Toxicity of soil leaching liquor from coking plant in developmental zebrafish embryos/larvae model.

The coking industry in China is the largest coke supplier in the world. Contaminated soil in industrial areas poses a serious threat to human and ecosystems. Most of the studies investigated the toxicity of soil from coking plant on soil microorganisms, while the toxic effects of soil leaching liquor on aquatics are limited. In this study, the composition of soil leaching liquor from a coking plant in Taiyuan (TY) was analyzed, and the developmental toxicity on zebrafish was evaluated. The results showed that a total of 91 polycyclic aromatic hydrocarbons were detected in the leaching liquor, followed by phenols and benzene series. The leaching liquor induced developmental impairment in zebrafish larvae, including delayed incubation, deficits in locomotor behavior, vascular and cardiac dysplasia, and impaired neurodevelopment. The results of metabolomics analysis showed that TY soil leaching liquor induced significant metabolic profile disturbances in zebrafish embryos/larvae. The developmental toxicity of the leaching liquor metabolic disorders may be associated with the leaching liquor-induced abnormalities in zebrafish embryonic development. Metabolic pathways were identified by arginine and proline metabolism, phosphotransferase system, starch and sucrose metabolism, steroid biosynthesis, beta-alanine metabolism, and nucleotide metabolism pathways.

Protective effects of cyanidin-3-O-glucoside on BPA-induced neurodevelopmental toxicity in zebrafish embryo model.

Bisphenol A (BPA) is ubiquitous in the environment and poses a threat to wildlife and human health. It has been reported that BPA may cause the neurotoxicity during gestational and neonatal periods. Cyanidin-3-O-glucoside (C3G) is one of the most abundant anthocyanins that has shown multiple bio-functions. In this study, the protective effects and possible mechanism of C3G against BPA-induced neurodevelopment toxicity in zebrafish embryos/larvae were studied. The results showed that co-exposure of C3G (25 µg/mL) significantly attenuated BPA-induced deficit in locomotor behavior and restored the BPA-induced aberrant changes in brain morphology of zebrafish larvae. Further studies showed that the defects of central nervous development and the downregulated neurogenesis relative genes induced by BPA were significantly counteracted by co-exposure with 5 µg/mL of C3G. In addition, C3G (25 µg/mL) mitigated the decline of glutathione (GSH) content and enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), attenuated oxidative stress and cell apoptosis induced by BPA in zebrafish. The enhancements of the expression of genes involved in the Nrf2-ARE pathway (Nrf2, HO-1, NQO1, GCLC, and GCLM) were also observed by co-exposure of C3G. The results indicate that C3G exerts protective effects on BPA-induced neurodevelopmental toxicity through improving transcription of neurogenesis related genes, enhancing antioxidative defense system and reducing cell apoptosis by regulation of apoptotic genes in zebrafish larvae. The results suggest that anthocyanins may play important role against the exogenous toxicity for vertebrates.

Sulfation of O-glycans on Mucin-type Proteins From Serous Ovarian Epithelial Tumors.

Despite sulfated O-linked glycans being abundant on ovarian cancer (OC) glycoproteins, their regulation during cancer development and involvement in cancer pathogenesis remain unexplored. We characterized O-glycans carrying sulfation on galactose residues and compared their expression with defined sulfotransferases regulated during OC development. Desialylated sulfated oligosaccharides were released from acidic glycoproteins in the cyst fluid from one patient with a benign serous cyst and one patient with serous OC. Oligosaccharides characterized by LC-MSn were identified as core 1 and core 2 O-glycans up to the size of decamers and with 1 to 4 sulfates linked to GlcNAc residues and to C-3 and/or C-6 of Gal. To study the specificity of the potential ovarian sulfotransferases involved, Gal3ST2 (Gal-3S)-, Gal3ST4 (Gal-3S)-, and CHST1 (Gal-6S)-encoding expression plasmids were transfected individually into CHO cells also expressing the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIg G2b) fusion protein and the human core 2 transferase (GCNT1). Characterization of the PSGL-1/mIg G2b O-glycans showed that Gal3ST2 preferentially sulfated Gal on the C-6 branch of core 2 structures and Gal3ST4 preferred Gal on the C-3 branch independently if core-1 or -2. CHST1 sulfated Gal residues on both the C-3 (core 1/2) and C-6 branches of core 2 structures. Using serous ovarian tissue micro array, Gal3ST2 was found to be decreased in tissue classified as malignant compared with tissues classified as benign or borderline, with the lowest expression in poorly differentiated malignant tissue. Neither Gal3ST4 nor CHST1 was differentially expressed in benign, borderline, or malignant tissue, and there was no correlation between expression level and differentiation stage. The data displays a complex sulfation pattern of O-glycans on OC glycoproteins and that aggressiveness of the cancer is associated with a decreased expression of the Gal3ST2 transferase.

