Bacillus pumilus TS1 alleviates Salmonella Enteritidis-induced intestinal injury in broilers.

BACKGROUND: In the current context of reduced and limited antibiotic use, several pathogens and stressors cause intestinal oxidative stress in poultry, which leads to a reduced feed intake, slow or stagnant growth and development, and even death, resulting in huge economic losses to the poultry breeding industry. Oxidative stress in animals is a non-specific injury for which no targeted drug therapy is available; however, the health of poultry can be improved by adding appropriate feed additives. Bacillus pumilus, as a feed additive, promotes growth and development and reduces intestinal oxidative stress damage in poultry. Heat shock protein 70 (HSP70) senses oxidative damage and repairs unfolded and misfolded proteins; its protective effect has been widely investigated. Mitogen-activated protein kinase/protein kinase C (MAPK/PKC) and hypoxia inducible factor-1 alpha (HIF-1α) are also common proteins associated with inflammatory response induced by several stressors, but there is limited research on these proteins in the context of poultry intestinal Salmonella Enteritidis (SE) infections. In the present study, we isolated a novel strain of Bacillus pumilus with excellent performance from the feces of healthy yaks, named TS1. To investigate the effect of TS1 on SE-induced enteritis in broilers, 120 6-day-old white-feathered broilers were randomly divided into four groups (con, TS1, SE, TS1 + SE). TS1 and TS1 + SE group chickens were fed with 1.4 × 107 colony-forming units per mL of TS1 for 15 days and intraperitoneally injected with SE to establish the oxidative stress model. Then, we investigated whether TS1 protects the intestine of SE-treated broiler chickens using inflammatory cytokine gene expression analysis, stress protein quantification, antioxidant quantification, and histopathological analysis. RESULTS: The TS1 + SE group showed lower MDA and higher GSH-Px, SOD, and T-AOC than the SE group. TS1 alleviated the effects of SE on intestinal villus length and crypt depth. Our results suggest that SE exposure increased the expression of inflammatory factors (IL-1ß, IL-6, TNF-α, IL-4, and MCP-1), p38 MAPK, and PKCß and decreased the expression of HSP60, HSP70, and HIF-1α, whereas TS1 alleviated these effects. CONCLUSIONS: Bacillus pumilus TS1 alleviated oxidative stress damage caused by SE and attenuated the inflammatory response in broilers through MAPK/PKC regulation of HSPs/HIF-1α.

[Application of glycan microarrays in cancer research].

Glycosylation is one of the common post-translational modifications of proteins to regulate the ability of tumor invasion, metastasis and tumor heterogeneity by interacting with glycan-binding proteins such as lectins and antibodies. Glycan microarray can be constructed by chemical synthesis, chemical-enzyme synthesis or natural glycan releasing. Glycan microarray is an essential analytical tool to discover the interaction between glycan and its binding proteins. Here we summarize the standard techniques to construct glycan microarray for the application in cancer vaccine, monoclonal antibody and diagnostic markers.

Autoantibody signature in hepatocellular carcinoma using seromics.

BACKGROUND: Alpha-fetoprotein (AFP) is a widely used biomarker for hepatocellular carcinoma (HCC) early detection. However, low sensitivity and false negativity of AFP raise the requirement of more effective early diagnostic approaches for HCC. METHODS: We employed a three-phase strategy to identify serum autoantibody (AAb) signature for HCC early diagnosis using protein array-based approach. A total of 1253 serum samples from HCC, liver cirrhosis, and healthy controls were prospectively collected from three liver cancer centers in China. The Human Proteome Microarray, comprising 21,154 unique proteins, was first applied to identify AAb candidates in discovery phase (n = 100) and to further fabricate HCC-focused arrays. Then, an artificial neural network (ANN) model was used to discover AAbs for HCC detection in a test phase (n = 576) and a validation phase (n = 577), respectively. RESULTS: Using HCC-focused array, we identified and validated a novel 7-AAb panel containing CIAPIN1, EGFR, MAS1, SLC44A3, ASAH1, UBL7, and ZNF428 for effective HCC detection. The ANN model of this panel showed improvement of sensitivity (61.6-77.7%) compared to AFP (cutoff 400 ng/mL, 28.4-30.7%). Notably, it was able to detect AFP-negative HCC with AUC values of 0.841-0.948. For early-stage HCC (BCLC 0/A) detection, it outperformed AFP (cutoff 400 ng/mL) with approximately 10% increase in AUC. CONCLUSIONS: The 7-AAb panel provides potentially clinical value for non-invasive early detection of HCC, and brings new clues on understanding the immune response against hepatocarcinogenesis.

