iTRAQ Proteomics Research Articles

Identification of targets of JS-K against HBV-positive human hepatocellular carcinoma HepG2.2.15 cells with iTRAQ proteomics

JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors. We have discovered that JS-K was effective against Hepatitis B virus (HBV)-positive HepG2.2.15 cells. This study used iTRAQ to identify differentially expressed proteins following JS-K treatment of HepG2.2.15 cells. Silenced Transgelin (shTAGLN-2.15) cells were constructed, and the cell viability was analyzed by the CCK8 assay after treatment with JS-K. There were 182 differentially expressed proteins in JS-K treated-HepG2.2.15 cells; 73 proteins were up-regulated and 109 proteins were down-regulated. These proteins were categorized according to GO classification. KEGG enrichment analysis showed that Endocytosis, Phagosome and Proteoglycans were the most significant pathways. RT-PCR confirmed that the expression levels of TAGLN, IGFBP1, SMTN, SERPINE1, ANXA3, TMSB10, LGALS1 and KRT19 were significantly up-regulated, and the expression levels of C5, RBP4, CHKA, SIRT5 and TRIM14 were significantly down-regulated in JS-K treated-HepG2.2.15 cells. Western blotting confirmed the increased levels of USP13 and TAGLN proteins in JS-K treated-HepG2.2.15 cells. Molecular docking revealed the binding of JS-K to TAGLN and shTAGLN-2.15 cells were resistant to JS-K cytotoxicity, suggesting that TAGLN could be an important target in JS-K anti-HBV-positive liver cancer cells. These proteomic findings could shed new insights into mechanisms underlying the effect of JS-K against HBV-related HCC.

Elevated Serum FGG Levels Prognosticate and Promote the Disease Progression in Prostate Cancer.

Castration-resistant prostate cancer (CRPC) threatens the health of men in general and no effective therapeutics currently exists for the treatment of CRPC. It is therefore of great importance to find a novel molecule that can be a biomarker and a therapeutic target for CRPC. First, we found that the serum fibrinogen gamma (FGG) levels in patients with CRPC were significantly higher than those with localized prostate cancer (PCa) through iTRAQ proteomics and ELISA experiments. Immunohistochemistry, quantitative real-time polymerase chain reaction and western blot also showed an increase of FGG expression in CRPC tissues and cells. Then we proved the proliferation, invasion and migration ability of CRPC cells were significantly reduced after FGG knockdown. The number of apoptotic cells increased at least sixfold after FGG silencing, and was observed in conjunction with an upregulation of p53, caspase 3, clea-caspase 3, and Bax, and a downregulation of Bcl2 and survivin. FGG knockdown in DU145 cells resulted in smaller xenografts than control cells in a mouse model. and we established that FGG is modulated by IL-6 which was increased in CRPC patients via phosphorylation of STAT3. The data suggests that FGG may be a potential therapeutic target and prognostic marker for CRPC.

Proteomic analysis of hemocyte reveals the immune regulatory mechanisms after the injection of corticosteroid-releasing hormone in mud crab Scylla Paramamosain

