Absolute Quantitation Analysis Research Articles

Quantitative proteomic analysis reveals the mechanisms of polymyxin B toxicity to Escherichia coli

Polymyxin B is increasingly employed all over the world to treat patients who affected by multidrug-resistant Gram-negative bacteria. Although the mechanism of resistance to polymyxin B is well known, the metabolic role of bacteria in stress response to polymyxin B remains an important task and may help to better understand polymyxin B-related stress response. In this study, the proteome changes of Escherichia coli (E. coli) continuously induced in concentrations of 1.0 mg/L and 10.0 mg/L polymyxin B were revealed. Compared to E. coli (PMB0), E. coli exposed to polymyxin B at 1.0 mg/L (PMB1) and 10.0 mg/L (PMB10) resulted in 89 and 314 differentially expressed proteins (DEPs), respectively. Such differences related to fatty acid degradation, quorum sensing and two-component regulatory system pathways. Based on absolute quantitative (iTRAQ) proteomics analysis, this study comprehensively studied the changes of E. coli proteome in culture with concentrations of 1.0 mg/L and 10.0 mg/L polymyxin B through confocal laser scanning microscopy observation, cell viability detection and reactive oxygen species analysis. The results showed that E. coli cultured at concentration of 10.0 mg/L polymyxin B increased the expression levels of multidrug-resistant efflux transporters and efflux pump membrane transporters, which might further improve the pathogens of polymyxin B-resistant bacteria lastingness and evolution. It has emerged globally to resist polymyxin B. The reuse of polymyxin B should be aroused public attention to avoid causing more serious environmental pollution. These findings could provide new insights into polymyxin B-related stress.

Chinese Liquor Fermentation: Identification of Key Flavor-Producing Lactobacillus spp. by Quantitative Profiling with Indigenous Internal Standards.

Identifying the functional microbes in spontaneous food fermentation is important for improving food quality. To identify the key flavor producers in Chinese liquor fermentation, we propose a novel quantitative microbiome profiling method that uses indigenous internal standards to normalize high-throughput amplicon sequencing results. We screened Lactobacillus acetotolerans and Lactobacillus jinshani as indigenous internal standards based on their high distribution frequencies and relative abundances. After determining the absolute abundance of indigenous internal standards using quantitative PCR with species-specific primers, the liquor-fermented bacterial community and its dynamics were better characterized by internal standards normalization. Based on quantitative microbiome profiling, we identified that Lactobacillus was a key flavor producer correlated with eight flavor compounds. Metatranscriptomic analysis indicated that Lactobacillus was active in transcribing genes involving the biosynthesis of flavor compounds and their precursors. This work has developed a novel and extensible absolute quantification method for microbiota that will alleviate concerns in the statistical analyses based on relative microbiome profiling, and shed insights into the function of Lactobacillus in food fermentation. It can potentially be applied to other microbial ecology studies.IMPORTANCE In this study, we developed a novel strategy using indigenous internal standards to normalize the high-throughput amplicon sequencing results. We chose two Lactobacillus species as indigenous internal standards and characterized the absolute abundance of the bacterial community. Further, we identified Lactobacillus as the key flavor producer using quantitative microbiome profiling combined with multivariate statistics and metatranscriptomic analysis. This work developed a novel strategy for absolute quantitative abundance analysis of microbiota and expanded our understanding of the role of Lactobacillus in food fermentation.

Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization

The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-l-lysine, o,o’-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2–1000 pmol for parent amino acids, 80 fmol–20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o’-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid.

Discovering Therapeutic Protein Targets for Bladder Cancer Using Proteomic Data Analysis.