Formulation and characterization of voriconazole nanospray dried powders.

Purpose: Voriconazole nanoparticles (API-NPs) were prepared by nanospray drying to improve the solubility of voriconazole and reduce its interindividual variability.Methods: The preparation procedure was optimized by central composite design-response surface methodology. The properties of the nanoparticles were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) analyses. The solubility, dissolution, and stability of the API-NPs were determined experimentally. The pharmacokinetics were assessed based on rat plasma levels of voriconazole. An acute oral toxicity test of the API-NPs was performed in mice.Results: The powers were formulated using cetyltrimethylammonium chloride (CTAC) as the carrier material. SEM and particle size results showed that the API-NPs had a narrow particle size distribution. The XRD, DSC, and FTIR analyses show a decrease in crystallinity and a polymorphic transformation of the nanoparticles after nanospray drying. The solubility in water was approximately 15 times higher than that of voriconazole. The API-NP tablets exhibited significantly higher plasma exposure, namely, longer acting times and lower variability. The acute administration of voriconazole showed no toxic histopathological effects on organ tissue.Conclusion: The solubility of voriconazole was greatly improved, it showed higher bioavailability and safety, and the interindividual variability in voriconazole pharmacokinetics was reduced by nanospray drying.

Higher matrix stiffness as an independent initiator triggers epithelial-mesenchymal transition and facilitates HCC metastasis.

BACKGROUND: Increased liver stiffness exerts a detrimental role in driving hepatocellular carcinoma (HCC) malignancy and progression, and indicates a high risk of unfavorable outcomes. However, it remains largely unknown how liver matrix stiffness as an independent cue triggers epithelial-mesenchymal transition (EMT) and facilitates HCC metastasis. METHODS: Buffalo rat HCC models with different liver stiffness backgrounds and an in vitro Col I-coated cell culture system with tunable stiffness were used in the study to explore the effects of matrix stiffness on EMT occurrence and its underlying molecular mechanism. Clinical significance of liver stiffness and key molecules required for stiffness-induced EMT were validated in HCC cohorts with different liver stiffness. RESULTS: HCC xenografts grown in higher stiffness liver exhibited worse malignant phenotypes and higher lung metastasis rate, suggesting that higher liver stiffness promotes HCC invasion and metastasis. Cell tests in vitro showed that higher matrix stiffness was able to strikingly strengthen malignant phenotypes and independently induce EMT occurrence in HCC cells, and three signaling pathways converging on Snail expression participated in stiffness-mediated effect on EMT including integrin-mediated S100A11 membrane translocation, eIF4E phosphorylation, and TGF ß1 autocrine. Additionally, the key molecules required for stiffness-induced EMT were highly expressed in tumor tissues of HCC patients with higher liver stiffness and correlated with poor tumor differentiation and higher recurrence. CONCLUSIONS: Higher matrix stiffness as an initiator triggers epithelial-mesenchymal transition (EMT) in HCC cells independently, and three signaling pathways converging on Snail expression contribute to this pathological process. This work highlights a significant role of biomechanical signal in triggering EMT and facilitating HCC invasion and metastasis.

N-glycopeptide Signatures of IgA2 in Serum from Patients with Hepatitis B Virus-related Liver Diseases.