Corticosteroid-releasing hormone (CRH) is a crucial neuroendocrine-immune factor regulating the immune response of Scylla paramamosain. To understand the regulatory mechanisms of CRH in S. paramamosain, the hemolymph of S. paramamosain with injection of CRH (1.5 ng/crab) at 24 h were chosen to perform proteomic analysis in this study. Furthermore, quantitative real-time PCR (RT-PCR) method was used to validate the accuracy of proteomic data at 24 h after CRH injection. The proteomic data showed that 255 DEPs were identified, in which 231 and 24 were up- or down-regulated, respectively. Besides, the results of enriched pathways showed that the DEPs were involved in signaling pathways, cellular immunity, humoral immunity and the response of immune related processes. These results revealed that CRH promoted the activation of signal transduction, regulated immune systems and antioxidation, and enhanced the immune related processes (such as protein synthesis, protein transport, carbohydrate mobilization and energy redistribution). These findings will benefit to foster the understanding on the effects of glucocorticoids on neuroendocrine-immune (NEI) networks of crustacean, and supply a substantial material and foundation for further researching of the NEI response. SignificanceCorticotrophin-releasing hormone (CRH) is a 41-amino acid neuropeptide and has been preliminarily studied in aquatic animals. CRH can regulate many important physiological activities comprising protein synthesis, energy metabolism, growth, breeding and behavior in fish, which play an important roles in neuroendocrine-immune (NEI) regulatory network of fish. The neuroendocrine system of crustacean has a primary research, that inspired by fish NEI network. Despite the research on the neuroendocrine system in crustacean has rapidly increased in recent years, our understanding of the regulation between neuroendocrine system and immune system in crustacean is still limited. The research on the strategy of NEI network in crustaceans becomes a significant issue.In the present study, the isobaric tags for relative and absolute quantification (iTRAQ) technology approach were applied to examine the NEI network of Scylla Paramamosain. control group and treatment group (CRH: 1.5 ng/crab) were settled for the iTRAQ experiment, and sampled at 24 h after CRH injection. The study aimed to gain knowledge on the immune response in Scylla Paramamosain after CRH injection and identify related differentially expressed proteins (DEPs) of the crab. The results of this study provide a preliminary resource for analysis the immune mechanism for crustaceans.In general, our work represents the first report of the utilization of the iTRAQ proteomics method for the study of NEI regulatory network in Scylla Paramamosain after CRH injection. We identified a number of DEPs involved in diverse pathways including immune signaling pathways, cellular immunity, humoral immunity, immune related process. These results demonstrated a very complex network involving immune and multiple related metabolic pathways in hemocytes of Scylla Paramamosain and will be of great value in understanding the crab neuroendocrine-immune immune mechanism.

Combination of RNA-Seq transcriptomics and iTRAQ proteomics reveal the mechanism involved in fresh-cut yam yellowing

The aim of this study was to examine the regulation of transcriptomics and proteomics related to the yellowing of fresh-cut yams after storage. The comparison of yellow fresh-cut yam (YFY) vs. white fresh-cut yam (control) revealed 6894 upregulated and 6800 downregulated differentially expressed genes along with 1277 upregulated and 677 downregulated differentially expressed proteins. The results showed that the total carotenoids, flavonoids, and bisdemethoxycurcumin in YFY were higher than in the control due to the significant up-regulation of critical genes in the carotenoid biosynthesis pathway, flavonoid biosynthesis pathway, and stilbenoid, diarylheptanoid, and gingerol biosynthesis pathway. In addition, the tricarboxylic acid cycle and phenylpropanoid biosynthesis were both enhanced in YFY compared to the control, providing energy and precursors for the formation of yellow pigments. The results suggest that the synthesis of yellow pigments is regulated by critical genes, which might explain the yellowing of fresh-cut yam after storage.

Discovery of Novel Protein Biomarkers in Urine for Diagnosis of Urothelial Cancer Using iTRAQ Proteomics

Urothelial carcinoma (UC) is the ninth most prevalent malignancy worldwide. Noninvasive and efficient biomarkers with high accuracy are imperative for the surveillance and diagnosis of UC. CKD patients were enrolled as a control group in this study for the discovery of highly specific urinary protein markers of UC. An iTRAQ-labeled quantitative proteomic approach was used to discover novel potential markers. These markers were further validated with 501 samples by ELISA assay, and their diagnostic accuracies were compared to those of other reported UC markers. BRDT, CYBP, GARS, and HDGF were identified as novel urinary UC biomarkers with a high discrimination ability in a population comprising CKD and healthy subjects. The diagnostic values of the four novel UC markers were better than that of a panel of well-known or FDA-approved urinary protein markers CYFR21.1, Midkine, and NUMA1. Three of our discovered markers (BRDT, HDGF, GARS) and one well-known marker (CYFR21.1) were finally selected and combined as a marker panel having AUC values of 0.962 (95% CI, 0.94-0.98) and 0.860 (95% CI, 0.83-0.89) for the discrimination between UC and normal groups and UC and control (healthy + CKD) groups, respectively.

Novel Long Noncoding RNA 005620 Induces Epirubicin Resistance in Triple-Negative Breast Cancer by Regulating ITGB1 Expression.

