iTRAQ Proteomics Research Articles

ITRAQ proteome analysis reveals the underling mechanisms of foliage zinc-spraying to improve photosynthetic capacity and seed yields of Peaonia ostii ‘Fengdan’

Zinc (Zn) deficiency is a significant nutritional limitation to crop yield globally, particularly in calcareous soil environments. Tree peony of Peaonia ostii ‘Fengdan’ is regarded as an oil crop due to its seeds rich in alpha-linolenic acid, a beneficial compound for health promotion. However, low seed yield remains a primary challenge in attaining sufficient seed oil from tree peony. In this study, Zn fertilization was applied to soil or foliage of P. ostii ‘Fengdan’ in the growth period before fruit development. Our findings reveal that foliar Zn-spraying, as opposed to soil application, proves to be a more effective method for augmenting seed yield, Zn accumulation and photosynthetic capacity in ‘Fengdan’. Comparative analyses of the leaf proteome of ‘Fengdan’ using iTRAQ profiling under foliar Zn-spraying identified 115 differentially expressed proteins (DEPs), including 36 upregulated proteins, which likely contribute to the observed increase in seed yields of ‘Fengdan’ caused by foliage Zn-spraying. Specifically, Zn2+ stimulation of phosphatidylinositol signaling initiates a cascade of metabolic regulations. Firstly, ATP synthesis promotes leaf photosynthetic capacity, facilitated by improved sucrose metabolism through upregulated pullulanase and 1,4-alpha-glucan-branching enzyme. Furthermore, lipid synthesis and transport are facilitated by upregulated lipoyl synthase and plastid lipid-associated proteins. Additionally, DEPs involved in secondary metabolism are upregulated in the production of various metabolites conducive to ‘Fengdan’ growth. Overall, our results demonstrate that foliage Zn-spraying enhances seed yield in P. ostii ‘Fengdan’ by elevating Zn content and secondary metabolite synthesis in leaves, thereby augmenting leaf photosynthetic capacity and lipid synthesis. This study provides an effective way to increase seed yield of tree peony by exogenous Zn application.

Proteomics Analysis of the Protective Effect of Polydeoxyribonucleotide Extracted from Sea Cucumber (Apostichopus japonicus) Sperm in a Hydrogen Peroxide-Induced RAW264.7 Cell Injury Model.

Sea cucumber viscera contain various naturally occurring active substances, but they are often underutilized during sea cucumber processing. Polydeoxyribonucleotide (PDRN) is an adenosine A2A receptor agonist that activates the A2A receptor to produce various biological effects. Currently, most studies on the activity of PDRN have focused on its anti-inflammatory, anti-apoptotic, and tissue repair properties, yet relatively few studies have investigated its antioxidant activity. In this study, we reported for the first time that PDRN was extracted from the sperm of Apostichopus japonicus (AJS-PDRN), and we evaluated its antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and hydroxyl radical scavenging assays. An in vitro injury model was established using H2O2-induced oxidative damage in RAW264.7 cells, and we investigated the protective effect of AJS-PDRN on these cells. Additionally, we explored the potential mechanism by which AJS-PDRN protects RAW264.7 cells from damage using iTRAQ proteomics analysis. The results showed that AJS-PDRN possessed excellent antioxidant activity and could significantly scavenge DPPH, ABTS, and hydroxyl radicals. In vitro antioxidant assays demonstrated that AJS-PDRN was cytoprotective and significantly enhanced the antioxidant capacity of RAW264.7 cells. The results of GO enrichment and KEGG pathway analysis indicate that the protective effects of AJS-PDRN pretreatment on RAW264.7 cells are primarily achieved through the regulation of immune and inflammatory responses, modulation of the extracellular matrix and signal transduction pathways, promotion of membrane repair, and enhancement of cellular antioxidant capacity. The results of a protein-protein interaction (PPI) network analysis indicate that AJS-PDRN reduces cellular oxidative damage by upregulating the expression of intracellular selenoprotein family members. In summary, our findings reveal that AJS-PDRN mitigates H2O2-induced oxidative damage through multiple pathways, underscoring its significant potential in the prevention and treatment of diseases caused by oxidative stress.

ITRAQ proteomics analysis of placental tissue with gestational diabetes mellitus.