N-glycosylation alteration has been reported in liver diseases. Characterizing N-glycopeptides that correspond to N-glycan structure with specific site information enables better understanding of the molecular pathogenesis of liver damage and cancer. Here, unbiased quantification of N-glycopeptides of a cluster of serum glycoproteins with 40-55 kDa molecular weight (40-kDa band) was investigated in hepatitis B virus (HBV)-related liver diseases. We used an N-glycopeptide method based on 18O/16O C-terminal labeling to obtain 82 comparisons of serum from patients with HBV-related hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Then, multiple reaction monitoring (MRM) was performed to quantify N-glycopeptide relative to the protein content, especially in the healthy donor-HBV-LC-HCC cascade. TPLTAN205ITK (H5N5S1F1) and (H5N4S2F1) corresponding to the glycopeptides of IgA2 were significantly elevated in serum from patients with HBV infection and even higher in HBV-related LC patients, as compared with healthy donor. In contrast, the two glycopeptides of IgA2 fell back down in HBV-related HCC patients. In addition, the variation in the abundance of two glycopeptides was not caused by its protein concentration. The altered N-glycopeptides might be part of a unique glycan signature indicating an IgA-mediated mechanism and providing potential diagnostic clues in HBV-related liver diseases.

Glycoprofiling of Early Gastric Cancer Using Lectin Microarray Technology.

BACKGROUND: Recently, studies have reported that protein glycosylation plays an important role in the occurrence and development of cancer. Gastric cancer is a common cancer with high morbidity and mortality owing to most gastric cancers are discovered only at an advanced stage. Here, we aim to discover novel specific serum glycanbased biomarkers for gastric cancer. METHODS: A lectin microarray with 50 kinds of tumor-associated lectin was used to detect the glycan profiles of serum samples between early gastric cancer and healthy controls. Then lectin blot was performed to validate the differences. RESULTS: The result of the lectin microarray showed that the signal intensities of 13 lectins showed significant differences between the healthy controls and early gastric cancer. Compared to the healthy, the normalized fluorescent intensities of the lectins PWA, LEL, and STL were significantly increased, and it implied that their specifically recognized GlcNAc showed an especially elevated expression in early gastric cancer. Moreover, the binding affinity of the lectins EEL, RCA-II, RCA-I, VAL, DSA, PHA-L, UEA, and CAL were higher in the early gastric cancer than in healthy controls. These glycan structures containing GalNAc, terminal Galß 1-4 GlcNAc, Tri/tetraantennary N-glycan, ß-1, 6GlcNAc branching structure, α-linked fucose residues, and Tn antigen were elevated in gastric cancer. While the two lectins CFL GNL reduced their binding ability. In addition, their specifically recognized N-acetyl-D-galactosamine structure and (α-1,3) mannose residues were decreased in early gastric cancer. Furthermore, lectin blot results of LEL, STL, PHA-L, RCA-I were consistent with the results of the lectin microarray. CONCLUSIONS: The findings of our study clarify the specific alterations for glycosylation during the pathogenesis of gastric cancer. The specific high expression of GlcNAc structure may act as a potential early diagnostic marker for gastric cancer.

Identification of CD14 as a potential biomarker of hepatocellular carcinoma using iTRAQ quantitative proteomics.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors without effective diagnostic biomarkers. This study intended to dynamically analyze serum proteomics in different pathological stages of liver diseases, and discover potential diagnostic biomarkers for early HCC. Patients with hepatitis B virus (HBV) infection, liver cirrhosis (LC), or HCC together with healthy controls (HC) were enrolled. Proteins differentially expressed between groups were screened using isobaric tagging for relative and absolute quantitation (iTRAQ), and promising HCC biomarker candidates were subjected to bioinformatics analysis, including K-means clustering, gene ontology (GO) and string network analysis. Potential biomarkers were validated by Western blotting and enzyme-linked immunosorbent assay (ELISA), and their diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis. Finally, 93 differentially expressed proteins were identified, of which 43 differed between HBV and HC, 70 between LC and HC, and 51 between HCC and HC. Expression levels of gelsolin (GELS) and sulfhydryl oxidase 1 (QSOX1) varied with disease state as follows: HC < HBV < LC < HCC. The reverse trend was observed with CD14. These iTRAQ results were confirmed by Western blotting and ELISA. Logistic regression and ROC curve analysis identified the optimal cut-off for alpha-fetoprotein (AFP), CD14 and AFP/CD14 was 191.4 ng/mL (AUC 0.646, 95%CI 0.467-0.825, sensitivity 31.6%, specificity 94.4%), 3.16 ng/mL (AUC 0.760, 95%CI 0.604-0.917, sensitivity 94.7%, specificity 50%) and 0.197 ng/mL (AUC 0.889, 95%CI 0.785-0.993, sensitivity 84.2%, specificity 83.3%) respectively. In conclusion, Assaying CD14 levels may complement AFP measurement for early detection of HCC.

