Comparative Proteomics Reveals the Spoilage-Related Factors of Shewanella putrefaciens Under Refrigerated Condition

Shewanella putrefaciens is a microorganism with strong spoilage potential for aquatic products. This study aimed to investigate the potential spoilage factors of S. putrefaciens by comparative proteomic analysis. The spoilage potential of two strains of S. putrefaciens (00A and 00B) isolated from chilled spoiled bigeye tuna was investigated. The results of total volatile basic nitrogen (TVB-N), trimethylamine (TMA) in fish inoculated with S. putrefaciens, extracellular protease activity of S. putrefaciens, and degradation of fish proteins indicated that the spoilage potential of S. putrefaciens 00A was much higher than that of 00B. Fish proteins are usually degraded by spoilage microorganism proteases into small molecular peptides and amino acids, which are subsequently degraded into spoilage metabolites in bacterial cells, leading to deterioration of fish quality. Thus, proteomic analysis of the extracellular and intracellular proteins of 00A vs. 00B was performed. The results indicated that the intracellular differentially expressed protein (IDEP) contained 243 upregulated proteins and 308 downregulated proteins, while 78 upregulated proteins and 4 downregulated proteins were found in the extracellular differentially expressed protein (EDEP). GO annotation revealed that IDEP and EDEP were mainly involved in cellular and metabolic processes. KEGG annotation results showed that the upregulated proteins in IDEP were mainly involved in sulfur metabolism, amino acid metabolism, and aminoacyl-tRNA biosynthesis, while downregulated proteins were related to propanoate metabolism. In contrast, EDEP of KEGG annotation was mainly involved in ribosomes, quorum sensing, and carbohydrate metabolism. Proteins associated with spoilage containing sulfur metabolism (sulfite reductase, sulfate adenylyltransferase, adenylyl-sulfate kinase), amino acid metabolism (biosynthetic arginine decarboxylase, histidine ammonia-lyase), trimethylamine metabolism (trimethylamine-N-oxide reductase), and extracellular proteins (ATP-dependent Clp protease proteolytic subunit) were identified as upregulated. These proteins may play a key role in the spoilage potential of S. putrefaciens. These findings would contribute to the identification of key spoilage factors and understanding of the spoilage mechanism of microorganisms.

Comprehensive Analysis Reveals the Potential Regulatory Mechanism Between Ub–Proteasome System and Cell Cycle in Colorectal Cancer

The ubiquitin (Ub)–proteasome system (UPS) is an important regulatory component in colorectal cancer (CRC), and the cell cycle is also characterized to play a significant role in CRC. In this present study, we firstly identified UPS-associated differentially expressed genes and all the differentially expressed protein-coding genes in CRC through three differential analyses. UPS-associated genes were also further analyzed via survival analysis. A weighted gene co-expression network analysis (WGCNA) was used to identify the cell cycle-associated genes. We used protein–protein interaction (PPI) network to comprehensively mine the potential mechanism of the UPS–cell cycle regulatory axis. Moreover, we constructed a signature based on UPS-associated genes to predict the overall survival of CRC patients. Our research provides a novel insight view of the UPS and cell cycle system in CRC.

The Alterations in Methylene Blue/Light-Treated Frozen Plasma Proteins Revealed by Proteomics

Introduction: The aim of this study was to investigate the modified proteins in methylene blue/light-treated frozen plasma (MB-FP) compared with fresh frozen plasma (FFP) in order to gain a better application of MB/light-treated plasma in clinic transfusion. Methods: MB-FP and FFP were collected from Changchun central blood station, and a trichloroacetic acid/acetone precipitation method was used to remove albumin for the enrichment of lower abundance proteins. The plasma protein in MB-FP and FFP were separated using two-dimensional gel electrophoresis (2-DE) and the differentially expressed protein spots were analyzed using mass spectrometry. Finally, the differentially expressed proteins were tested using Western blot and enzyme-linked immunosorbent assay (ELISA). Results: Approximately 14 differentially expressed protein spots were detected in the MB-FP, and FFP was chosen as the control. After 2-DE comparison analysis and mass spectrometry, 8 significantly differentially expressed protein spots were identified, corresponding to 6 different proteins, including complement C1r subcomponent (C1R), inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4), keratin, type II cytoskeletal 1 (KRT1), hemopexin (HPX), fibrinogen gamma chain (FGG), and transthyretin (TTR). Western blot showed no significant difference in the expression level of KRT1 between MB-FP and FFP (p > 0.05). Both Western blot and ELISA indicated that the level of HPX was significantly higher in FFP than in MB-FP (p < 0.05). Conclusion: This comparative proteomics study revealed that some significantly modified proteins occur in MB-FP, such as C1R, ITI-H4, KRT1, HPX, FGG, and TTR. Our findings provide more theoretical data for using MB-FP in transfusion medicine. However, the relevance of the data for the transfusion of methylene blue/light-treated plasma remains unclear. The exact modification of these proteins and the effects of these modified proteins on their functions and their effects in clinical plasma infusion need to be further studied.