Triple-negative breast cancer (TNBC) is often treated with anthracyclines (e.g., epirubicin or doxorubicin), but very little is known about anthracycline resistance, especially epirubicin resistance in TNBC. To identify novel long noncoding RNAs (lncRNAs) involved in epirubicin resistance in TNBC, we established a new TNBC MDA-MB-231 cell line that was resistant to epirubicin (Epi-R). A total of 12 differentially expressed lncRNAs were identified using RNA sequencing analysis of Epi-R cells. Among these lncRNAs, we found a novel intronic lncRNA, lnc005620, was highly expressed in Epi-R cells and human TNBC tissues. Further gain- and loss-of-function studies demonstrated that lnc005620 played an oncogenic role and partially abrogated the effects of epirubicin on TNBC cells. Using iTRAQ proteomics analysis, we found that three members of the integrin family, integrin β4, integrin β1 and integrin α6, were all upregulated in Epi-R MDA-MB-231 cells. Integrin β1, encoded by the ITGB1 gene, was validated to be a downstream target of lnc005620 in Epi-R MDA-MB-231 cells. Our study demonstrates that novel lnc005620 promotes TNBC progression and chemoresistance to epirubicin via integrin β1 both in vitro and in vivo and provides a promising therapeutic target for TNBC patients in terms of enhancing the benefits of epirubicin treatment.

ITRAQ proteome analysis of the antifungal mechanism of citral on mycelial growth and OTA production in Aspergillus ochraceus.

Aspergillus ochraceus causes food spoilage and produces mycotoxin ochratoxin A (OTA) during storage of agricultural commodities. In this study, citral was used to inhibit A. ochraceus growth and OTA accumulation, proteomic analysis was employed to verify the mechanism of citral. Citral was found to significantly inhibit fungal growth and mycotoxin production in A. ochraceus. Specifically, 75, 125, 150 and 200 μL L-1 citral suppressed mycelial growth by 33%, 46%, 50% and 100%, respectively. Additionally, 75 μL L-1 citral inhibited OTA accumulation by 25%. Proteomic analysis was performed to elucidate the inhibitory mechanism of citral on mycelial growth and OTA production at subinhibitory concentrations (75 μL L-1 ). Proteomics analysis identified 2646 proteins in A. ochraceus fc-1, of which 218 were differentially expressed between control and 75 μL L-1 citral treatment samples. Differentially expressed proteins were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of biological process, cellular component and molecular function terms. Potential factors affecting mycelial growth and OTA production were analysed, and OTA production was revealed to be a complex process involving many associated factors related to various processes including nutrient intake, sterol biosynthesis, ribosome biogenesis, energy metabolism, oxidative stress and amino acid metabolism. In addition, citral at 75 μL L-1 down-regulated OTA biosynthetic genes including pks and nrps, but slightly up-regulated the global regulatory factors veA, velB and laeA. The findings further demonstrate the potential of citral for the preservation of grains and other agricultural products, and provide new insight into its antifungal mechanisms at subinhibitory concentrations. © 2021 Society of Chemical Industry.

Integrated RNA-seq and Proteomic Studies Reveal Resource Reallocation towards Energy Metabolism and Defense in Skeletonema marinoi in Response to CO2 Increase.