Bladder cancer accounts for almost 54% of urinary system cancer and is the second most frequent cause of death in genitourinary malignancies after prostate cancer. About 70% of bladder tumors are non-muscle-invasive, and the rest are muscle-invasive. Recurrence of the tumor is the common feature of bladder cancer. Chemotherapy is a conventional treatment for MIBC, but it cannot improve the survival rate of these patients sufficiently. Therefore, researchers must develop new therapies. Antibody-based therapy is one of the most important strategies for the treatment of solid tumors. Selecting a suitable target is the most critical step for this strategy. The aim of this study is to detect therapeutic cell surface antigen targets in bladder cancer using data obtained by proteomic studies. Isobaric tag for relative and absolute quantitation (iTRAQ) analysis had identified 131 overexpressed proteins in baldder cancer tissue and reverse-phase proteomic array (RPPA) analysis had been done for 343 tumor tissues and 208 antibodies. All identified proteins from two studies (131+208 proteins) were collected and duplicates were removed (331 unique proteins). Gene ontology study was performed using gene ontology (GO) and protein analysis through evolutionary relationships (PANTHER) databases. The Human Protein Atlas database was used to search the protein class and subcellular location of membrane proteins obtained from the PANTHER analysis. Membrane proteins that could be suitable therapeutic targets for bladder cancer were selected. These included: Epidermal growth factor receptor (EGFR), Her2, Kinase insert domain receptor (KDR), Heat shock protein 60 (HSP60), HSP90, Transferrin receptor (TFRC), Activin A Receptor Like Type 1 (ACVRL1), and cadherin 2 (CDH2). Monoclonal antibodies against these proteins or their inhibitors were used for the treatment of different cancers in preclinical and clinical trials. These monoclonal antibodies and inhibitor molecules and also their combination can be used for the treatment of bladder cancer.

ITRAQ-based proteomics reveals the potential mechanism of fluoride-induced myocardial contraction function damage.

Fluorosis is a worldwide public health problem, and its adverse effects on the heart have been confirmed by many studies. Abnormal myocardial contractions are often associated with impairment of cardiac function as a cause or consequence. We designed two-part experiments to search for biomarkers and clarify the underlying molecular mechanism of fluoride on myocardial contraction. First, we used Pressure-volume Loop analysis to evaluate changes in myocardial function indexes with multiple fluoride exposure levels in mice (0, 30, 70, and 150mg/L) exposed for 4 weeks. The results showed that fluoride exposure affects the heart pump function and reduces cardiac contractility. Then, we established a rat model of fluoride exposure (0, 30, 60, and 90mg/L) for 6 months to carry out proteomic analysis of fluoride-induced myocardial contractile injury. Hematoxylin-eosin (H&E) staining was used to determine the severity of myocardial injury, and myocardial tissue samples were submitted for isobaric tags for relative and absolute quantitation (ITRAQ) analysis. A total of 1607 proteins were successfully identified with 294 differentially expressed proteins (DEPs) in fluoride treated groups. According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, 12 DEPs were confirmed to be involved in pathways related to myocardial contraction. Furthermore, we constructed a protein-protein interaction (PPI) network for these 12 core DEPs to illustrate the role and location of each DEP in the myocardial contraction pathway. The results of this study are helpful for identify a potential mechanism and biomarkers of fluoride-induced myocardial contraction function damage, moreover, which can provide a new insight into the heart toxicity of fluoride in animals at the proteomics level.

Ribonucleoside Diphosphate Reductase Plays an Important Role in Patulin Degradation by Enterobacter cloacae subsp. dissolvens.

Patulin contamination is a worldwide concern due to its significant impact on human health. Several yeast strains have been screened for patulin biodegradation; however, little information is available on bacterial strains and their mechanism of degradation. In the present study, we isolated a bacterial strain TT-09 and identified it as Enterobacter cloacae subsp. dissolvens based on the BioLog system and 16S rDNA phylogenetic analysis. The strain was demonstrated to be able to transform patulin into E-ascladiol. Isobaric tags for relative and absolute quantitation and reverse transcription quantitative polymerase chain reaction analyses provided evidence that ribonucleoside diphosphate reductase (NrdA), an important enzyme involved in DNA biosynthesis, plays a crucial role in patulin degradation. Deletion of nrdA resulted in a total loss in the ability to degrade patulin in TT-09. These results indicate a new function for NrdA in mycotoxin biodegradation. The present study provides evidence for understanding a new mechanism of patulin degradation and information that can be used to develop new approaches for managing patulin contamination.