miR-29a-3p-dependent COL3A1 and COL5A1 expression reduction assists sulforaphane to inhibit gastric cancer progression.

The antitumor properties of cruciferous vegetables are mainly due to their high content of isothiocyanates, and sulforaphane (SFA) is the most well-known compound. The aim of this study was to determine the mechanism of SFA inhibiting gastric cancer (GC) progression. After verifying SFA suppressing GC growth in vivo, we utilized the GSE79973 and GSE118916 datasets to identify the GC development signatures that overlap with the RNA-seq analysis in SFA-treated AGS cells. GSEA of the RNA-seq data indicated that SFA regulation of GC progression was related to extracellular matrix and collagens; thus, we identified COL3A1 and COL5A1 as the targets of SFA, which functioned as oncogenes. We found positive correlations between COL3A1 and COL5A1 expression in GC cells, and confirmed that miR-29a-3p is the common regulator of their expression. RNA immunoprecipitation assays based on Ago2, Dicer, and exportin-5 showed that SFA could promote mature miR-29a-3p generation. We also proved that SFA inactivated the Wnt/ß-catenin pathway in GC cells in a miR-29a-3p-dependent manner. Overall, SFA boosts miR-29a-3p maturation to downregulate COL3A1 and COL5A1 and inactivate the Wnt/ ß -catenin pathway to suppress GC progression.

Treatment of particle/gas partitioning using level III fugacity models in a six-compartment system.

In this paper, two level III fugacity models are developed and applied using an environmental system containing six compartments, including air, aerosols, soil, water, suspended particulate matters (SPMs), and sediments, as a "unit world". The first model, assumes equilibrium between air and aerosols and between water and SPMs. These assumptions lead to a four-fugacity model. The second model removes these two assumptions leading to a six-fugacity model. The two models, compared using four PBDE congeners, BDE-28, -99, -153, and -209, with a steady flux of gaseous congeners entering the air, lead to the following conclusions. 1. When the octanol-air partition coefficient (KOA) is less than 1011.4, the two models produce similar results; when KOA > 1011.4, and especially when KOA > 1012.5, the model results diverge significantly. 2. Chemicals are in an imposed equilibrium in the four-fugacity model, but in a steady state and not necessary an equilibrium in the six-fugacity model, between air and aerosols. 3. The results from the six-fugacity model indicate an internally consistent system with chemicals in steady state in all six compartments, whereas the four-fugacity model presents an internally inconsistent system where chemicals are in equilibrium but not a steady state between air and aerosols. 4. Chemicals are mass balanced in air and aerosols predicted by the six-fugacity model but not by the four-fugacity model. If the mass balance in air and aerosols is achieved in the four-fugacity model, the condition of equilibrium between air and aerosols will be no longer valid.

Identifying the p65-Dependent Effect of Sulforaphene on Esophageal Squamous Cell Carcinoma Progression via Bioinformatics Analysis.

