BackgroundProteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly.ResultsThe proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone.ConclusionsThe acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.
Some heart failure (HF) patients develop ventilatory oscillation which is composed of exercise periodic breathing (EPB) and sleep apnea. The ventilatory oscillation is associated with exercise intolerance. This study employed an integrated monitoring system to elucidate the way of central and peripheral hemodynamic adaption responding to exercise. This study recruited 157 HF patients to perform exercise testing using a bicycle ergometer. A noninvasive bio-reactance device was adopted to measure cardiac hemodynamics, whereas a near-infrared spectroscopy (NIRS) was used to assess perfusion and O2 extraction in the frontal cerebral lobe (FC) and vastus lateralis muscle (VL) during exercise respectively. Furthermore, quality of life (QoL) was measured with the Short Form-36 (SF-36) and the Minnesota Living with Heart Failure questionnaires (MLHFQ). The patients were divided into an EPB group (n = 65) and a non-EPB group (n = 92) according to their ventilation patterns during testing. Compared to their non-EPB counterparts, the patients with EPB exhibited 1) impaired aerobic capacity with a smaller peak oxygen consumption (VO2peak) and oxygen uptake efficiency slopes; 2) impaired circulatory and ventilatory efficiency with relatively high cardiac output and ventilation per unit workload; 3) impaired ventilatory/hemodynamic adaptation in response to exercise with elevated deoxyhemoglobin levels in the FC region; and 4) impaired QoL with lower physical component scores on the SF-36 and higher scores on the MLHFQ. In conclusion, EPB may reduce circulatory-ventilatory-hemodynamic efficiency during exercise, thereby impairing functional capacity in patients with HF.
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