Rising atmospheric CO2 concentrations are causing ocean acidification (OA) with significant consequences for marine organisms. Because CO2 is essential for photosynthesis, the effect of elevated CO2 on phytoplankton is more complex and the mechanism is poorly understood. Here we applied RNA-seq and iTRAQ proteomics to investigate the impacts of CO2 increase (from ∼400 to 1000 ppm) on the temperate coastal marine diatom Skeletonema marinoi We identified 32,389 differentially expressed genes (DEGs) and 1,826 differentially expressed proteins (DEPs) from elevated CO2 conditions, accounting for 48.5% of total genes and 25.9% of total proteins we detected, respectively. Elevated pCO2 significantly inhibited the growth of S marinoi, and the 'omic' data suggested that this might be due to compromised photosynthesis in the chloroplast and raised mitochondrial energy metabolism. Furthermore, many genes/proteins associated with nitrogen metabolism, transcriptional regulation, and translational regulation were markedly up-regulated, suggesting enhanced protein synthesis. In addition, S marinoi exhibited higher capacity of ROS production and resistance to oxidative stress. Overall, elevated pCO2 seems to repress photosynthesis and growth of S marinoi, and through massive gene expression reconfiguration induce cells to increase investment in protein synthesis, energy metabolism and antioxidative stress defense, likely to maintain pH homeostasis and population survival. This survival strategy may deprive this usually dominant diatom in temperate coastal waters of its competitive advantages in acidified environments.Importance Rising atmospheric CO2 concentrations are causing ocean acidification with significant consequences for marine organisms. Chain-forming centric diatoms of Skeletonema is one of the most successful groups of eukaryotic primary producers with widespread geographic distribution. Among the recognized 28 species, S. marinoi can be a useful model for investigating the ecological, genetic, physiological, and biochemical characteristics of diatoms in temperate coastal regions. In this study, we found that the elevated pCO2 seems to repress photosynthesis and growth of S. marinoi, and through massive gene expression reconfiguration induce cells to increase investment in protein synthesis, energy metabolism and antioxidative stress defense, likely to maintain pH homeostasis and population survival. This survival strategy may deprive this usually dominant diatom in temperate coastal waters of its competitive advantages in acidified environments.

Bacterial ClpP Protease Is a Potential Target for Methyl Gallate.

Methyl gallate (MG) is an effective microbicide with great potential application in the integrated management of plant diseases and an important potential drug for clinical application. However, its target remains unknown. This study conducted a transposon sequencing (Tn-seq) under MG treatment in plant pathogenic bacterium Ralstonia solanacearum. Tn-seq identified that the mutation of caseinolytic protease proteolytic subunit gene clpP significantly increased the resistance of R. solanacearum to MG, which was validated by the in-frame gene deletion. iTRAQ (isobaric tags for relative and absolute quantitation) proteomics analysis revealed that chemotaxis and flagella associated proteins were the major substrates degraded by ClpP under the tested condition. Moreover, sulfur metabolism-associated proteins were potential substrates of ClpP and were upregulated by MG treatment in wild-type R. solanacearum but not in clpP mutant. Furthermore, molecular docking confirmed the possible interaction between MG and ClpP. Collectively, this study revealed that MG might target bacterial ClpP, inhibit the activity of ClpP, and consequently disturb bacterial proteostasis, providing a theoretical basis for the application of MG.

Amynthas corticis genome reveals molecular mechanisms behind global distribution

Earthworms (Annelida: Crassiclitellata) are widely distributed around the world due to their ancient origination as well as adaptation and invasion after introduction into new habitats over the past few centuries. Herein, we report a 1.2 Gb complete genome assembly of the earthworm Amynthas corticis based on a strategy combining third-generation long-read sequencing and Hi-C mapping. A total of 29,256 protein-coding genes are annotated in this genome. Analysis of resequencing data indicates that this earthworm is a triploid species. Furthermore, gene family evolution analysis shows that comprehensive expansion of gene families in the Amynthas corticis genome has produced more defensive functions compared with other species in Annelida. Quantitative proteomic iTRAQ analysis shows that expression of 147 proteins changed in the body of Amynthas corticis and 16 S rDNA sequencing shows that abundance of 28 microorganisms changed in the gut of Amynthas corticis when the earthworm was incubated with pathogenic Escherichia coli O157:H7. Our genome assembly provides abundant and valuable resources for the earthworm research community, serving as a first step toward uncovering the mysteries of this species, and may provide molecular level indicators of its powerful defensive functions, adaptation to complex environments and invasion ability.

ITRAQ analysis reveals the effect of gabD and sucA gene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis

Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.