Understanding the mechanism by which sulforaphene (SFE) affects esophageal squamous cell carcinoma (ESCC) contributes to the application of this isothiocyanate as a chemotherapeutic agent. Thus, we attempted to investigate SFE regulation of ESCC characteristics more deeply. We performed gene set enrichment analysis (GSEA) on microarray data of SFE-treated ESCC cells and found that differentially expressed genes are enriched in TNFα_Signaling_via_the_NFκB_Pathway. Coupled with the expression profile data from the GSE20347 and GSE75241 datasets, we narrowed the set to 8 genes, 4 of which (C-X-C motif chemokine ligand 10 (CXCL10), TNF alpha induced protein 3 (TNFAIP3), inhibin subunit beta A (INHBA), and plasminogen activator, urokinase (PLAU)) were verified as the targets of SFE. RNA-sequence (RNA-seq) data of 182 ESCC samples from The Cancer Genome Atlas (TCGA) were grouped into two phenotypes for GSEA according to the expression of CXCL10, TNFAIP3, INHBA, and PLAU. The enrichment results proved that they were all involved in the NFκB pathway. ChIP-seq analyses obtained from the Cistrome database indicated that NFκB-p65 is likely to control the transcription of CXCL10, TNFAIP3, INHBA, and PLAU, and considering TNFAIP3 and PLAU are the most significantly differentially expressed genes, we used chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) to verify the regulation of p65 on their expression. The results demonstrated that SFE suppresses ESCC progression by down-regulating TNFAIP3 and PLAU expression in a p65-dependent manner.

Identification by mass spectrometry and immunoblotting of xenogeneic antigens in the N- and O-glycomes of porcine, bovine and equine heart tissues.

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by ß-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

Glyco-engineered cell line and computational docking studies reveals enterotoxigenic Escherichia coli CFA/I fimbriae bind to Lewis a glycans.

We have previously reported clinical data to suggest that colonization factor I (CFA/I) fimbriae of enterotoxigenic Escherichia coli (ETEC) can bind to Lewis a (Lea), a glycan epitope ubiquitous in the small intestinal mucosa of young children (<2 years of age), and individuals with a genetic mutation of FUT2. To further elucidate the physiological binding properties of this interaction, we engineered Chinese Hamster Ovary (CHO-K1) cells to express Lea or Leb determinants on both N- and O-glycans. We used our glyco-engineered CHO-K1 cell lines to demonstrate that CfaB, the major subunit of ETEC CFA/I fimbriae, as well as four related ETEC fimbriae, bind more to our CHO-K1 cell-line expressing Lea, compared to cells carrying Leb or the CHO-K1 wild-type glycan phenotype. Furthermore, using in-silico docking analysis, we predict up to three amino acids (Glu25, Asn27, Thr29) found in the immunoglobulin (Ig)-like groove region of CfaB of CFA/I and related fimbriae, could be important for the preferential and higher affinity binding of CFA/I fimbriae to the potentially structurally flexible Lea glycan. These findings may lead to a better molecular understanding of ETEC pathogenesis, aiding in the development of vaccines and/or anti-infection therapeutics.

Rational Selection of the 3D Structure of Biomacromolecules for Molecular Docking Studies on the Mechanism of Endocrine Disruptor Action.

Molecular modeling has become an essential tool in predicting and simulating endocrine disrupting effects of chemicals. A key prerequisite for successful application of molecular modeling lies in the correctness of 3D structure for biomacromolecules to be simulated. To date, there are several databases that can provide the experimentally-determined 3D structures. However, commonly, there are many challenges or disadvantageous factors, e.g., (a) lots of 3D structures for a given biomacromolecular target in the protein database; (b) the quality variability for those structures; (c) belonging to different species; (d) mutant amino acid residue in key positions, and so on. Once an inappropriate 3D structure of a target biomacromolecule was selected in molecular modeling, the accuracy and scientific nature of the modeling results could be inevitably affected. In this article, based on literature survey and an analysis of the 3D structure characterization of biomacromolecular targets belonging to the endocrine system in protein databases, six principles were proposed to guide the selection of the appropriate 3D structure of biomacromolecules. The principles include considering the species diversity, the mechanism of action, whether there are mutant amino acid residues, whether the number of protein chains is correct, the degree of structural similarity between the ligand in 3D structure and the target compounds, and other factors, e.g., the experimental pH conditions of the structure determined process and resolution.