Gestational diabetes mellitus is an endocrine and metabolic disorder that appears for the first time during pregnancy and causes varying degrees of short- and/or long-term effects on the mother and child. The etiology of the disease is currently unknown and isobaric tags for relative and absolute quantitation proteomics approach, the present study attempted to identify potential proteins in placental tissues that may be involved in the pathogenesis of GDM and adverse foetal pregnancy outcomes. Pregnant women with GDM hospitalised were selected as the experimental group, and pregnant women with normal glucose metabolism as the control group. The iTRAQ protein quantification technology was used to screen the differentially expressed proteins between the GDM group and the normal control group, and the differentially expressed proteins were analysed by GO, KEGG, PPI, etc., and the key proteins were subsequently verified by western blot. Based on the proteomics of iTRAQ, we experimented with three different samples of placental tissues from GDM and normal pregnant women, and the total number of identified proteins were 5906, 5959, and 6017, respectively, which were similar in the three different samples, indicating that the results were reliable. Through the Wayne diagram, we found that the total number of proteins coexisting in the three groups was 4475, and 91 differential proteins that could meet the quantification criteria were strictly screened, of which 32 proteins were up-regulated and 59 proteins were down-regulated. By GO enrichment analysis, these differential proteins are widely distributed in extracellular membrane-bounded organelle, mainly in extracellular exosome, followed by intracellular vesicle, extracellular organelle. It not only undertakes protein binding, protein complex binding, macromolecular complex binding, but also involves molecular biological functions such as neutrophil degranulation, multicellular organismal process, developmental process, cellular component organization, secretion, regulated exocytosis. Through the analysis of the KEGG signaling pathway, it is found that these differential proteins are mainly involved in HIF-1 signaling pathway, Glycolysis/Gluconeogenesis, Central carbon metabolism in cancer, AMPK signaling pathway, Proteoglycans in cancer, Protein processing in endoplasmic reticulum, Thyroid cancer, Alcoholism, Glucagon signaling pathway. This preliminary study helps us to understand the changes in the placental proteome of GDM patients, and provides new insights into the pathophysiology of GDM.

SpSIZ1 from hyperaccumulator Sedum plumbizincicola orchestrates SpABI5 to fine-tune cadmium tolerance.

Sedum plumbizincicola is a renowned hyperaccumulator of cadmium (Cd), possesses significant potential for eco-friendly phytoremediation of soil contaminated with Cd. Nevertheless, comprehension of the mechanisms underpinning its Cd stress response remains constrained, primarily due to the absence of a comprehensive genome sequence and an established genetic transformation system. In this study, we successfully identified a novel protein that specifically responds to Cd stress through early comparative iTRAQ proteome and transcriptome analyses under Cd stress conditions. To further investigate its structure, we employed AlphaFold, a powerful tool for protein structure prediction, and found that this newly identified protein shares a similar structure with Arabidopsis AtSIZ1. Therefore, we named it Sedum plumbizincicola SIZ1 (SpSIZ1). Our study revealed that SpSIZ1 plays a crucial role in positively regulating Cd tolerance through its coordination with SpABI5. Overexpression of SpSIZ1 significantly enhanced plant resistance to Cd stress and reduced Cd accumulation. Expression pattern analysis revealed higher levels of SpSIZ1 expression in roots compared to stems and leaves, with up-regulation under Cd stress induction. Importantly, overexpressing SpSIZ1 resulted in lower Cd translocation factors (Tfs) but maintained relatively constant Cd levels in roots under Cd stress, leading to enhanced Cd stress resistance in plants. Protein interaction analysis revealed that SpSIZ1 interacts with SpABI5, and the expression of genes responsive to abscisic acid (ABA) through SpABI5-dependent signaling was significantly up-regulated in SpSIZ1-overexpressing plants with Cd stress treatment. Collectively, our results illustrate that SpSIZ1 interacts with SpABI5, enhancing the expression of ABA downstream stress-related genes through SpABI5, thereby increasing Cd tolerance in plants.

Differentially Expressed Proteins in the Serum of Elderly Patients Who Experienced Perioperative Neurocognitive Disorders Following Transurethral Resection of the Prostate.