ITRAQ-based proteomic profiling reveals protein alterations after traumatic brain injury and supports thyroxine as a potential treatment

Traumatic brain injury (TBI) is a primary cause of disability and death across the world. Previously, RNA analysis was widely used to study the pathophysiological mechanisms underlying TBI; however, the relatively low correlation between the transcriptome and proteome revealed that RNA transcription abundance does not reliably predict protein abundance, which led to the emergence of proteomic research. In this study, an iTRAQ proteomics approach was applied to detect protein alterations after TBI on a large scale. A total of 3937 proteins were identified, and 146 proteins were significantly changed after TBI. Moreover, 23 upregulated proteins were verified by parallel reaction monitoring (PRM), and fold changes in 16 proteins were consistent with iTRAQ outcomes. Transthyretin (Ttr) upregulation has been demonstrated at the transcriptional level, and this study further confirmed this at the protein level. After treatment with thyroxine (T4), which is transported by Ttr, the effects of T4 on neuronal histopathology and behavioral performance were determined in vivo (TBI + T4 group). Brain edema was alleviated, and the integrity of the blood brain barrier (BBB) improved. Escape latency in the Morris water maze (MWM) declined significantly compared with the group without T4 treatment. Modified neurological severity scores (mNSS) of the TBI + T4 group decreased from day 1 to day 7 post-TBI compared with the TBI + saline group. These results indicate that T4 treatment has potential to alleviate pathologic and behavioral abnormalities post-TBI. Protein alterations after T4 treatment were also detected by iTRAQ proteomics. Upregulation of proteins like Lgals3, Gfap and Apoe after TBI were reversed by T4 treatment. GO enrichment showed T4 mainly affected intermediate filament organization, cholesterol transportation and axonal regeneration. In summary, iTRAQ proteomics provides information about the impact of TBI on protein alterations and yields insight into underlying mechanisms and pathways involved in TBI and T4 treatment. Finally, Ttr and other proteins identified by iTRAQ may become potential novel treatment targets post-TBI.

Analysis of Differentially Expressed Proteins Involved in miR-449a for Hepatocellular Carcinoma by iTRAQ Proteomics.

To investigate the relationship between down-regulation of miR-449a and prognosis of hepatocellular carcinoma (HCC) and to elucidate the potential target proteins of miR-449a. The expression of miR-449a in 142 HCC tissues was detected by RT-PCR. The correlation between down-regulation of miR-449a and prognosis of HCC was statistically analyzed during clinical follow-up. The Bel-7042 HCC cell line in miR-449a-mimic and miR-449a-inhihbitor model was used, and the potential target protein of miR-449a was screened by isobaric tags for relative and absolute quantitation (iTRAQ) technology. miR-449a was significantly down-regulated in HCC tissues, which was significantly associated with post-operative metastasis (p < 0.0001) and recurrence (p < 0.0001). The median overall survival time in the low-expression group of miR-449a was significantly lower than that of the high-expression group (19 months vs. 37 months, p = 0.001). In addition, the tumor-free survival time of the low-expression group was significantly lower than that of the high-expression group (14 months vs. 24 months, p = 0.001). iTRAQ analysis screened out 137 differential proteins, among which 88 were up-regulated and 49 were down-regulated. GO clustering, KEGG pathway, and STRING analysis were performed, suggesting that these differential proteins have complicated functions, such as ATP binding, metal ion binding, RNA binding, human papillomavirus infection, and Epstein-Barr virus infection. miR-449a was negatively correlated with HCC prognosis. The differential proteins screened by iTRAQ can provide the basis for studying the target proteins regulated by miR-449a and understanding the pathogenesis of HCC.

The Screening of Ovarian Candidate Proteomics Biomarkers of Dolang Sheep at Different Physiological Periods Based on iTRAQ Technology

To screen the differentially expressed ovary proteins in Dolang sheep with oestrum, anestrus and pregnancy using isobaric tag for relative and absolute quantitation (iTRAQ) technology, which will provide the basis for the research of regulation and identification of oestrum, the diagnosis of early pregnancy. In total, we identified 4847 proteins, 470 of which were significantly different proteins. Compared with anestrus, 18 proteins were up and 102 proteins were down in oestrum, 20 proteins were up and 60 proteins were down in pregnancy; Compared with oestrum, 50 proteins were up and 24 proteins were down during pregnancy. Such as cyclin-Y isoformX1, cleavages timulation factor subunit 1 isoform X1, follistatin-related protein 1 isoform X2, nicotinate phosphoribosyl transferase isoform X1 at up-regulated, hemopexin isoform X1, apolipoprotein A-II, nucleophosmin at down-regulated, which were related to hormones generation, regulation of oestrum, zygote production and apoptosis, early embryo attachment and embryo development. Of which, cyclin-Y isoform X1 and nicotinate phosphoribosyl transferase isoform X1 significant increase in pregnancy, compared with oestrum and anestrus. The study&nbsp;identified&nbsp;different proteins at oestrum and pregnancy of Dolang sheep based on iTRAQ proteomics technology, which provide new thoughts and methods to further exploring the candidate biomarkers of sheep estrous identification and diagnosis of early gestation.