Screening infant spasmodic differentially expressed protein based on iTRAQ technique

Objective To screen serum biomarkers of infantile spasm by iTRAQ technique. Methods Fifteen cases of infants with infantile spasm and 15 cases of healthy infants were selected to find the plasma differential proteins by isobaric tag for relative and absolute quantitation(iTRAQ) technique and analysis of the biological information, then candidate proteins with significant differences were screened out. Results Compared with the control group, 59 different proteins were obtained in the experimental group with a multiple of 1.2 times, which were involved in biological processes such as cytoskeleton organization, integrin activation and maintenance of protein location in cells.Five disease-related protein markers were selected and discussed. Conclusion Differences in protein expression were found in plasma between infants with infantile spasm and normal controls.It may provide a new direction for the research of infant spasm.The research of cytoskeleton protein may become the breach of the infant spasm pathogenesis.New potential biomarkers, such as serine/threonine protein kinase and dopamine beta hydroxylase may provide a new direction for diagnosis and treatment of infant spasm. Key words: Infantile spasm; Isobaric tag for relative and absolute quantitation; Biomarker

Comparison of the protein acetylome of endothelial cells upon shear flow and resveratrol treatment.

Posttranslational acetylation/deacetylation known as the acetylome is important in regulating protein activity. Shear flow (SF) and resveratrol (RSV) are two stimuli that represent physical and chemical signal separately. The acetylome co-regulated by these two stimuli remain unclear. Human umbilical cord vein endothelial cells (HUVECs) were subjected to either SF of 12 dynes/cm² or 10 μM RSV. The purified acetylated peptides were labeled by isobaric tags for relative and absolute quantitation (iTRAQ) analysis. The signaling cascades of the identified acetylome were predicted by ingenuity pathway analysis (IPA). Co-immunoprecipitation was applied to confirm the acetylation status of proteins. Five groups of proteins showed an increased acetylation upon SF and RSV treatment. After algorithm, 628 proteins with increased acetylation and 22 proteins with decreased acetylation were identified in the SF acetylome. For the acetylome regulated by RSV, 145 proteins with increased acetylation and 23 proteins with decreased acetylation were identified. Compared these two acetylomes, 129 proteins with increased acetylation and 2 proteins with decreased acetylation were co-regulated by both SF and RSV treatments. IPA analysis showed that this co-regulated acetylome was involved in heat shock response, and the signals of eNOS, STAT3, JAK/STAT and ERK/MAPK. Co-immunoprecipitation analysis further confirmed the acetylated status of mitochondrial HSP60 and mitochondrial citrate synthase. This study indicated that physical signal is more complicated than chemical signal in the case of acetylome. The co-regulated proteins are worthy for further study in discussing synergetic effect between physical and chemical signal in cardioprotection.

Insights on bio-degumming of kenaf bast based on metagenomic and proteomics

BackgroundMicrobes play important roles in kanef-degumming. This study aims at identifying the key candidate microbes and proteins responsible for the degumming of kenaf bast (Hibiscus cannabinus). Kenaf bast was cut into pieces and immersed into microbia fermentation liquid collected from different sites. Fermentation liquid samples were collected at 0, 40, 110 and 150 h and then subjected to the 16S/18S rRNA sequencing analysis and isobaric tag for relative and absolute quantitation (iTRAQ) analysis. The microbial (bacterial and fungal) diversity and the differentially expressed proteins/peptides (DEPs) were identified.ResultsWith the prolonged degumming time, the weight loss rate increased, the bacterial diversity was decreased. [Weeksellaceae], Enterobacteriaceae and Moraxellaceae were rapidly increased at 0~40 h, and then decreased and were gradually replaced by Bacteroidaceae from 40 h to 150 h. Similarly, Chryseobacterium and Dysgonomonas were gradually increased at 0~110 h and then decreased; Acinetobacter and Lactococcus were increased at 0~40 h, followed by decrease. Bacteroides was the dominant genus at 150 h. Sequencing 18S rRNA-seq showed the gradually decreased Wallemia hederae and increased Codosiga hollandica during degumming. iTRAQ data analysis showed Rds1, and pyruvate kinase I was decreased and increased in the kanef-degumming, respectively. Other DEPs of ferredoxin I, superoxide dismutase and aconitatehydratase were identified to be related to the Glyoxylate and dicarboxylate metabolism (ko00630).ConclusionsBacteria including Chryseobacterium, Dysgonomonas, Acinetobacter, Lactococcus and Bacteroidesand fungi like Wallemia hederae and Codosiga hollandica are key candidate microbes for kanef degumming.

Protein expression profile changes of lung tissue in patients with pulmonary hypertension.