Perioperative neurocognitive disorders (PND) are a group of prevalent neurological complications that often occur in elderly individuals following major or emergency surgical procedures. The etiologies are not fully understood. This study endeavored to investigate novel targets and prediction methods for the occurrence of PND. A total of 229 elderly patients diagnosed with prostatic hyperplasia who underwent transurethral resection of the prostate (TURP) combined with spinal cord and epidural analgesia were included in this study. The patients were divided into two groups, the PND group and non-PND group, based on the Z-score method. According to the principle of maintaining consistency between preoperative and intraoperative conditions, three patients from each group were randomly chosen for serum sample collection. isobaric tags for relative and absolute quantification (iTRAQ) proteomics technology was employed to analyze and identify the proteins that exhibited differential expression in the serum samples from the two groups. Bioinformatics analysis was performed on the proteins that exhibited differential expression. Among the 1101 serum proteins analyzed in the PND and non-PND groups, eight differentially expressed proteins were identified in PND patients. Of these, six proteins showed up-regulation, while two proteins showed down-regulation. Further bioinformatics analysis of the proteins that exhibited differential expression revealed their predominant involvement in cellular biological processes, cellular component formation, as well as endocytosis and phagocytosis Additionally, these proteins were found to possess the RING domain of E3 ubiquitin ligase. The iTRAQ proteomics technique was employed to analyze the variation in protein expression in serum samples from patients with PND and those without PND. This study successfully identified eight proteins that exhibited differential expression levels between the two groups. Bioinformatics analysis indicates that proteins exhibiting differential expression are primarily implicated in the biological processes associated with microtubules. Investigating the microtubule formation process as it relates to neuroplasticity and synaptic formation may offer valuable insights for enhancing our comprehension and potential prevention of PND. Registered (ChiCTR2000028836). Date (20190306).

Extracellular matrix (ECM) pathway involved in skin immune response of Cynoglossus semilaevis upon Vibrio vulnificus infection

Half-smooth tongue sole (Cynoglossus semilaevis) is regarded as a significant commercial marine fish species in China, and frequent outbreaks of vibriosis has led to substantial economic losses. In this study, we investigated the molecular mechanisms underlying the involvement of the extracellular matrix (ECM) signaling pathway in the skin immune response of C. semilaevis infected with Vibrio vulnificus. The results showed that most differentially expressed proteins (DEPs) were identified through iTRAQ proteome analysis, and ten ECM-related proteins were screened at 12 and 36 ​h post-infection (hpi). Notably, several DEPs associated with ECM including HSPG, FN, COCH, LAMB1, LAMC1, TSPN, COL6A1, AMBP, HX and ITGA6 were tested for their expression at the transcriptional level during V. vulnificus infection using qRT-PCR assay. The analysis of protein-protein interaction (PPI) showed that the integrin ITGA6 exhibited obvious interactions among ECM-related DEPs. Furthermore, the spatio-temporal expression of the Csitga6 gene was highest in the skin, gill, muscle, and spleen, but lower in the liver, kidney and intestine. To our knowledge, this is the first report on the involvement of ECM pathway in the skin immune response to V. vulnificus infection, and provides a reference for further study of the ECM mechanism in the mucosal immune response of marine fish.

ITRAQ-based proteomics revealed the acaricidal mechanism of methyl isoeugenol against Dermatophagoides farinae

ABSTRACT Dermatophagoides farinae is a major source of allergens that are the cause of allergic diseases in humans. Methyl isoeugenol (MIE) is a naturally occurring compound in plants and its strong acaricidal toxicity has been established; however, the mechanism behind its acaricidal properties remains unclear. In this study, to investigate the action mechanism of MIE on D. farinae the isobaric tags for relative and absolute quantification (iTRAQ) proteomics method is used. Protein samples were extracted from D. farinae before and after 24 h of MIE treatment for iTRAQ technique analysis. Bioinformatics analysis revealed that most differentially expressed proteins were located in the cytoplasm, impacting cytoplasmic function. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed significant enrichment of the differential proteins in the cellular autophagy signalling pathway. The autophagy-related proteins Autophagy-Related Protein 3 (ATG3) and Cathepsin L (CTSL) were up-regulated, disrupting the autophagic process. We found that the main action mechanism of MIE against D. farinae might be cellular autophagy. The transcriptional validation of six differential proteins demonstrated consistent expression trends with the proteomics data. This study reveals the acaricidal mechanism of MIE for the first time and lays the foundation for further research on D. farinae control.

Serum proteins differentially expressed in gestational diabetes mellitus assessed using isobaric tag for relative and absolute quantitation proteomics.

