5,568 research outputs found

    A Novel Gaussian Extrapolation Approach for 2D Gel Electrophoresis Saturated Protein Spots

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    Analysis of images obtained from two-dimensional gel electrophoresis (2D-GE) is a topic of utmost importance in bioinformatics research, since commercial and academic software available currently has proven to be neither completely effective nor fully automatic, often requiring manual revision and refinement of computer generated matches. In this work, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, the algorithm reveals overexposed areas, where spots may be truncated, and plateau regions caused by smeared and overlapping spots. Next, it reconstructs the correct distribution of pixel values in these overexposed areas and plateau regions, using a two-dimensional least-squares fitting based on a generalized Gaussian distribution. Pixel correction in saturated and smeared spots allows more accurate quantification, providing more reliable image analysis results. The method is validated for processing highly exposed 2D-GE images, comparing reconstructed spots with the corresponding non-saturated image, demonstrating that the algorithm enables correct spot quantificatio

    A Novel Gaussian Extrapolation Approach for 2D Gel Electrophoresis Saturated Protein Spots

    Get PDF
    Analysis of images obtained from two-dimensional gel electrophoresis (2D-GE) is a topic of utmost importance in bioinformatics research, since commercial and academic software available currently has proven to be neither completely effective nor fully automatic, often requiring manual revision and refinement of computer generated matches. In this work, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, the algorithm reveals overexposed areas, where spots may be truncated, and plateau regions caused by smeared and overlapping spots. Next, it reconstructs the correct distribution of pixel values in these overexposed areas and plateau regions, using a two-dimensional least-squares fitting based on a generalized Gaussian distribution. Pixel correction in saturated and smeared spots allows more accurate quantification, providing more reliable image analysis results. The method is validated for processing highly exposed 2D-GE images, comparing reconstructed spots with the corresponding non-saturated image, demonstrating that the algorithm enables correct spot quantification

    A novel Gaussian fitting approach for 2D gel electrophoresis saturated protein spots

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    Analysis of 2D-GE images is a hot topic in bioinformatics research, since currently available commercial and academic software has proven to be not really effective and not completely automatic, often requiring manual revision of spots detection and refinement of computer generated matches. In this work, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, it reveals overexposed areas where spots may be truncated, and plateau regions caused by smeared and overlapped spots. As next, the correct distribution of pixel values in the overexposed areas and plateau regions is recovered by a two-dimensional fitting based on a generalized Gaussian distribution approximating the spots volume. Pixel correction according to the generalized Gaussian curve in saturated and smeared spots allows more accurate quantifications, providing more reliable image analysis results. As validation, we process highly exposed 2D-GE image, containing saturate spots, with respect to the corresponding non-saturated image, confirming that the method can effectively fix the saturated spots and enable correct spots quantification

    Proteomic studies of pseudomonas putida KT2440 in carcinogenicity screening via 2-dimensional gel electrophoresis

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    This project is part of a pilot study designed to establish a two dimensional gel electrophoresis-based protocol, which can be utilized in screening mutagencity/carcinogencity of uncharacterized substances. Currently, the Ames test is the most commonly used method, which is solely based on mutation at the genome level. In contrast to the genome, which is static, protein expression in a cell under a given set of conditions (the proteome) is dynamic. A screening method based on the proteome rather than the genome may prove more reliable and quantitative in predicting mutagenicity and carcinogenicity. In this project, Pseudomonas putida KT2440 was employed as the biological model because its genome has been completely sequenced and the strain is already known to be able to grow on several aromatic carbon sources. Two carbon sources were selected, succinic acid and phenylethylamine. The strain grew in the non-aromatic and aromatic carbon source respectively. Proteins were extracted at a fixed growth stage (mid-log phase) so that protein expression is consistent. Protein expression was then analyzed by two dimensional gel electrophoresis, with the hope of identifying proteins that are expressed in response to phenylethylamine ? the proteomic signature for phenylethylamine. In total, six signature protein spots were found. In addition to identifying these signature protein spots, a major goal of this project (to establish standard protocols for protein expression and analysis) was accomplished. Based on the results obtained in this project, several directions for further improvement in later stages of the procedure are also discussed

    Palmitate-induced lipotoxicity alters acetylation of multiple proteins in clonal β cells and human pancreatic islets

