Deep Coverage Tissue and Cellular Proteomics Revealed IL-1ß Can Independently Induce the Secretion of TNF-Associated Proteins from Human Synoviocytes.

Synovitis is a key contributor to the inflammatory environment in osteoarthritis (OA) joints. Currently, the biological therapy of OA is not satisfactory in multiple single-target trials on anti-TNF agents, or IL-1 antagonists. Systems biological understanding of the phosphorylation state in OA synovium is warranted to direct further therapeutic strategies. Therefore, in this study, we compared the human synovial phosphoproteome of the OA with the acute joint fracture subjects. We found that OA synovium had significantly more phosphoproteins, and 82 phosphoproteins could only be specifically found in all the OA samples. Differentially expressed proteins of the OA synovium were focusing on endoplasmic reticulum-/Golgi-associated secretion and negative regulation of cell proliferation, which was verified through an IL-1ß-treated human synoviocyte (HS) in vitro model. With data-independent acquisition-based mass spectrometry, we found that IL-1ß could induce HS to secrete proteins that were significantly associated with the endosomal/vacuolar pathway, endoplasmic reticulum/Golgi secretion, complement activation, and collagen degradation. Especially, we found that while specifically suppressing HS endocytosis, IL-1ß could activate the secretion of 25 TNF-associated proteins, and the change of SERPINE2 and COL3A1 secretion was verified by immunoblotting. In conclusion, our results suggest that OA synovium has a polarized phosphoproteome to inhibit proliferation and maintain active secretion of HS, whereas IL-1ß alone can transform HS to produce a synovitis-associated secretome, containing numerous TNF-associated secretory proteins in a TNF-independent mode.

Phosphoproteome Characterization of Human Colorectal Cancer SW620 Cell-Derived Exosomes and New Phosphosite Discovery for C-HPP.

Identification of all phosphorylation forms of known proteins is a major goal of the Chromosome-Centric Human Proteome Project (C-HPP). Recent studies have found that certain phosphoproteins can be encapsulated in exosomes and function as key regulators in tumor microenvironment, but no deep coverage phosphoproteome of human exosomes has been reported to date, which makes the exosome a potential source for the new phosphosite discovery. In this study, we performed highly optimized MS analyses on the exosomal and cellular proteins isolated from human colorectal cancer SW620 cells. With stringent data quality control, 313 phosphoproteins with 1091 phosphosites were confidently identified from the SW620 exosome, from which 202 new phosphosites were detected. Exosomal phosphoproteins were significantly enriched in the 11q12.1-13.5 region of chromosome 11 and had a remarkably high level of tyrosine-phosphorylated proteins (6.4%), which were functionally relevant to ephrin signaling pathway-directed cytoskeleton remodeling. In conclusion, we here report the first high-coverage phosphoproteome of human cell-secreted exosomes, which leads to the identification of new phosphosites for C-HPP. Our findings provide insights into the exosomal phosphoprotein systems that help to understand the signaling language being delivered by exosomes in cell-cell communications. The mass spectrometry proteomics data have been deposited to the ProteomeXchange consortium with the data set identifier PXD004079, and iProX database (accession number: IPX00076800).

[A troponin detection-combined study of rabbit experiment for evaluating cardiac fatigue].

The objective of this study is to combine troponin and indicators of cardiac acoustics for synthetically evaluating cardiac fatigue of rabbits, analyzing exercise-induced cardiac fatigue (EICF) and exercise-induced cardiac damage (EICD). New Zealand white rabbits were used to conduct a multi-step swimming experiments with load, reaching an exhaustive state for evaluating if the amplitude ratio of the first to second heart sound (S1/S2) and heart rate (HR) during the exhaustive exercise would decrease or not and if they would be recovered 24-48 h after exhaustive exercise. The experimental end point was to complete 3 times of exhaustions or death from exhaustion. Circulating troponin I (cTnI) were detected from all of the experimental rabbits at rest [(0. 02±0. 01) ng/mL], which, in general, indicated that there existed a physiological release of troponin. After the first exhaustive swim, cTnI of the rabbits increased. However, with 24-hour rest, S1/S2, HR, and cTnI of the tested rabbits all returned toward baseline levels, which meant that the experimental rabbits experienced a cardiac fatigue process. After repeated exhaustion, overloading phenomena were observed, which led to death in 3 out of 11 rabbits, indicating their cardiac damage; the troponin elevation under this condition could be interpreted by pathological release. Evaluation of myocardial damage can not be based on the troponin levels alone, but can only be based on a comprehensive analysis.

[Preliminary study of rabbit experiment modality for evaluating cardiac fatigue].

This paper presents a preliminary study of rabbit experiment modality incorporating a new indicator for evaluating cardiac function changes, providing a basis for subsequent study of cardiac fatigue. Using only biochemical indicators, such as troponins, is difficult to make a distinction between exercise-induced cardiac fatigue (EICF) and exercise-induced cardiac damage (EICD). Therefore, some new indicators are needed to evaluate cardiac fatigue synthetically. In our study, we used New Zealand white rabbits to conduct a multi-step swimming experiments with load. We made the rabbits reach an exhaustive state to evaluate whether the amplitude ratio of the first to second heart sound (S1/S2) and heart rate (HR) during the exhaustive exercise would be decreased and whether they would be able to recover after the exhaustive exercise for 24 hours. During the first phase of swimming, S1/S2 and HR were increased, and then decreased at exhaustive state. They were recovered after the exhaustive exercise for 24 hours. Overloading led to deaths of three rabbis, and new phenomena from overloading and related to this kind of death were observed. The experiments proved that Multi-steps swimming experiments with loads by using New Zealand white rabbit is useful for studying cardiac fatigue and premonition of sudden cardiac death.