As a well-known fact to the public, gestational diabetes mellitus (GDM) could bring serious risks for both pregnant women and infants. During this important investigation into the linkage between GDM patients and their altered expression in the serum, proteomics techniques were deployed to detect the differentially expressed proteins (DEPs) of in the serum of GDM patients to further explore its pathogenesis, and find out possible biomarkers to forecast GDM occurrence. To investigation serum proteins differentially expressed in GDM were assessed using isobaric tag for relative and absolute quantitation (iTRAQ) proteomics and bioinformatics analyses. Subjects were divided into GDM and normal control groups according to the IADPSG diagnostic criteria. Serum samples were randomly selected from four cases in each group at 24-28 wk of gestation, and the blood samples were identified by applying iTRAQ technology combined with liquid chromatography-tandem mass spectrometry. Key proteins and signaling pathways associated with GDM were identified by bioinformatics analysis, and the expression of key proteins in serum from 12 wk to 16 wk of gestation was further verified using enzyme-linked immunosorbent assay (ELISA). Forty-seven proteins were significantly differentially expressed by analyzing the serum samples between the GDM gravidas as well as the healthy ones. Among them, 31 proteins were found to be upregulated notably and the rest 16 proteins were downregulated remarkably. Bioinformatic data report revealed abnormal expression of proteins associated with lipid metabolism, coagulation cascade activation, complement system and inflammatory response in the GDM group. ELISA results showed that the contents of RBP4, as well as ANGPTL8, increased in the serum of GDM gravidas compared with the healthy ones, and this change was found to initiate from 12 wk to 16 wk of gestation. GDM symptoms may involve abnormalities in lipid metabolism, coagulation cascade activation, complement system and inflammatory response. RBP4 and ANGPTL8 are expected to be early predictors of GDM.

Mesenchymal Stem Cells Accelerate Recovery of Acetic Acid-Induced Chronic Gastric Ulcer by Regulating Ekt/Akt/TRIM29 Axis.

Chronic gastric ulcer (CGU), a prevalent digestive disease, has a high incidence and is seriously harmful to human health. Mesenchymal stem cells (MSCs) have been proven to have beneficial therapeutic effects in many human diseases. Here, a CGU model induced by acetic acid in mice was used to evaluate the repair effects and potential mechanism of human umbilical cord-derived MSCs (hUC-MSCs) and hUC-MSCs derived conditioned medium (hUC-MSC-CM). We found that hUC-MSCs and hUC-MSC-CM treatment significantly repaired morphological characteristics of CGU, improved proliferation and decreased apoptosis of gastric cells, and promoted the generation of new blood vessels in granulation tissues. In addition, we could detect the homing of MSCs in gastric tissue, and MSCs may differentiate into Lgr5-positive cells. As well as this, in vitro experiments showed that hUC-MSC-CM could promote cell proliferation, stimulate cell cycle progression, and reduce the incidence of apoptosis. The transcriptome of cells and the iTRAQ proteome of gastric tissues suggest that MSCs may play a therapeutic role by increasing the expression of TRIM29. Additionally, it was found that knocking down TRIM29 significantly decreased the ameliorative effects of hUC-MSC-CM on cell apoptosis. As a result of further molecular experiments, it was found that TRIM29 is capable of phosphorylating Erk/Akt in specific cell type. As a whole, it appears that hUC-MSCs can be an effective therapeutic approach for promoting gastric ulcer healing and may exert therapeutic effects in the form of paracrine and differentiation into gastric cells.

Comparative Analysis of Schistosoma japonicum from Pairing-to-Sexual Maturation based on iTRAQ Proteomics

Objective: Schistosomiasis, which is caused by the schistosome worm, poses significant health challenges. Understanding the sexual development and maturation of schistosomes would provide valuable insight for preventing the transmission of schistosomiasis and pathologic damage to the host. Methods: Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics was performed to monitor the dynamic proteomic profiles in Schistosoma japonicum during development from pairing-to-maturation. RNA interference (RNAi) experiments were used to elucidate sex-biased gene function. Results: Of the 2927 identified proteins, 58.6% showed differential expression after comparing sexes and developmental stages. Both male and female worms displayed a similar number of gender-differentially expressed proteins after pairing. However, these proteins exhibited significant gender-specific functions, with reproduction central in females, while males were enriched in metabolic processes. Females exhibited 73% of their time-variant protein expression during 22-26 days post-infection (dpi), while males had 62% during 18-22 dpi, indicating earlier maturation in males. Functional analysis revealed different peptidases expressed during male and female development and maturation. Sex-biased SjU2AF exhaustion led to worm development delay, abnormal reproductive organ development, and death. Conclusions: Comparative proteomics enhances our understanding of mechanisms underlying schistosome maturation and reveals a new potential target for chemotherapy and vaccines.