BackgroundPulmonary hypertension occurs in approximately 1% of the global population, and the prognosis for such patients may be poor. However, the mechanisms underlying the development of this disease remain unclear. Thus, understanding the development of pulmonary hypertension and finding new therapeutic targets and approaches are important for improved clinical outcomes.MethodsLung tissue specimens were collected from six patients with atrial septal defect and pulmonary hypertension (all women, with a mean age of 46.5 ± 4.7 years, and their condition could not be corrected with an internal medical occlusion device) and from nine control patients with lung cancer who underwent lobectomy (six men and three women, with a mean age of 56.7 ± 1.7 years). Isobaric tags for relative and absolute quantitation and liquid chromatography tandem mass spectrometry analyses were used to detect protein expression levels.ResultsWe found 74 significantly upregulated and 88 significantly downregulated differentially expressed proteins between control and pulmonary hypertensive lung tissue specimens. Gene ontology analyses identified the top 20 terms in all three categories, that is, biological process, cellular component, and molecular function. Kyoto Encyclopedia of Genes and Genomes and protein–protein interaction analyses determined the top 10 signaling pathways and found that the six hub proteins associated with the differentially expressed upregulated proteins (PRKAA1, DHPR, ACTB, desmin, ACTG1, and ITGA1) were all involved in hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and dilated cardiomyopathy.ConclusionOur results identified protein expression profile changes in lung tissue derived from patients with pulmonary hypertension, providing potential new biomarkers for clinical diagnosis and prognosis for patients with pulmonary hypertension and offering candidate protein targets for future therapeutic drug development.

Exploration of targets regulated by miR-125b in porcine adipocytes.

MicroRNA (miRNA) has been proved to play a key role in lipid metabolism. In our previous study, miR-125b was validated to be differentially expressed in preadipocytes and adipocytes, which was also proved to involve in lipid metabolism. To explore the comprehensive targets of miR-125b in adipocytes, isobaric tag for relative and absolute quantitation (iTRAQ) analysis was performed to obtain differentially expressed proteins in adipocytes comparing negative control (NC) and miR-125b mimic, combining with digital gene expression (DGE) profiling of mRNA incorporated into RNA-induced silencing complex (RISC) pulled down by biotinylated miR-125b mimic and targets prediction of miR-125b by three algorithms, acyl-CoA dehydrogenase short chain (ACADS) and mitochondrial trans-2-enoyl-CoA reductase (MECR) were screened out as miR-125b direct targets. Luciferase reporter assay further validated that miR-125b mimic significantly inhibited the luciferase activity by targeting wild type (WT) 3'-UTR compared with NC. qPCR analysis of ACADS and MECR mRNA from adipose tissues of miR-125b knockout (KO) mice further confirmed the inhibition of miR-125b on ACADS and MECR expressions. Here we report miR-125b play a vital role in maintaining homeostasis of fatty acid metabolism by targeting key enzyme ACADS and MECR in the process of fatty acid elongation and degradation.

Proteomic Studies for the Investigation of γ-Globin Induction by Decitabine in Human Primary Erythroid Progenitor Cultures.

Reactivation of γ-globin is considered a promising approach for the treatment of β-thalassemia and sickle cell disease. Therapeutic induction of γ-globin expression, however, is fraught with lack of suitable therapeutic targets. The aim of this study was to investigate the effects that treatment with decitabine has on the proteome of human primary erythroid cells from healthy and thalassemic volunteers, as a means of identifying new potential pharmacological targets. Decitabine is a known γ-globin inducer, which is not, however, safe enough for clinical use. A proteomic approach utilizing isobaric tags for relative and absolute quantitation (iTRAQ) analysis, in combination with high-pH reverse phase peptide fractionation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), was employed to investigate the effects of decitabine treatment. Bioinformatics analysis making use of the Database for Annotation, Visualization and Integrated Discovery (DAVID) was employed for functional annotation of the 192 differentially expressed proteins identified. The data are available via ProteomeXchange with identifier PXD006889. The proteins fall into various biological pathways, such as the NF-κB signaling pathway, and into many functional categories including regulation of cell proliferation, transcription factor and DNA binding, protein stabilization, chromatin modification and organization, and oxidative stress proteins.

Quantitative analysis of proteins related to chemoresistance to paclitaxel and carboplatin in human SiHa cervical cancer cells via iTRAQ.