Adverse effects of Δ9-tetrahydrocannabinol on neuronal bioenergetics during postnatal development.

Ongoing societal changes in views on the medical and recreational roles of cannabis increased the use of concentrated plant extracts with a Δ9-tetrahydrocannabinol (THC) content of more than 90%. Even though prenatal THC exposure is widely considered adverse for neuronal development, equivalent experimental data for young age cohorts are largely lacking. Here, we administered plant-derived THC (1 or 5 mg/kg) to mice daily during P5–P16 and P5–P35 and monitored its effects on hippocampal neuronal survival and specification by high-resolution imaging and iTRAQ proteomics, respectively. We found that THC indiscriminately affects pyramidal cells and both cannabinoid receptor 1+ (CB1R)+ and CB1R– interneurons by P16. THC particularly disrupted the expression of mitochondrial proteins (complexes I–IV), a change that had persisted even 4 months after the end of drug exposure. This was reflected by a THC-induced loss of membrane integrity occluding mitochondrial respiration and could be partially or completely rescued by pH stabilization, antioxidants, bypassed glycolysis, and targeting either mitochondrial soluble adenylyl cyclase or the mitochondrial voltage-dependent anion channel. Overall, THC exposure during infancy induces significant and long-lasting reorganization of neuronal circuits through mechanisms that, in large part, render cellular bioenergetics insufficient to sustain key developmental processes in otherwise healthy neurons.

Effect and mechanism of total flavonoids of Lichi Semen on CCl_4-induced liver fibrosis in rats, and prediction of Q-marker

This paper was to investigate the effect of total flavonoids of Lichi Semen(TFL) on carbon tetrachloride(CCl_4)-induced liver fibrosis in rats, analyze and predict its mechanism of action and potential quality markers(Q-marker). Firstly, male SD rats were taken and injected subcutaneously with a 40% CCl_4-vegetable oil solution twice a week for 8 consecutive weeks to establish a rat model of liver fibrosis. The rats with liver fibrosis were randomly divided into model group, silybin group(43.19 mg·kg~(-1)), Fuzheng Huayu Capsules group(462.75 mg·kg~(-1)), and TFL groups(100 mg·kg~(-1) and 25 mg·kg~(-1)), with normal rats as a blank group, 10 rats in each group. Except for the blank group, the rats in the other groups were subcutaneously injected with 40% CCl_4-vegetable oil solution of a maintenance dose, once a week. The rats in various treatment groups received corresponding doses of drugs, while the rats in the blank group and model group received the same volume of normal saline once a day for 4 weeks. At the end of the experiment, blood was collected from the abdominal aorta and the liver tissues were collected. The levels of total bilirubin(TBiL), direct bilirubin(DBiL), indirect bilirubin(IBiL), alanine aminotransferase(ALT), and aspartate aminotransferase(AST) in serum were detected by using an automatic biochemical detector. Masson staining was used to observe the histopathological changes of rat liver. Then, the chemical compositions of TFL were collected, and the action targets of these chemical compositions were predicted through SWISS database and reverse molecular docking server(DRAR-CPI). After screening of disease targets of liver fibrosis by Gene Cards database, the protein-protein interaction was analyzed with use of STRING database, and GO(gene ontology) analysis and KEGG(Kyoto encyclopedia of genes and genomes) enrich analysis were also carried out. Moreover, an iTRAQ proteomics technology was used to determine protein expression in liver tissues of rats in TFL, model and blank groups to verify the targets. Furthermore, Cytoscape software was used to establish and visualize the network of chemical components, targets and pathways, and predict the potential Q-marker of TFL. The results showed that the levels of TBiL, DBiL, IBiL, ALT, and AST in the model group were significantly higher than those in the blank normal group(P&lt;0.05), and the above levels in the treatment groups were lower than those in the model group, but with no significant differences. Masson staining showed that the liver damage and the degree of fibrosis were severe in the model group, and were relieved to different degrees in the treatment groups. Then, 74 chemical components were screened, which could act on 865 targets such as EGFR and SRC, participating in the regulation of cancer pathways, PI3 K-Akt signaling pathway, HIF-1 signaling pathway and other signaling pathways closely related to liver fibrosis. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin showed the highest correlation with liver fibrosis-related targets and pathways. Proteomics results showed that a total of 18 proteins among the 45 proteins predicted by internet pharmacology were identified, among which 6 proteins were significantly expressed, including 5 up-regulated proteins and 1 down-regulated protein. The protein expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1 was significantly returned to a normal state in the TFL treatment groups. In conclusion, TFL may demonstrate the anti-hepatic fibrosis and potential hepatoprotective effects by regulating the expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1, which may be associated with the regulation of multiple signaling pathways related to liver fibrosis such as PI3 K-Akt pathway. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin could be regarded as potential Q-markers of TFL for quality control.