Exploring the anti-glioma mechanism of the active components of Cortex Periplocae based on network pharmacology and iTRAQ proteomics in vitro.

The active components of Cortex Periplocae (CP) exert antitumor properties in many cancers. However, little is known about their effects on glioma or the related underlying mechanisms. The study investigated the underlying mechanism of CP in treating glioma. The U251 and TG905 cells were treated with an ethanol extract from CP. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8) and a colony formation assay. The flow cytometric analysis was applied to explore the induction of cell cycle arrest and apoptosis. The expression levels of cell cycleand apoptosis-associated proteins were measured with western blot. A network pharmacology method was performed to predict the potential mechanism underlying the effects of the active components of CP on glioma. Then, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics analysis was used to verify the differentially expressed proteins and pathways in order to reveal the underlying mechanisms. Furthermore, to determine the iTRAQ results, 6 candidate proteins were chosen for quantification using parallel reaction monitoring (PRM). The CP extract inhibited the proliferation of U251 and TG905 cells and induced cell cycle arrest and apoptosis. There are 16 active compounds of CP. The antitumor mechanism of CP may be related to the apoptosis pathway, p53 signaling pathway, PI3K-AKT pathway, or transcriptional misregulation in cancer pathway. Six proteins (HSP90AB1, TOP2A, ATP1A1, TGFβ1, ATP1B1, and TYMS) were determined to be key factors involved in regulating CP in glioma. Our research revealed the underlying mechanism of CP in treating glioma using integrated network pharmacology and iTRAQ-based quantitative proteomics technology.

ITRAQ-based proteomics identifies proteins associated with betaine accumulation in Lycium barbarum L

In order to better understand the mechanism of betaine accumulation in Lycium barbarum L. (LBL), we used iTRAQ (Isotope relative and absolute quantitative labeling) proteomics to screen and identify differentially abundant proteins (DAPs) at five stages (S1-young fruit stage, S2-green fruit stage, S3-early yellowing stage, S4-late yellowing stage, S5-ripening stage). A total of 1799 DAPs and 171 betaine-related DAPs were identified, and phosphatidylethanolamine N-methyltransferase (NMT), choline monooxygenase (CMO), and betaine aldehyde dehydrogenase (BADH) were found to be the key enzymes related to betaine metabolism. These proteins are mainly involved in carbohydrates, amino acids and their derivatives, fatty acids, carboxylic acids, photosynthesis and photoprotection, isoquinoline alkaloid biosynthesis, peroxisomes, and glycine, serine, and threonine metabolism. Three of the key enzymes were also up- and down-regulated to different degrees at the mRNA level. The study provide new insights into the of mechanism of betaine accumulation in LBL. SignificanceBetaine, a class of naturally occurring, water-soluble alkaloids, has been found to be widespread in animals, higher plants, and microbes. In addition to being an osmotic agent, betaine has biological functions such as hepatoprotection, neuroprotection, and antioxidant activity. Betaine metabolism (synthesis and catabolism) is complexly regulated by developmental and environmental signals throughout the life cycle of plant fruit maturation. As a betaine-accumulating plant, little has been reported about the regulatory mechanisms of betaine metabolism during the growth and development of Lycium barbarum L. (LBL) fruit. Therefore, this study used iTRAQ quantitative proteomics technology to investigate the abundance changes of betaine-related proteins in LBL fruit, screen and analyze the differential abundance proteins related to betaine metabolism, and provide theoretical references for the in-depth study of the mechanism of betaine metabolism in LBL fruit.

Proteomic analysis reveals oxidative stress-induced activation of Hippo signaling in thiamethoxam-exposed Drosophila

Thiamethoxam (THIA) is a widely used neonicotinoid insecticide. However, the toxicity and defense mechanisms activated in THIA-exposed insects are unclear. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) proteomics technology to identify changes in protein expression in THIA-exposed Drosophila. We found that the antioxidant proteins Cyp6a23 and Dys were upregulated, whereas vir-1 was downregulated, which may have been detoxification in response to THIA exposure. Prx5 downregulation promoted the generation of reactive oxygen species. Furthermore, the accumulation of reactive oxygen species led to the induction of antioxidant defenses in THIA-exposed Drosophila, thereby enhancing the levels of oxidative stress markers (e.g., superoxide dismutase, glutathione S-transferase, and glutathione) and reducing catalase expression. Furthermore, the Hippo signaling transcription coactivator Yki was inactivated by THIA. Our results suggesting that Hippo signaling may be necessary to promote insect survival in response to neonicotinoid insecticide toxicity.