CHI3L1 Inhibits Expression of NIS by Activating MEK/ERK1/2 Signal Pathway in Thyroid Cancer

Purpose To study the differentially expressed protein between thyroid cancer and radioactive iodine refractory differentiated thyroid cancer (RR-DTC), which presents highly aggressive and unfavorable prognosis. Meanwhile, to search for a potential radiotherapeutic target for patients suffer from RR-DTC. Methods Totally 6 metastatic lymph nodes of RR-DTC and DTC were collected during lymph node dissection for proteomics studies. Immunohistochemical staining was performed to verify the expression of Chitinase-3-like 1 (CHI3L1) in RR-DTC and DTC tissues. Western blotting was used to detect the expression of sodium-iodine symporter (NIS), MEK, and ERK1/2 in CHI3L1 over-expression stable transfectants, control stable transfectants, and PTC-K1 cells. Results CHI3L1 was demonstrated to be significantly up-regulated in RR-DTC by immunohistochemical staining. Besides, CHI3L1 over expression would inhibit expression of sodium-iodine symporter, a key protein for iodine accumulation, by activating MEK/ERK1/2 signal pathway. Conclusion CHI3L1 might be a potential molecular target for RR-DTC due to its over expression in RR-DTC and membrane location.

Differential Proteomic Identification and Bioinformatics Analysis of Femoral Neck in Elderly Female Patients with Hyperuricaemia

Background: Serum uric acid (UA) is positively correlated with bone mineral density (BMD). However, the mechanism by which serum UA affects BMD remains unclear. Objective: The aim was carried out to search for the functional proteins related to serum UA and femoral neck BMD to better understand the pathophysiological mechanism of osteoporosis. Materials and methods: In this study, patients in the UA group (hyperuricaemia combined with femoral neck fracture) and the control group (normal uricaemia combined with femoral neck fracture) were selected according to the inclusion criteria. Total protein was extracted from the femoral neck of each patient. Fluorescence differential gel electrophoresis was used to separate the total proteins, and the differentially expressed protein spots were detected by image analysis. After enzyme digestion, peptide mass fingerprinting and database searches were performed to identify the differentially expressed proteins. DAVID software and Kyoto Encyclopedia of Genes and Genomes (KEGG) data were used for enrichment analysis of the screened differential proteins. Results: After mass spectrometry and database searching, 66 differentially expressed protein spots were identified between the UA group and the control group. Most differentially expressed proteins functioned in cytoskeleton formation, energy metabolism, or signal transduction. They were mainly involved in 50 biological processes, including peroxisome proliferator-activated receptor (PPAR) signalling and fatty acid metabolism. PPARγ and PLIN1 were subject to Western blotting analysis detection; results were consistent with the Label-Free result. Conclusion: Based on an analysis of the biological information, these proteins may be associated with the incidence and progression of the femoral neck bone tissues of hyperuricaemia patients.

Nitrogen Transfer Play an Important Role of Differentially Expressed Protein of Roots in Eucalyptus Urophylla × E. grandis and Dalbergia Odorifera Nursery Seedlings Intercropping System

Background: The mixing of Eucalyptus with N2-fixing trees is a frequently successful and sustainable cropping practice. In this study, we evaluated nitrogen transfer and conducted a proteomic analysis on seedlings of Eucalyptus and a N2-fixing tree, Dalbergia (D.) odorifera, from intercropping and monocropping systems, to elucidate the physiological effecting and the molecular mechanisms on N transfer of Eucalyptus mixed with D. odorifera. Results: We demonstrated the following: (1) Nitrogen transfer occurred from D. odorifera to E. urophylla × E. grandis by 14.6 %. Interspecific facilitation of N uptake and root growth in E. urophylla × E. grandis was increased in the intercropping system, but the root growth and N absorption of D. odorifera were inhibited. (2) Among differentially expressed proteins was greater than 1.5 times, E. urophylla × E. grandis were found to be up-regulated several proteins for the nitrogen assimilation and enhancing the nitrogen competition, such as the proteins related to tricarboxylic acid/organic transformation, nitrogen metabolism and nitrogen assimilation; it also up-regulated the expression of stress resistant protein for its adaptability. However, the assimilation and metabolism of nitrogen was promoted in D. odorifera through the up-regulation of amino acid metabolism related proteins, and increased the key enzyme abundance of glycolysis pathway in D. odorifera. (3) Importantly, E. urophylla × E. grandis was the beneficiary in the process of N transfer, there were more different proteins involved in the synthesis pathway than that of the metabolic pathway, but there were more functional proteins involved in metabolic degradation in D. odorifera. Additionaly, the two groups of nitrogen compound transporter were found in E. urophylla × E. grandis, i.e. the molecular mechanism of the N transfer from D. odorifera to E. urophylla × E. grandis was explained by proteomics. Conclusions: Our study suggests that N transfer occurred from D. odorifera to E. urophylla × E. grandis and it was affected by the variations in the differentially expressed protein. We anticipate the result can be verified in field experiments for the sustainable development of Eucalyptus plantations.