PSIX-29 JAK2-STAT3 Pathway Mediated Satellite Cell Apoptosis to Govern Skeletal Muscle Growth with Lysine

Abstract Apoptosis is programmed cell death that can be stimulated by external stress or nutrition restrictions. Lysine (Lys) is an essential amino acid for pig growth, and the relationship between Lys deficiency caused apoptosis and inhibition of skeletal muscle growth remains unknown. The objective of this study was to investigate whether apoptosis could be regulated by Lys supplementation and the potential mechanism. In current work, 30 male Duroc × Landrace × Large weaned piglets were divided randomly into 3 groups: control group (Lys 1.30%), Lys deficiency group (Lys 0.86%), and Lys rescue group (Lys 0.86%, 0-14d; 1.30%,15–28 d). The experiment lasted for 28 days, and on the morning of 29 d, piglets were slaughtered to collect samples. Isobaric tag for relative and absolute quantification (iTRAQ) proteomics analysis of the longissimus dorsi muscle showed that Janus family tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT) pathway was involved in Lys deficiency-induced apoptosis and inhibited skeletal muscle growth. Meanwhile, western blotting results of the longissimus dorsi muscle demonstrated that Lys deficiency caused apoptosis (P &amp;lt; 0.05) with the JAK2-STAT3 pathway inhibition (P &amp;lt; 0.05). Interestingly, apoptosis was suppressed (P &amp;lt; 0.05), and the JAK2-STAT3 pathway was reactivated (P &amp;lt; 0.05) after Lys re-supplementation in longissimus dorsi muscle. In addition, results of satellite cells (SCs) isolated from the longissimus dorsi muscle of 5-day-old Landrace piglets showed that Lys deficiency-induced apoptosis (P &amp;lt; 0.05) was mediated by the JAK2-STAT3 pathway inhibition (P &amp;lt; 0.05). Moreover, the JAK2-STAT3 pathway was reactivated (P &amp;lt; 0.05) by Lys re-supplementation and suppressed apoptosis in SCs (P &amp;lt; 0.05), and this effect was blocked (P &amp;lt; 0.05) after SCs treated with AG-490 (a specific inhibitor of JAK2). Collectively, Lys inhibited apoptosis in SCs to govern skeletal muscle growth via the JAK2-STAT3 pathway.

