive rate than normal database searching using statistical search engines, and identified more oxidatively modified peptides. A major advantage was that additional modifications could be searched after data collection, and multiple modifications on a single peptide identified. The oxPTMs present on albumin and ApoB-100 have potential as indicators of oxidative damage in ageing or inflammatory diseases.

The use of curriculum modifications and instructional accommodations to provide access for middle school students with autism to the general curriculum

The number of students identified as having autism increased by 500% in the past 10 years (United States Government Accountability Office, 2005). All students with disabilities are required to be placed in least restrictive environments and to be given access to the general curriculum in the major subjects of math, reading, writing, and science as mandated by federal legislation such as the Individuals with Disabilities Education Act (IDEA, 2004) and No Child Left Behind (NCLB, 2001). As a result of this legislation, an increasing number of students with autism are being educated in inclusive classrooms. Most studies on general education access and curriculum modifications and/or instructional accommodations center on students with intellectual disabilities (e.g. Soukup, Wehmeyer, Bashinski, & Boviard, 2007; Wehmeyer, Lattin, Lapp-Rincker, & Agran, 2003). Wehmeyer et al. (2003) and Soukup et al. (2007) found included students with intellectual disabilities had more access to the general curriculum than mostly self-contained students. This meant included students were more likely to be working on the general curriculum as mandated by NCLB than those in only self-contained classrooms. This study builds and expands the research of Wehmeyer et al., as well as Soukup et al., by examining how students with autism are given access to the general curriculum through curriculum modifications and instructional accommodations used by general education teachers in three schools. This investigation focused on nine inclusive classrooms for students with autism using a parallel mixed methods design (Newman, Newman, & Newman, 2011). Classroom observations using both an IEP related checklist and field notes, teacher interviews, an archival document review of the Individual Education Plan (IEP) for the selected students with autism were performed. Findings of this study were organized by interview questions and subsequent coding categories. Quantitative data were organized in a nominal scale. Participants asserted that their middle school students with autism functioned well in their classrooms, occasionally exhibiting behavioral differences. Most instructional accommodations on IEPs were being implemented by participants, and participants often provided additional instructional accommodations not mandated by the IEP. The majority of participants credited county workshops for their knowledge of instructional accommodations.

Études des modifications de la réponse apoptotique induites par le virus de l'hépatite C dans des foies normaux et infectés et dans la lignée cellulaire HuH7 contenant ou non un réplicon sous-génomique du virus

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Modifications de l'expression génique du système nerveux central lors de l'insuffisance hépatique aiguë : rôle dans les mécanismes pathophysiologiques responsables de l'œdème cérébral

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.