VDAC1 and RhoA in Plasma Exosomes Influence the Severity of Adolescent Idiopathic Scoliosis

Background: Adolescent idiopathic scoliosis (AIS) is the most common spine deformity, but biomarkers for its condition are lacking. Rhodopsin A (RhoA) and voltage-dependent anion-selective channel 1 (VDAC1) in plasma exosomes were defined as differentially expressed proteins between AIS patients and healthy controls. The purpose of this study was to assess exosomes as biomarkers for the occurrence and progression of AIS. Methods：We recruited 10 AIS patients and 8 healthy controls to detect expressed proteins from plasma by liquid chromatography coupled to tandem mass spectrometry. Plasma samples were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Pathway analysis identified that the VDAC1 and RhoA proteins were alterations expressed in the AIS patients, with the most different alteration was found in extracellular exosomes. Ultracentrifugation was carried out to isolate exosomes from plasma. Verification of the most differentially expressed protein was accessed by Western blot analysis and bioinformatics analysis was performed to predict the pathway of it.Results: 42 of significantly differentially expressed proteins were found in all subjects, and 17 proteins had significant difference. The differentially expressed proteins were enriched in plasma exosomes, and some proteins, such as FN1, were upregulated and others, such as VDAC1, RhoA and AHNAK, were downregulated in the AIS patients. Furthermore, ultracentrifugation was carried out to isolate exosomes from plasma, and RhoA and VDAC1 proteins in plasma exosomes were verified to downregulate by western blot. KEGG signaling pathways were used to predict potential pathways involved in the RhoA and VDAC1 proteins in the AIS patients. We found that the RhoA protein influences AIS probably through the chemokine signaling pathway, platelet activation and cAMP signaling pathway, and the VDAC1 protein is a key factor that participates in the necroptosis pathway, acting on the development of AIS.Conclusions: Consequently, this study mapped a profile of plasma protein, found the differentially expressed protein in AIS, which indicating that plasma exosomes, as a novel biomarker with high specificity, could be associated with the severity of AIS.

An Increase in Liver Polyamine Concentration Contributes to the Tryptophan-Induced Acute Stimulation of Rat Hepatic Protein Synthesis

Tryptophan has a unique role as a nutritional signaling molecule that regulates protein synthesis in mouse and rat liver. However, the mechanism underlying the stimulating actions of tryptophan on hepatic protein synthesis remains unclear. Proteomic and metabolomic analyses were performed to identify candidate proteins and metabolites likely to play a role in the stimulation of protein synthesis by tryptophan. Overnight-fasted rats were orally administered L-tryptophan and then sacrificed 1 or 3 h after administration. Four differentially expressed protein spots were detected in rat liver at 3 h after tryptophan administration, of which one was identified as an ornithine aminotransferase (OAT) precursor. OAT is the main catabolic enzyme for ornithine, and its expression was significantly decreased by tryptophan administration. The concentration of ornithine was increased in the liver at 3 h after tryptophan administration. Ornithine is a precursor for polyamine biosynthesis. Significantly increased concentrations of polyamines were found in the liver at 3 h after administration of tryptophan. Additionally, enhanced hepatic protein synthesis was demonstrated by oral administration of putrescine. We speculate that the increase in ornithine level through suppression of OAT expression by tryptophan administration may lead to accelerated polyamine synthesis, thereby promoting protein synthesis in the liver.

Comparative proteome analysis of the spinal dural arteriovenous fistula arterial draining vein with label-free quantitative proteomics

Background: Spinal dural arteriovenous fistula (SDAVF) is the most common spinal vascular shunt lesion. Although pathological changes in the SDAVF draining vein (SDAVF-DV) have been elucidated, protein changes remain enigmatic. We investigated protein changes in the SDAVF-DV.Methods: Three SDAVF-DV samples were collected, and superficial temporal artery (STA) and superficial temporal vein (STV) samples were used as controls. After quantification and enzymolysis of the proteins, label-free quantitative proteomics was performed, and the peptide mixture was fractionated and analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify the differentially expressed proteins. Bioinformatics analysis of the differentially expressed proteins was also performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network analyses.Results: Compared with the STA, the SDAVF-DV had 195 upregulated proteins and 303 downregulated proteins. GO analysis showed that the most differential GO terms in each category were the adenylate cyclase-modulating G protein-coupled receptor signalling pathway, U6 snRNP and SH3 domain binding. KEGG pathway analysis showed that the most differentially expressed protein pathway was focal adhesion. Compared with the STV, the SDAVF-DV had 158 upregulated proteins and 362 downregulated proteins. GO analysis showed that the most differential GO terms in each category were lamellipodium assembly, U6 snRNP, and SH3 domain binding. KEGG pathway analysis showed that the most differentially expressed protein pathway was dilated cardiomyopathy. The PPI analysis revealed PPIs among the top 300 proteins.Conclusions: We demonstrated that the SDAVF-DV showed specific protein expression changes under long-period venous hypertension. The results of the present study will provide insights into the pathogenesis of SDAVF formation at the protein level. The proteomic results provide a scientific foundation for further study to explore the pathophysiological mechanism of SDAVF.