S100A6 promotes proliferation of intrahepatic cholangiocarcinoma cells via the activation of the p38/MAPK pathway.

AIM: We explored the expression of S100A6 and its role in intrahepatic cholangiocarcinoma (ICC). METHODS: The expression of S100A6 in ICC samples was detected by immunohistochemistry. In vitro experiments, we silenced and overexpressed S100A6 to investigate its role in cell functions. RESULTS: The expression of S100A6 was markedly increased in ICC tissues and cell lines. S100A6 overexpression was an independent risk factor for patients' survival. Silencing S100A6 resulted in a suppression of proliferation and p38/MAPK activity, while overexpressing S100A6 caused a promotion of proliferation and p38/MAPK. DISCUSSION:  S100A6 participated in the proliferation of ICC cells and correlated with a more aggressive behavior of ICC. Conclusion: S100A6 may serve as a novel prognostic marker and a potential therapeutic target for ICC patients.

Protein glycosylation in viral hepatitis-related HCC: Characterization of heterogeneity, biological roles, and clinical implications.

Protein glycosylation is one of the most frequent and well-known posttranslational modifications, playing important roles in physiopathological processes. Glycosylation is catalyzed by enzymatic additions of heterogeneous glycans with specific linkage of monosaccharides and, in a preformed fashion, to specific amino acids within glycoproteins. Altered glycan macroheterogeneity and microheterogeneity have been reported in glycoproteins produced mainly by the liver, facilitating tumorigenesis, progression, and metastasis. Characterizing the heterogeneity and biological functions of glycans in liver diseases would lead to a better understanding of the molecular pathogenesis of liver damage and cancer, providing novel diagnostic, prognostic, and therapeutic clues. Technical advances in glycoproteomics and glycomics have allowed a more comprehensive and deeper understanding of disease-related glycosylation events. Here, we briefly review the structural diversity of glycans in viral hepatitis -related hepatocellular carcinoma, discuss their roles in regulating the initiation and development of liver cancer, and introduce potential clinical applications.

Sorafenib induced alteration of protein glycosylation in hepatocellular carcinoma cells.

Sorafenib is a multikinase inhibitor and is effective in treating hepatocellular carcinoma (HCC). However, it remains unknown whether sorafenib induces the alteration of protein glycosylation. The present study treated HCC MHCC97L and MHCC97H cells with a 50% inhibitory concentration of sorafenib. Following this treatment, alteration of protein glycosylation was detected using a lectin microarray. Compared with the controls, the binding capacity of glycoproteins extracted from sorafenib-treated HCC cells to the lectins Bauhinia purpurea lectin, Dolichos biflorus agglutinin, Euonymus europaeus lectin, Helix aspersa lectin, Helix pomatia lectin, Jacalin, Maclura pomifera lectin and Vicia villosa lectin were enhanced; while, the binding capacities to the lectins Caragana arborescens lectin, Lycopersicon esculentum lectin, Limulus polyphemus lectin, Maackia amurensis lecin I, Phaseolus vulgaris leucoagglutinin, Ricinus communis agglutinin 60, Sambucus nigra lectin and Solanum tuberosum lectin were reduced (spot intensity median/background intensity median ≥2, P<0.05). This difference in glycoprotein binding capacity indicates that cells treated with sorafenib could increase α-1,3GalNAc/Gal, ß-1,3 Gal, GalNAcα-Ser/Thr(Tn) and α-GalNAc structures and decrease GlcNAc, sialic acid, tetra-antennary complex-type N-glycan and ß-1,4Gal structures. These results were additionally confirmed by lectin blotting. Expression levels of signaling molecules including erythroblastosis 26-1 (Ets-1), extracellular signal-related kinases (ERK) and phosphorylated-ERK were measured by western blotting. There was a reduction in the expression of Ets-1 and ERK phosphorylation in sorafenib or 1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene treated cells suggesting that sorafenib may reduce the expression levels of Ets-1 by blocking the Ras/Raf/mitogen activated protein kinase signaling pathway. In the present study, it was clear that sorafenib could inhibit the proliferation of HCC cells and alter protein glycosylation. The findings of this study may lead to providing a novel way of designing new anti-HCC drugs.