ObjectiveThis study aimed to identify proteins related to paclitaxel and carboplatin chemoresistance in cervical cancer.MethodsQuantitative proteomic analysis was performed on normal SiHa cells and those treated with paclitaxel and carboplatin for 14 days, with isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to identify related processes and differentially expressed proteins.ResultsA total of 67 and 96 differentially expressed proteins were identified in the paclitaxel- and carboplatin- treated groups, respectively. GO and KEGG enrichment analyses identified 53 (43 upregulated and 10 downregulated) and 85 differentially expressed proteins (70 upregulated and 15 downregulated) in the paclitaxel- and carboplatin-treated groups, respectively. The cell counting kit-8 results revealed that APOA1 was overexpressed in both the paclitaxel- and carboplatin- resistant SiHa cells compared with the control cells. Immunohistochemistry showed that APOA1 was highly expressed in the paclitaxel- and carboplatin- resistant squamous cell carcinoma of the cervix.ConclusionThis study is the first to use iTRAQ to identify paclitaxel- and carboplatin- resistance proteins in cervical cells. We identified several proteins previously unassociated with paclitaxel and carboplatin resistance in cervical cancer, thereby expanding our understanding of paclitaxel and carboplatin resistance mechanisms. Moreover, these findings indicate that the APOA1 protein could serve as a potential marker for monitoring and predicting paclitaxel and carboplatin resistance levels.

Isobaric tags for relative and absolute quantitation-based quantitative proteomic analysis of X-linked inhibitor of apoptosis and H2AX in etoposide-induced renal cell carcinoma apoptosis.

Background:X-linked inhibitor of apoptosis (XIAP) is a vital factor in the anti-apoptosis mechanism of tumors and is highly expressed in renal cell carcinoma (RCC). However, the mechanism through which XIAP regulates DNA damage repair is unknown. This study investigated the regulatory mechanism of XIAP in etoposide-induced apoptosis in two Caki-1 cell lines with high or low XIAP expression.Methods:The two cell lines were established using RNA interference technology. The differentially expressed proteins in the two cell lines were globally analyzed through an isobaric tags for relative and absolute quantitation-based quantitative proteomics approach. Proteomic analysis revealed 255, 375, 362, and 5 differentially expressed proteins after 0, 0.5, 3, and 12 h of drug stimulation, respectively, between the two cell lines. The identified differentially expressed proteins were involved in numerous biological processes. In addition, the expression of histone proteins (H1.4, H2AX, H3.1, H3.2, and H3.3) was drastically altered, and the effects of XIAP silencing were accompanied by the marked downregulation of H2AX. Protein-protein interactions were assessed and confirmed through immunofluorescence and Western blot analyses.Results:The results suggested that XIAP may act as a vital cell signal regulator that regulates the expression of DNA repair-related proteins, such as H2AX, and influences the DNA repair process.Conclusions:Given these functions, XIAP may be the decisive factor in determining the sensitivity of RCC cell apoptosis induction in response to chemotherapeutic agents.

MTORC1 Mediates Lysine-Induced Satellite Cell Activation to Promote Skeletal Muscle Growth.

As the first limiting amino acid, lysine (Lys) has been thought to promote muscle fiber hypertrophy by increasing protein synthesis. However, the functions of Lys seem far more complex than that. Despite the fact that satellite cells (SCs) play an important role in skeletal muscle growth, the communication between Lys and SCs remains unclear. In this study, we investigated whether SCs participate directly in Lys-induced skeletal muscle growth and whether the mammalian target of rapamycin complex 1 (mTORC1) pathway was activated both in vivo and in vitro to mediate SC functions in response to Lys supplementation. Subsequently, the skeletal muscle growth of piglets was controlled by dietary Lys supplementation. Isobaric tag for relative and absolute quantitation (iTRAQ) analysis showed activated SCs were required for longissimus dorsi muscle growth, and this effect was accompanied by mTORC1 pathway upregulation. Furthermore, SC proliferation was governed by medium Lys concentrations, and the mTORC1 pathway was significantly enhanced in vitro. After verifying that rapamycin inhibits the mTORC1 pathway and suppresses SC proliferation, we conclude that Lys is not only a molecular building block for protein synthesis but also a signal that activates SCs to manipulate muscle growth via the mTORC1 pathway.