Comparative iTRAQ proteomics identified proteins in fresh and frozen thawed yak spermatozoa

The changes in semen and cryodamage after the cryopreservation process negatively affect sperm function and motility. However, possible proteomic alterations of yak semen during cryopreservation have not yet been achieved. In this study, we compared proteomes of fresh and frozen thawed yak sperm using iTRAQ combined with LC-MS/MS proteome approach. Totally, 2064 proteins were quantitatively identified, including 161 in fresh sperm that showed significant differences compared to frozen thawed sperm. According to the Gene ontology (GO) enrichment analysis, differentially expressed proteins (DEPs) are predominantly associated with spermatogenesis, tricarboxylic acid cycle, ATP synthesis, and differentiation biological process. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEPs were mainly involved in metabolic pathways related to pyruvate metabolism, carbon metabolism, glycolysis/gluconeogenesis, together with the citrate (TCA) cycle. In the analysis of the protein-protein interaction (PPI) network, 15 potential proteins (PDHB, DLAT, PDHA2, PGK1, TP5C1, etc.) that could be related to the sperm quality of the yaks were obtained. Furthermore, 6 DEPs were validated by parallel reaction monitoring (PRM), confirming that the iTRAQ data were reliable. These results indicate that cryopreservation alters the proteome of yak sperm, which is possibly related to cryodamage and fertilization ability.

Glaucomatous aqueous humor vesicles are smaller and differ in composition compared to controls

Cells communicate with each other using vesicles of varying sizes, including a specific repertoire known as exosomes. We isolated aqueous humor (AH)-derived vesicles using two different methods: ultracentrifugation and an exosome isolation kit. We confirmed a unique vesicle size distribution in the AH derived from control and primary open-angle glaucoma (POAG) patients using various techniques, including Nanotracker, dynamic light scattering, atomic force imaging, and electron microscopy. Bonafide vesicle and/or exosome markers were present by dot blot in both control and POAG AH-derived vesicles. Marker levels differed between POAG and control samples, while non-vesicle negative markers were absent in both. Quantitative labeled (iTRAQ) proteomics showed a reduced presence of a specific protein, STT3B, in POAG compared to controls, which was further confirmed using dot blot, Western blot, and ELISA assays. Along the lines of previous findings with AH profiles, we found vast differences in the total phospholipid composition of AH vesicles in POAG compared to controls. Electron microscopy further showed that the addition of mixed phospholipids alters the average size of vesicles in POAG. We found that the cumulative particle size of type I collagen decreased in the presence of Cathepsin D, which normal AH vesicles were able to protect against, but POAG AH vesicles did not. AH alone had no effect on collagen particles. We observed a protective effect on collagen particles with an increase in artificial vesicle sizes, consistent with the protective effects observed with larger control AH vesicles but not with the smaller-sized POAG AH vesicles. Our experiments suggest that AH vesicles in the control group provide greater protection for collagen beams compared to POAG, and their increased vesicle sizes are likely contributing factors to this protection.

ITRAQ proteome analysis of insight into TLR20.2 functions through IFN1 signaling exerts regulatory effects on GCRV replication

Maternal immunization is an efficacious way to protect offspring from pathogen infections. To clarify the immune-enhancing mechanism of maternal immunization in grass carp, the iTRAQ assay was applied to screen differentially expressed proteins (DEPs) among eggs produced from maternal-immunized and non-immunized female grass carp. A total of 138 DEPs were obtained and TLR20.2 was the sole identified consensus DEP among the maternal-immunized and non-immunized groups. In general, the gene and protein expressions of TLR20.2 in the egg cell and early developmental stage embryos could be enhanced by maternal-immunized treatment. The mRNA levels of tlr20.2 were up-regulated in the liver, head kidney, and spleen in response to grass carp reovirus (GCRV) infection. Mechanistically, TLR20.2 functions through the IFN1 signaling and displayed inhibitory functions on GCRV replication by remarkably down-regulation of vp2 and vp7 expressions. The HSP90 was identified to be one of the proteins that interacted with TLR20.2, which coexpressed with TLR20.2 and mainly concentrated around the central veins. HSP90 promotes GCRV replication by inhibiting the irf3, irf7, and ifn1 expressions, which resulted in remarkably increased vp2 and vp7 gene expressions. The HMGB1a and HMGB1b could bind to the TLR20–2 promoter, and showed positive effects on tlr20.2 expression by significantly promoting its promoter activity (P < 0.01). Moreover, HMGB1a and HMGB1b exerted negative effects on HSP90 expression and thus the GCRV replication. Overall, this study provided molecular evidence that maternal immunization treatment protect against GCRV infection through the enhancement of TLR20.2 expression, and the HMGB1-TLR20.2-HSP90-IFN axis confers regulatory effects on GCRV replication. The results are novel findings to help elucidate the resistance-enhancing mechanism in fish species.