Assessment of Hepatocellular Carcinoma Metastasis Glycobiomarkers Using Advanced Quantitative N-glycoproteome Analysis.

Hepatocelluar carcinoma (HCC) is one of the most common malignant tumors with high incidence of metastasis. Glycosylation is involved in fundamental molecular and cell biology process occurring in cancer including metastasis formation. In this study, lectin microarray, lectin blotting, lectin affinity chromatography and tandem 18O stable isotope labeling coupled with liquid chromatography-mass spectrometer (LC-MS) analysis were applied to quantify the changes in N-glycosite occupancy for HCC metastasis serum. Firstly, lectin microarray was used to screen glycoforms and Phaseolus vulgaris Leucoagglutinin (PHA-L) reactive structure (ß1,6-GlcNAc branched N-glycan) was found to be increased significantly in HCC patients with metastasis compared with those with non-metastasis. Then, PHA-L affinity glycoproteins were enriched followed by N-glycosite occupancy measurement with strategy of tandem 18O stable isotope labeling. 11 glycoproteins with significantly changed N-glycosite occupancy were identified, they were associated with cell migration, invasion and adhesion through p38 mitogen-activated protein kinase signaling pathway and nuclear factor kappa B signaling pathway. Quantification of N-glycosite occupancy for PHA-L reactive glycoproteins could help to discover important glycoproteins of potential clinically significance in terms of HCC etiology. Also, understanding of N-glycosite occupancy alterations will aid the characterization of molecular mechanism of HCC metastasis as well as establishment of novel glycobiomarkers.

Diagnosis of AFP-negative early-stage hepatocellular carcinoma using Fuc-PON1.

Our previous study demonstrated that Fuc-PON1 (the ratio of fucosylated serum paraoxonase 1 to the total serum serum paraoxonase 1) was increased significantly in hepatocellular carcinoma (HCC) patients with low AFP levels. Herein, a separate cohort of AFP-negative (AFP-) early HCC patients was studied to validate the diagnostic potential of Fuc-PON1. Aleuria aurantia lectin (AAL) ELISA and protein ELISA were measured simultaneously to calculate PON1 fucosylation at its protein level. Lens culinaris agglutinin reactive AFP (AFP-L3) and glypican-3 (GPC3) concentrations of the same specimens were also evaluated. The AUC was 0.78 (95% CI 0.704 to 0.852) for Fuc-PON1, with sensitivity of 62.2%, specificity of 67.7%, and accuracy of 64.5%. However, concentration alterations of AFP-L3 and GPC3 in AFP-HCC patients were not remarkable. The results of the present study provided confirmed evidences for clinical application of Fuc-PON1, which demonstrated its superior diagnosis potential for distinguishing AFP- early HCC from AFP- liver cirrhosis (LC) patients.

Quantification of N-glycosylation site occupancy status based on labeling/label-free strategies with LC-MS/MS.

Protein N-glycosylation plays important roles in physiological and pathological processes. Characterizing the site-specific N-glycosylation including N-glycan macroheterogeneity (glycosylation site occupancy) and microheterogeneity (site-specific glycan structure) is important for understanding of glycoprotein biosynthesis and function. N-Glycan macroheterogeneity is a physiological property of glycoprotein and the technical obstacles have restricted research into the regulation and functions of this heterogeneity. Quantification of N-glycosylation site occupancy would uncover the critical role of macroheterogeneity in a variety of biological properties. Liquid chromatography (LC)- mass spectrometry (MS)-based quantification is emerging as a powerful tool for glycosylation characterization. This review summarizes the labeling and label-free quantitative MS approaches for quantifying N-glycosylation site occupancy, including its quantification for target glycoproteins in recent years.