Identification of urinary candidate biomarkers of cisplatin-induced nephrotoxicity in patients with carcinoma

Nephrotoxicity limits the use of cisplatin (CP) in cancer therapy; however, current clinical measures of renal health do not reflect low degrees of kidney injury. Therefore, discovering new biomarkers for CP-induced acute kidney injury (AKI) is essential for monitoring kidney health during therapy. To identify novel candidate biomarkers in urine for reduced renal function due to CP therapy, we conducted a pilot study on cancer patients eligible for CP treatment. Urine from 30 patients was collected before (baseline) and after 3d of intravenous CP infusion. Urine samples were subjected to Isobaric Tag for Relative Absolute Quantitation (iTRAQ) analysis. Biological roles and pathways for the proteins with altered urine concentrations were identified using bioinformatic tools ITRAQ analysis detected 1411 proteins, 12 of which showed significantly altered levels. Growth differentiation factor-15 (GDF15), leucine-rich alpha-2-glycoprotein 1 (LRG1), and secreted phosphoprotein 1 (SPP1/ osteopontin, OPN) were identified as potential candidate markers by proteomic analysis and were validated by ELISA in another 30 patients and in a CP-induced AKI mouse model. Therefore, GDF15, LRG1, and SPP1 may be applied as novel candidate urinary markers of kidney injury after cisplatin treatment. These findings may facilitate the development of new methods to monitor kidney function, particularly in CP-based chemotherapy. SignificanceCisplatin is pivotal for cancer treatment; however, nephrotoxicity limits its use. Clinical measures for renal health are not reflective of early signs of acute kidney injury. Thus, new indicators of the state of renal health following cisplatin treatment will have to be discovered. This study reports the use of proteomics to mine for candidate markers of kidney injury in the urine of patients undergoing treatment with cisplatin. Results were experimentally validated using patient urine and a mouse model of cisplatin-induced acute kidney injury. The novel candidate biomarkers reported in this study may be used for the non-invasive monitoring of renal health and in mitigating the side-effects of cisplatin during the course of cancer treatment.

Dissection of the molecular targets and signaling pathways of Guzhi Zengsheng Zhitongwan based on the analysis of serum proteomics

BackgroundGuzhi Zengsheng Zhitongwan (GZZSZTW) is an effective formula of traditional Chinese herbal medicine and has been widely applied in the treatment of joint diseases for many years. The aim of this study was to dissect the molecular targets and signaling pathways of Guzhi Zengsheng Zhitongwan based on the analysis of serum proteomics.MethodsThe Chinese herbs of GZZSZTW were immersed in 5 l distilled water and boiled with reflux extraction method. The extract was filtered, concentrated and freeze-dried. The chemical profile of GZZSZTW extract was determined by high-performance lipid chromatography (HPLC). The 7-week old Sprague-Dawley (SD) rats in GZZSZTW groups were received oral administration at doses of 0.8, 1.05, and 1.3 g/kg per day and the rats in blank group were fed with drinking water. Serum samples were collected from the jugular veins. Primary chondrocyte viability was evaluated by CCK-8 assay. A full spectrum of the molecular targets and signaling pathways of GZZSZTW were investigated by isobaric tags for relative and absolute quantitation (iTRAQ) analysis and a systematic bioinformatics analysis accompanied with parallel reaction monitoring (PRM) and siRNA validation.ResultsGZZSZTW regulated a series of functional proteins and signaling pathways responsible for cartilage development, growth and repair. Functional classification analysis indicated that these proteins were mainly involved in the process of cell surface dynamics. Pathway analysis mapped these proteins into several signalling pathways involved in chondrogenesis, chondrocyte proliferation and differentiation, and cartilage repair, including hippo signaling pathway, cGMP-PKG signaling pathway, cell cycle and calcium signaling pathway. Protein–protein interaction analysis and siRNA knockdown assay identified an interaction network consisting of TGFB1, RHO GTPases, ILK, FLNA, LYN, DHX15, PKM, RAB15, RAB1B and GIPC1.ConclusionsOur results suggest that the effects of GZZSZTW on treating joint diseases might be achieved through the TGFB1/RHO interaction network coupled with other proteins and signaling pathways responsible for cartilage development, growth and repair. Therefore, the present study has greatly expanded our knowledge and provided scientific support for the underlying therapeutic mechanisms of GZZSZTW on treating joint diseases. It also provided possible alternative strategies for the prevention and treatment for joint diseases by using traditional Chinese herbal formulas.