Heterotypic cell adhesion assay for the study of cell adhesion inhibition.

CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its ligand CD58 on antigen-presenting cells. Interaction between CD2 and CD58 or leukocyte function-associated antigen-3 (LFA-3) helps to optimize immune recognition facilitating contact between T lymphocytes and antigen-presenting cells. Modulation or inhibition of this interaction has been shown to be therapeutically useful in the treatment of autoimmune diseases. Antibodies and small molecules including peptides have been designed to modulate or disrupt the cell adhesion interactions due to CD2 and CD58. E-rosetting assay is a widely used method applied in the study of the modulation of CD2-CD58 interaction, which is either labor-intensive or radio-hazardous. In this chapter, we describe two methods that are used to study cell adhesion inhibition: (a) E-rosetting Assay and (b) Lymphocyte-epithelial assay. The second method, lymphocyte-epithelial assay, is a rapid and sensitive heterotypic cell adhesion assay for studying cell adhesion inhibition. The method relies on the CD2 expression on the surface of Jurkat cells and the CD58 expression on the surface of Caco-2 cells, which were confirmed by flow cytometry and ELISA studies respectively. This heterotypic cell adhesion assay described typically takes less than 4 h to perform, allows the evaluation of inhibitory activity of peptides/small molecules to modulate CD2-CD58 interaction in real cell system.

Evidence for no relevance of anti-major histocompatibility complex class I-related chain a antibodies in liver transplantation.

The polymorphic major histocompatibility complex class I-related chain A (MICA) antigen is being increasingly recognized as a potential target molecule for immune cells during allograft rejection. Here we studied whether MICA is a target antigen for antibodies in liver transplant patients. Eighty-four patients were investigated for the presence of MICA antibodies before and after liver transplantation with MICA-transfected cells and flow cytometry. MICA typing was performed by polymerase chain reaction. Expression of MICA in liver cells was determined by reverse-transcription polymerase chain reaction, Western blotting, and flow cytometry. Liver biopsy specimens from liver transplant patients were examined for MICA expression. A total of 22 of 84 (26%) patients had MICA antibodies either pre-transplant (8/84, 9.5%) or post-transplant (14/84, 17%). No correlation between rejection frequencies (14/22, 63%) or other clinical parameters was observed in patients with MICA antibody versus those without MICA antibody (29/62, 47% P = not significant). We found weak messenger RNA expression for MICA in liver cells but no protein or cell surface expression. In addition, no MICA expression in liver biopsy sections from liver transplant patients was observed at any time point, including rejections. Thus, our preliminary results demonstrate no causal relationship between the presence of MICA antibodies and liver allograft rejections. Therefore, it is likely that MICA may not be an important target antigen during liver allograft rejections.

Anti endothelial cell autoantibodies selectively activate SAPK/JNK signalling in Wegener's granulomatosis.

The pathogenic role of anti-endothelial cell antibodies (AECA) in vascular injury is debated. It was previously shown that many patients with Wegener's granulomatosis (WG) have AECA that react with human kidney microvascular endothelial cells (EC). In addition, during active disease, renal endothelium strongly expresses the inflammatory molecules vascular adhesion protein-1 (VAP-1) and MHC class I-related antigen A (MICA). This study sought to determine whether AECA mediates this upregulation of VAP-1 and MICA and to define better the signaling pathways that are activated by these autoantibodies upon binding to EC in the kidney. Stimulation of human kidney microvascular EC with AECA IgG upregulated surface expression of MICA and VAP-1, elicited a rapid Ca2+ flux, induced high levels of the chemokines monocyte chemoattractant protein-1 and granulocyte chemotactic protein-2, induced specific phosphorylation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and the transcription factors c-Jun and activating transcription factor-2, and activated NF-kappaB. Specific inhibitors of SAPK/JNK significantly reduced AECA-induced chemokine production and phosphorylation of c-Jun and activating transcription factor-2 and abrogated protein expression of MICA but not VAP-1. In kidney sections from patients with WG, infiltrating cells that expressed the ligand for MICA (NKG2D+) were identified, as were CD8+ and 32 gamma delta+ T cells. In conclusion, AECA may be involved in the pathogenesis of WG, and the SAPK/JNK pathway and the endothelial inflammatory protein VAP-1 may be novel therapeutic targets for vasculitis.