Oct4 induces EMT through LEF1/ß-catenin dependent WNT signaling pathway in hepatocellular carcinoma.

Octamer 4 (Oct4), a member of the Pit-Oct-Unc transcription factor family required to maintain self-renewal and pluripotency of embryonic stem cells, has been previously identified to be associated with tumorigenesis and malignant transformation of numerous types of cancer including hepatocellular carcinoma (HCC). The present data shows that Oct4 enhances cancer stem cell properties and increases invasion ability in the Huh7 cell line. To increase understanding of the role of Oct4 in HCC, the present study used a functional genomics approach and analyzed the resulting transcriptional profiles to identify Oct4-dependent genes in Huh7. Affymetrix GeneChip Human genome U133 Plus 2.0 Arrays were used to determine differential gene expression profiles and then validated by quantitative polymerase chain reaction. The present study found that altered expression of 673 genes (fold-change ≥2) affected multiple signaling pathways linked with self-renew and metastasis. Among these differentially expressed genes, the present study noticed that the key component of the WNT signaling pathway lymphoid enhancer binding factor 1 (LEF1) and Twist Family BHLH transcription factor 1 were upregulated by Oct4, whilst cadherin 2 was downregulated. Additional studies found that the nuclear ß-catenin aggregation was increased in Oct4 overexpressed HCC cell lines. These results suggest that Oct4 regulates LEF1 to active LEF1/ß-catenin dependent WNT signaling pathway and promote epithelial-mesenchymal transition. The present findings provide novel mechanistic insight into an important role of Oct4 in HCC.

Aided Diagnosis of Hepatocellular Carcinoma Using Serum Fucosylated Haptoglobin Ratios.

Aberrant fucosylation plays a functional role in regulating ontogeny and celluar differentiation and are differentially regulated in cancerous condition, which could provide hallmarks for cancer diagnostics and surveillance. We previously developed a magnetic beads-based lectin ELISA system to measure fucosylated haptoglobin (Hp), which has been reported to be a cancer biomarker through a series of glycoproteomic analysis. In this study, serum fucosylated Hp ratios were measured using our ELISA kit in a separate cohort of 260 patients independently, including 130 healthy controls and 130 patients with hepatocellular carcinoma (HCC). Fucosylated Hp /Hp ratio (levels of fucosylated Hp /levels of protein Hp) and ELISA Index (OD value of fucosylated Hp /OD value of protein Hp) were calculated respectively to reflect Hp fucosylation level on its protein level. Our data showed that fucosylated Hp /Hp ratio (AUC=0.8449) and ELISA Index (AUC=0.8581) had better performance in distinguishing HCC from controls, which indicated that fucosylated Hp ratios could improve the diagnosis and prediction of HCC even with a low level of alpha-fetoprotein (AFP). Additionally, the combination analysis of AFP and fucosylated Hp ratios increased the AUC value for HCC diagnosis.

Reduced N-acetylglucosaminyltransferase III expression via Smad3 and Erk signaling in TGF-ß1-induced HCC EMT model.

N-acetylglucosaminyltransferase III (GnT-III), encoded by the MGAT3 glycogene, is thought to be a tumor metastatic suppressor. Our previous studies found that the mRNA expression level of GnT-III decreased in a hepatocyte growth factor (HGF)-induced hepatocellular carcinoma (HCC) epithelial-mesenchymal transition (EMT) model. However, the molecular mechanism of GnT-III expression is unclear. In the present study, we established an HCC EMT model using the classic inducer transforming growth factor-ß1 (TGF-ß1) and focused on the Smad3 and Erk signaling pathways. Results showed that GnT-III gene expression and its catalytic product bisecting GlcNAc structure decreased and phosphorylation of both Smad3 and Erk1/2 was up-regulated in TGF-ß1-treated MHCC97-L cells, while GnT-III mRNA expression and bisecting GlcNAc structure increased after blocking the activities of Smad3 and Erk signaling pathways. These findings suggested that GnT-III gene expression is modulated by the growth factor signaling pathways in the EMT model and that GnT-III may be a potential drug target for HCC treatment.