Methods in molecular biology
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Esophageal squamous cell carcinoma (ESCC) is a deadly disease, partly because it is often diagnosed late in disease stage. An accurate early diagnosis by endoscopy could detect advanced carcinoma as well as curable dysplasia and early ESCC. ⋯ Important progress has been made in high-quality endoscopic diagnosis, including magnifying endoscopy, narrowband imaging, and other image enhancement, as well as in techniques in endoscopic resection. These emerging techniques will aid the early diagnosis of ESCC that lead to higher chance of curing the cancer.
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The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. ⋯ The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.
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Immunohistochemistry is the identification of a cell protein by a specific antibody targeting that protein. It is the most common ancillary test to study the pathology of cancer. Immunohistochemical protein markers are used to differentiate poorly differentiated squamous cell carcinoma from poorly differentiated adenocarcinoma or neuroendocrine carcinomas. ⋯ Successful application of the immunochemistry depends on understanding the mechanisms and principles as well as the limitations of the procedure. Automation of the procedure by different models of automatic stainers is widely used in diagnostic laboratories. The use of autostainers streamlines the workflows and certainly reduces the labor, time, and cost of using immunohistochemistry in clinical and research settings.
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Mass spectrometry-based proteomics analysis could categorize proteins and study their interactions in large scale in human cancers. By this method, many proteins are upregulated or downregulated in esophageal squamous cell carcinoma (ESCC) when compared to nonneoplastic esophageal mucosae. ⋯ Different biological matrices such as pathological tissue, body fluids, and cancer cell lines-based proteomics have widely been used. Herein, we described cell line-based label-free shotgun proteomics (in-solution tryptic digestion) to identify the protein biomarkers differently expressed in ESCC.
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The third edition of "Plant Proteomics Methods and Protocols," with the title "Advances in Proteomics Techniques, Data Validation, and Integration with Other Classic and -Omics Approaches in the Systems Biology Direction," was conceived as being based on the success of the previous editions, and the continuous advances and improvements in proteomic techniques, equipment, and bioinformatics tools, and their uses in basic and translational plant biology research that has occurred in the past 5 years (in round figures, of around 22,000 publications referenced in WoS, 2000 were devoted to plants). The monograph contains 29 chapters with detailed proteomics protocols commonly employed in plant biology research. They present recent advances at all workflow stages, starting from the laboratory (tissue and cell fractionation, protein extraction, depletion, purification, separation, MS analysis, quantification) and ending on the computer (algorithms for protein identification and quantification, bioinformatics tools for data analysis, databases and repositories). ⋯ Unfortunately, only 10% of them kindly accepted. My gratitude to those who accepted our invitation but also to those who did not, as all of them have contributed to the plant proteomics field. I will enlist, in this introductory chapter, following my own judgment, some of the relevant papers published in the past 5 years, those that have shown us how to enhance and exploit the potential of proteomics in plant biology research, without aiming at giving a too exhaustive list.