Use of iTRAQ-based quantitative proteomic identification of CHGA and UCHL1 correlated with lymph node metastasis in colorectal carcinoma.

Metastatic dissemination of colorectal cancer (CRC), the third most common cancer type, is responsible for CRC deaths. Understanding the transition of lymph node metastasis (LNM) from Stage II to Stage III is beneficial in the prognosis and intervention of CRC. In this study, a quantitative proteomic survey was conducted to investigate the LNM-associated proteins and evaluate the clinicopathological characteristics of these target proteins in CRC. By using the LC-MS/MS iTRAQ technology, we analysed the proteomic changes between LMN II and LMN III. Fresh tumours from the CRC specimens consisting of 12 node-negative (Stage II) and 12 node-positive (Stage III) cases were analysed by LC-MS/MS iTRAQ proteome analysis. Subsequently, tissue microarray with immunohistochemistry staining was conducted to access the clinicopathological characteristics of these proteins in 116 paraffin-embedded CRC samples, each for non-LNM and LNM CRC. To study the effects of the differentially expressed proteins on the potential mechanism, Boyden chamber assay, flow cytometry and shRNA-based assessments were conducted to examine the role of the epithelial-mesenchymal transition (EMT) and the invasiveness of CRC cells and others in vivo xenograft mouse model experiments. Forty-eight proteins were found differentially expressed between non-LNM and LNM CRC tissues. Protein abundances of chromogranin-A (CHGA) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) were observed in node-positive CRC (p < 0.05). Knockdown of CHGA and UCHL1 significantly regulate cancer behaviours of HCT-116, including inhibition of cell migration, invasiveness, cell cycle G1/S arrest and reactive oxygen species (ROS) generation. Mechanistically, the CHGA and UCHL1 inactivation displayed decreased levels of UCH-L1, chromogranin A, β-catenin, cyclin E, twist-1/2, vimentin, MMP-9, N-cadherin and PCNA through the activation of the Rho-GTPase/AKT/NFκB pathways. Histone modification of H3K4 trimethylation of CHGA and UCHL1 promoter were increased to activate their transcription through the signalling transduction such as Rho-GTPase, AKT and NFκB pathways. Our results indicated that UCHL1 and chromogranin A are novel regulators in CRC lymph node metastasis to potentially provide new insights into the mechanism of CRC progression and serve as biomarkers for CRC diagnosis at the metastatic stage.

A modified natural small molecule inhibits triple-negative breast cancer growth by interacting with Tubb3

BackgroundTriple-negative breast cancer (TNBC) is a malignant tumor without specific therapeutic targets and a poor prognosis. Chemotherapy is currently the first-line therapeutic option for TNBC. However, due to the heterogeneity of TNBC, not all of TNBC patients are responsive to chemotherapeutic agents. Therefore, the demand for new targeted agents is critical. β-tubulin isotype III (Tubb3) is a prognostic factor associated with cancer progression, including breast cancer, and targeting Tubb3 may lead to improve TNBC disease control. Shikonin, the active compound in the roots of Lithospermun erythrorhizon suppresses the growth of various types of tumors, and its efficacy can be improved by altering its chemical structure. PurposeIn this work, the anti-TNBC effect of a shikonin derivative (PMMB276) was investigated, and its mechanism was also investigated. Study Design/MethodsThis study combines flow cytometry, immunofluorescence staining, immunoblotting, immunoprecipitation, siRNA silencing, and the iTRAQ proteomics assay to analyze the inhibition potential of PMMB276 on TNBC. In vivo study was performed, Balb/c female murine models with or without the small molecule treatments. ResultsHerein, we screened 300 in-house synthesized analogs of shikonin against TNBC and identified a novel small molecule, PMMB276; it suppressed cell proliferation, induced apoptosis, and arrested the cell cycle at the G2/M phase, suggesting that it could have a tumor suppressive role in TNBC. Tubb3 was identified as the target of PMMB276 using proteomic and biological activity analyses. Meanwhile, PMMB276 regulated microtubule dynamics in vitro by inducing microtubule depolymerization and it could act as a tubulin stabilizer by a different process than that of paclitaxel. Moreover, suppressing or inhibiting Tubb3 with PMMB276 reduced the growth of breast cancer in an experimental mouse model, indicating that Tubb3 plays a significant role in TNBC progression. ConclusionThe findings support the therapeutic potential of PMMB276, a Tubb3 inhibitor, as a treatment for TNBC. Our findings might serve as a foundation for the utilization of shikonin and its derivatives in the development of anti-TNBC.