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    Type 2 diabetes is characterized by progressive β cell dysfunction, with lipotoxicity playing a possible pathogenetic role. Palmitate is often used to examine the direct effects of lipotoxicity and it may cause mitochondrial alterations by activating protein acetylation. However, it is unknown whether palmitate influences protein acetylation in β cells. We investigated lysine acetylation in mitochondrial proteins from INS-1E β cells (INS-1E) and in proteins from human pancreatic islets (HPI) after 24 h palmitate exposure. First, we confirmed that palmitate damages β cells and demonstrated that chemical inhibition of deacetylation also impairs INS-1E function and survival. Then, by 2-D gel electrophoresis, Western Blot and Liquid Chromatography-Mass Spectrometry we evaluated the effects of palmitate on protein acetylation. In mitochondrial preparations from palmitate-treated INS-1E, 32 acetylated spots were detected, with 13 proteins resulting over-acetylated. In HPI, 136 acetylated proteins were found, of which 11 were over-acetylated upon culture with palmitate. Interestingly, three proteins, glutamate dehydrogenase, mitochondrial superoxide dismutase, and SREBP-1, were over-acetylated in both INS-1E and HPI. Therefore, prolonged exposure to palmitate induces changes in β cell protein lysine acetylation and this modification could play a role in causing β cell damage. Dysregulated acetylation may be a target to counteract palmitate-induced β cell lipotoxicity

    Dynamic cofilin phosphorylation in the control of lamellipodial actin homeostasis

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    During animal cell chemotaxis, signalling at the plasma membrane induces actin polymerisation to drive forward cell movement. Since the cellular pool of actin is limited, efficient protrusion formation also requires the coordinated disassembly of pre-existing actin filaments. To search for proteins that can monitor filamentous and globular actin levels to maintain the balance of polymerisation and disassembly, we followed changes in the proteome induced by RNA interference (RNAi)mediated alterations in actin signalling. This unbiased approach revealed an increase in the levels of an inactive, phosphorylated form of the actin-severing protein cofilin in cells unable to generate actin-based lamellipodia. Conversely, an increase in F-actin levels induced the dephosphorylation and activation of cofilin via activation of the Ssh phosphatase. Similarly, in the context of acute phosphoinositide 3-kinase (PI3K) signalling, dynamic changes in cofilin phosphorylation were found to depend on the Ssh phosphatase and on changes in lamellipodial Factin. These results indicate that changes in the extent of cofilin phosphorylation are regulated by Ssh in response to changes in the levels and/or organisation of F-actin. Together with the recent finding that Ssh phosphatase activity is augmented by F-actin binding, these results identify Ssh-dependent regulation of phosphorylated cofilin levels as an important feedback control mechanism that maintains actin filament homeostasis during actin signalling

    Power and limitations of electrophoretic separations in proteomics strategies

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    Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed

    Overexpression of Heat Shock Cognate Protein 71 kDa and Pyruvate Dehydrogenase in the Brain Tissue at the Early Stage of High Fat Diet Consumption

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    High-fat diet (HFD) increases the risk of obese, while obesity increases the prevalence of metabolic syndrome and non-communicable diseases. Therefore, it will be interesting to evaluate the changes in metabolic parameters and brain profile upon the early consumption of HFD. In this study, a total of 12 Sprague-Dawley male rats were divided into two groups (n = 6), each group was fed with normal diet and HFD (39% of total calories from fats), respectively, for 6 weeks consecutively. The body weight, blood glucose, cholesterol and triglyceride values were measured.  Furthermore, the brain and visceral adipose tissues were harvested at the end of the experiment. Protein was extracted from the brain tissue, and the protein extracts were separated by using two-dimensional gel electrophoresis and analyzed by liquid chromatography tandem mass spectrometric analysis (LC/MS/MS). In terms of food calorie, the rats fed with HFD consumed more energy than the rats fed with normal diet. Nevertheless, the blood triglyceride and cholesterol, and the visceral adipose tissue of both the HFD and normal diet fed rats were indifferent. At the molecular level, overexpression of stress proteins, namely heat shock cognate protein 71 kDa (Hsc70)  and pyruvate dehydrogenase were detected in brain tissue of HFD group. These results suggest that HFD intake causing significant change in brain proteins profile at the early phase of its consumption when no clear metabolic changes were observed. This showed that the brain was affected by HFD
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