Salud

URI permanente para esta comunidad

https://hdl.handle.net/10669/1424

Examinar

Examinando Salud por Materia "2D-GE"

Mostrando 1 - 2 de 2
  • Miniatura
    Ítem
    Two-dimensional gel electrophoresis (2D-GE) image analysis based on CellProfiler: Pseudomonas aeruginosa AG1 as model
    (2020) Molina Mora, José Arturo; Chinchilla Montero, Diana; Castro Peña, Carolina; García Santamaría, Fernando
    Two-dimensional gel electrophoresis (2D-GE) is an indispensable technique for the study of proteomes of biological systems, providing an assessment of changes in protein abundance under various experimental conditions. However, due to the complexity of 2D-GE gels, there is no systematic, automatic, and reproducible protocol for image analysis and specific implementations are required for each context. In addition, practically all available solutions are commercial, which implies high cost and little flexibility to modulate the parameters of the algorithms. Using the bacterial strain, Pseudomonas aeruginosaAG1 as a model, we obtained images from 2D-GE of periplasmic protein profiles when the strain was exposed to multiple conditions, including antibiotics. Then, we proceeded to implement and evaluate an image analysis protocol with open-source software, CellProfiler. First, a preprocessing step included a bUnwarpJ-Image pipeline for aligning 2D-GE images. Then, using CellProfiler, we standardized two pipelines for spots identification. Total spots recognition was achieved using segmentation by intensity, whose performance was evaluated when compared with a reference protocol. In a second pipeline with the same program, differential identification of spots was addressed when comparing pairs of protein profiles. Due to the characteristics of the programs used, our workflow can automatically analyze a large number of images and it is parallelizable, which is an advantage with respect to other implementations. Finally, we compared six experimental conditions of bacterial strain in the presence or absence of antibiotics, determining protein profiles relationships by applying clustering algorithms PCA (Principal Components Analysis) and HC (Hierarchical Clustering).
  • Miniatura
    Ítem
    Two-dimensional gel electrophoresis image analysis of two Pseudomonas aeruginosa clones
    (2022-11-17) Molina Mora, José Arturo; Chinchilla Montero, Diana; Castro Peña, Carolina; García Santamaría, Fernando
    A classical strategy to analyse the protein content of a biological sample is the two-dimensional gel electrophoresis (2D-GE). This technique separates proteins by both isoelectric point and molecular weight, and images are taken for subsequent analyses. However, analyses of 2D-GE images require standardized image analysis due to susceptibility of gels to get deformed, presence of overlapping spots and stripes, fuzzy and unstained spots, and others. This represent a difficulty for final users (researchers), which demand for free and user-friendly solutions. We have previously reported the standardization of a protocol to analyse 2D-GE images, and in the current study we applied it to two new bacterial isolates Pseudomonas aeruginosa C25 and C50. We first extracted periplasmic proteins after exposure to antibiotics, and we then run a 2D-GE analysis. Images were analysed using our standardized protocol, achieving the identification of protein spots using CellProfiler after pre-processing step. Comparison between strains was done using differential spot analysis, revealing a specific pattern in the protein expression between bacteria. These results will help to study the biological meaning of these strains using proteomic profiling under different conditions.