Salivary Biomarkers to Differentiate between Streptococcus pneumoniae and Influenza A Virus-Related Pneumonia in Children.

Community-acquired pneumonia (CAP) is common among children and can be fatal in certain conditions. In children, CAP can be caused by viral or bacterial infections. Identification of pathogens can help select appropriate therapeutic strategies. Salivary analysis may be a potential diagnostic tool because it is noninvasive, patient-friendly, and easy to perform in children. A prospective study was conducted in children with pneumonia admitted to a hospital. Salivary samples from patients with definite Streptococcus pneumoniae and influenza A strains were used for gel-free (isobaric tag for relative and absolute quantitation (iTRAQ)) proteomics. No statistically significant difference was detected in salivary CRP levels between Streptococcus pneumoniae and influenza A pneumonia in children. Several potential salivary biomarkers were identified using gel-free iTRAQ proteomics to differentiate pneumonia from Streptococcus pneumoniae or influenza A virus infections in pediatric patients. ELISA validated that Streptococcus pneumoniae group has a higher abundance of salivary alpha 1-antichymotrypsin than those in the influenza A group. Whether these salivary biomarkers can be used to distinguish other bacteria from viral pneumonia requires further verification.

Proteomic Analyses of Autologous Chondrocyte Implantation Plasma Highlight Cartilage Acidic Protein 1 as a Candidate for Preclinical Screening

Background: Stratification is required to ensure that only patients likely to benefit receive autologous chondrocyte implantation (ACI). It would be advantageous to identify biomarkers to predict ACI outcome that are measurable in blood, avoiding the need for an invasive synovial fluid harvest. Purpose: To assess if proteomic analyses can be used to identify novel candidate blood biomarkers in individuals who respond well or poorly to ACI. Study Design: Controlled laboratory study. Methods: Isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry was used to assess the proteome in plasma pooled from ACI responders (mean Lysholm improvement after ACI, 33; n = 10) or nonresponders (mean, −13; n = 10), collected at the time of surgery for cartilage harvest (stage 1) or implantation of culture-expanded chondrocytes (stage 2). An alternative proteomic method, label-free quantitation liquid chromatography–tandem mass spectrometry, was used to analyze plasma samples (majority matched to iTRAQ) individually. Differentially abundant proteins (±2.0-fold) were analyzed from both proteomic data sets, and markers of interest identified via pooled iTRAQ were validated via immunoassay of individual samples. Results: Protein differences could be detected in the plasma preoperatively between ACI responders and nonresponders (16 proteins; ≥±2.0-fold change; P < .05) using iTRAQ proteomics. The most pronounced plasma proteome shift was evident in response to stage 1 surgery in ACI nonresponders, with 48 proteins being differentially abundant between the procedures. Label-free quantitation liquid chromatography–tandem mass spectrometry analysis of these same plasma samples (nonpooled) resulted in very few proteins being identified that were significantly differentially abundant. However, this work highlighted cartilage acidic protein 1 as being increased preoperatively in nonresponders as compared with responders. Conclusions: This study is the first to use proteomic techniques to profile the plasma of individuals treated with ACI. Despite iTRAQ analysis of pooled plasmas indicating that there are differences in the plasma proteome between responders and nonresponders to ACI, these findings were not replicated when assessed using an alternative nonpooled technique. This study highlights some of the difficulties in profiling the plasma proteome in an attempt to identify novel biomarkers. Regardless, cartilage acidic protein 1 has been identified as a protein candidate, which is detectable in plasma and can predict outcome to ACI before treatment. Clinical Relevance: Candidate plasma protein biomarkers identified in this study have the potential to help determine which patients will be best suited to treatment with ACI.