Structure-based rational design of beta-hairpin peptides from discontinuous epitopes of cluster of differentiation 2 (CD2) protein to modulate cell adhesion interaction.

Modulation or inhibition of interaction of cluster of differentiation (CD) adhesion molecules CD2-CD58 has been shown to be therapeutically useful. The analysis of the crystal structure of CD2 complexed with CD58 was carried out to define the epitopes that are important for the interaction of the two proteins. The crystal structure of CD2 indicated that the interaction surface of CD2 with CD58 has two beta-strand structures (F and C strands) with charged residues. On the basis of the crystal structure of the complex CD2-CD58, we have designed beta-hairpin peptides from the beta-strand region of CD2 by conjugating the discontinuous sequences in the protein. The peptides were modeled by molecular dynamics simulation, and their inhibitory activities were evaluated in vitro using two heterotypic cell adhesion assays, E-rosetting and lymphocyte-epithelial cell adhesion assays. Results indicated that 12- and 14-residue conjugate cyclic peptides cKS12 and cDD14 exhibited 60% and 50% inhibition activity, respectively, at 90 microM.

Structure-activity studies of peptides from the "hot-spot" region of human CD2 protein: development of peptides for immunomodulation.

CD2 is a cell surface protein belonging to the immunoglobulin superfamily (IgSF) that plays a key role in mediating adhesion between human T-lymphocytes and target cells. The interaction between cell-adhesion molecules CD2 and CD58 is critical for immune response. Modulation or inhibition of these interactions has been shown to be therapeutically useful. Synthetic 12-mer linear and cyclic peptides and cyclic hexapeptides from the beta-turn and beta-strand region (hot spot) of human CD2 protein were designed to modulate CD2-CD58 interaction. The 12-amino acid synthetic cyclic peptides effectively blocked the interaction between CD2 and CD58 proteins as demonstrated by E-rosetting and heterotypic adhesion assays. NMR and molecular modeling studies indicated that these cyclic peptides exhibit beta-turn structure in solution and closely mimic the beta-turn structure of the surface epitopes of CD2 protein. The designed cyclic peptides with beta-turn structure have the ability to modulate CD2-CD58 interaction.

A novel, rapid and sensitive heterotypic cell adhesion assay for CD2-CD58 interaction, and its application for testing inhibitory peptides.

The immunoglobulin CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its receptor CD58 on antigen-presenting cells (APCs). Modulation or inhibition of this interaction has been shown to be therapeutically useful. E-rosetting assay is usually applied in the study of the modulation of CD2-CD58 interaction. In this study, we demonstrated a novel, rapid and sensitive heterotypic cell adhesion assay for CD2-CD58 interaction. The CD2 expression on the surface of Jurkat cells and the CD58 expression on the Caco-2 cells were confirmed by flow cytometry and ELISA studies, respectively. Then Jurkat cells were fluorescent-labeled with 2 microM of BCECF-AM for 45 min at 37 degrees C before adding to confluent Caco-2 monolayers cultured in 96-well culture dishes. After 30 min, non-adherent Jurkat cells were removed by washing with PBS, while the monolayer-associated Jurkat cells were lysed with 0.5 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantitated using a microplate fluorescence analyzer with BCECF's excitation maximum of 485 nm and emission maximum of 535 nm. This method was successfully applied for testing inhibitory peptides to CD2-CD58 interaction.