PSIX-30 mTORC1 Sensing Glutamate Signal to Promote Porcine Intestinal Development by Accelerating Intestinal Stem Cell Expansion

Abstract Mechanistic target of rapamycin complex 1 (mTORC1) coordinates cell growth and metabolism with environmental cues, such as amino acids, growth factors, and energy. Glutamate (Glu) is a primary metabolic fuel for the intestinal epithelium and extensively involves numerous physiological processes. Crypt intestinal stem cells (ISCs) driven intestinal epithelial renewal that needs a continuous energy supplement. However, the effects of Glu on the expansion of porcine ISCs and intestinal epithelial development remain unclear. Therefore, the objective of this study was to investigate the underlying mechanism that Glu promotes intestinal development. Firstly, a total of 14 weaned piglets (Duroc × Landrace × Large White) with similar body weight (BW) were randomly allocated into the control group, and the 1.0% Glu group with 7 replicates per group and 1 piglet per replicate. The experiment lasts for 21 days. The results showed that dietary Glu increased small intestinal weights, jejunal villus height, and the ratio of the villus height to the crypt depth. Moreover, dietary Glu promoted the proliferation and differentiation of intestinal epithelial cells. Subsequently, iTRAQ proteomics screening indicated that intestinal mTORC1 signaling may participate in Glu-stimulated intestinal epithelial development, which was confirmed by Western blotting. Meanwhile, the IR/IRS/PI3K/Akt pathway and EGFR/ERK pathway are the upstream of Glu-induced mTORC1 signaling activation, which verified in IPEC-J2 cell line and intestinal organoids. Furthermore, the in vivo and ex vivo crypt ISCs experiments showed that Glu accelerated ISC expansion as increased intestinal organoid forming efficiency and budding efficiency. Glu also promoted ISC self-renew and differentiated into various functional cells (enterocytes, goblet cells, enteroendocrine cells, Paneth cells). In summary, mTORC1 integrated Glu signal stimulated ISC expansion and ultimately promoted epithelial development.

Proteomic Profiling of the Liver, Hepatic Lymph Nodes, and Spleen of Buffaloes Infected with Fasciola gigantica.

In the present study, we used an isobaric tag for relative and absolute quantitation (iTRAQ) proteomics technology to characterize the differentially expressed proteins (DEPs) in the liver, hepatic lymph nodes (hLNs), and spleen of buffaloes infected with Fasciola gigantica (F. gigantica). We also used the parallel reaction monitoring (PRM) method to verify the expression levels of the DEPs in the three infected tissues. At three days post-infection (dpi), 225, 1821, and 364 DEPs were detected in the liver, hLNs, and spleen, respectively. At 42 dpi, 384, 252, and 214 DEPs were detected in the liver, hLNs, and spleen, respectively. At 70 dpi, 125, 829, and 247 DEPs were detected in the liver, hLNs, and spleen, respectively. Downregulation of metabolism was prominent in infected livers at all time points, and upregulation of immune responses was marked in the hLNs during early infection (three dpi); however, no changes in the immune response were detected at the late stages of infection (42 and 70 dpi). Compared to the hLNs, there was no significant upregulation in the levels of immune responses in the infected spleen. All the identified DEPs were used to predict the subcellular localization of the proteins, which were related to extracellular space and membrane and were involved in host immune responses. Further PRM analysis confirmed the expression of 18 proteins. These data provide the first simultaneous proteomic profiles of multiple organs of buffaloes experimentally infected with F. gigantica.

Expression and characterization of the serum proteome from yak induced into estrus by improved nutrition

Regulation of estrus plays a crucial role in the livestock industry. It is reported that providing better nutrition can induce early estrus in animals. However, little is known about the major endocrine and physiological mechanisms that could enhance estrus in anestrus animals. Hence in the current research two different groups of yaks, non-breeding season (February–June, NBS) estrus yaks as the experiment group and breeding season (July–September, BS) estrus animals as the control group were compared using the isobaric tags for relative and absolute quantitation (iTRAQ) technique. Study displayed that cold season supplementation significantly improved growth performance, serum biochemical indicators and reproductive hormone concentrations in yaks. We also identified 25 differentially expressed proteins in yak serum using iTRAQ proteomics. Go and KEGG analysis indicated that calcium signaling pathway and beta-alanine metabolism may be candidate pathways for seasonal estrus induced by nutrition. Differential protein expression was validated using parallel reaction monitoring (PRM). The results of this study initially identified A2M, IGF2, A1BG and APOA1 as candidate proteins for seasonal estrus induced by nutrition. Altogether, In conclusion, our results show that providing additional nutrients in the cold season can improve yak productivity and reproductive efficiency, and provide a new reference.