Comparative Proteome Analysis of Epicardial and Subcutaneous Adipose Tissues from Patients with or without Coronary Artery Disease.

Background and Aims Owing to its unique anatomical structure and metabolism, epicardial adipose tissue (EAT) has attracted amount of attention in coronary artery disease (CAD) research. Here, we analyzed differences in proteome composition in epicardial (EAT) and subcutaneous adipose tissues (SAT) from patients with or without CAD. Methods EAT and SAT samples were collected from 6 CAD patients and 6 non-CAD patients. Isobaric Tagging for Relative and Absolute Quantitation (iTRAQ) analysis combined with liquid chromatography tandem-mass spectrometry (LC-MS/MS) was performed to identify the differentially expressed proteins. Results In total, 2348 proteins expressed in EAT and 2347 proteins expressed in SAT were separately identified. 385 differentially expressed proteins were found in EAT and 210 proteins were found in SAT in CAD patients compared to non-CAD patients. Many proteins differentially expressed in EAT of CAD patients were involved in biological functions associated with CAD development such as cell-to-cell signaling and interaction, inflammatory response, and lipid metabolism. Differential expressions of proteins (MMP9, S100A9, and clusterin) in EAT or SAT were involved in several signaling pathways such as mitochondrial dysfunction, acute phase inflammation, and LXR/RXR activation, which was confirmed by western blotting, and similar results were obtained. Conclusions The largest profiles of differentially expressed proteins in EAT and SAT between CAD patients and non-CAD patients were identified. The significant signal pathways, mitochondrial dysfunction, and LXR/RXR activation, which differential proteins were involved in, were firstly found to play roles in EAT of CAD patients, and clusterin was firstly found to be upregulated in EAT of CAD patients.

Ac25 in Autographa californica multiple nucleopolyhedrovirus was crucial for progeny budded virion production

To analyze the effect of Ac25 on the proliferation of AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus) progeny virus and its function in virogenic stroma. AcMNPV is a model of baculovirus and is the most widely studied baculovirus. Ac25, as a single-stranded DNA-binding protein, is involved in viral genomic DNA replication. Viral proliferation assay showed that AcMNPV progeny virus could not be produced when Ac25 was knocked out, which indicated it was crucial for BV production. Absolute quantitative PCR analysis indicated that Ac25 was able to promote replication of the AcMNPV genome in host Sf9 cells. It was also found that Ac25 could increase the transcription level of 38k and vp39 late expression genes, and inhibit host cell proliferation. Ac25 is highly accumulated in the nucleus and promotes progeny virus production by stimulating viral genome replication and up-regulating the expression of late genes. Two potential applications of vAc-Ac25-EGFP were proposed: an improved bac-to-bac eukaryotic protein expression systems and biopesticides.

ITRAQ-based quantitative analysis reveals proteomic changes in Chinese cabbage (Brassica rapa L.) in response to Plasmodiophora brassicae infection

Clubroot disease is one of the major diseases affecting Brassica crops, especially Chinese cabbage (Brassica rapa L. ssp. pekinensis), which is known to be highly susceptible to the disease. In this study, the obligate biotrophic protist Plasmodiophora brassicae Woronin was used to infect the roots of Chinese cabbage seedlings. The disease symptoms were noticeable at 28 and 35 days after inoculation (DAI) in the susceptible (CM) line. Using isobaric tags for relative and absolute quantitation (iTRAQ) analysis, a total of 5,003 proteins of differential abundance were identified in the resistant/susceptible lines, which could be quantitated by dipeptide or polypeptide segments. Gene ontology (GO) analysis indicated that the differentially expressed proteins (DEPs) between the susceptible (CM) and resistant (CCR) lines were associated with the glutathione transferase activity pathway, which could catalyze the combination of glutathione and other electrophilic compounds to protect plants from disease. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEPs may be significantly enriched cytokinin signaling or arginine biosynthesis pathways, both of which are responses to stimuli and are plant defense reactions. The cytokinins may facilitate cell division in the shoot, resulting in the hypertrophy and formation of galls and the presentation of typical clubroot symptoms. In this study, the proteomic results provide a new perspective for creating germplasm resistance to P. brassicae, as well as a genetic basis for breeding to improve Chinese cabbage.