Coffee Leaf Tea Extracts Improve Hyperuricemia Nephropathy and Its Associated Negative Effect in Gut Microbiota and Amino Acid Metabolism in Rats.

Hyperuricemia nephropathy (HN) is a metabolic disease characterized by tubular damage, tubulointerstitial fibrosis, and uric acid kidney stones and has been demonstrated to be associated with hyperuricemia. Coffee leaf tea is drunk as a functional beverage. However, its prevention effects on HN remain to be explored. This study showed that coffee leaf tea extracts (TE) contain 19 polyphenols, with a total content of 550.15 ± 27.58 mg GAE/g. TE decreased serum uric acid levels via inhibiting XOD activities and modulating the expression of urate transporters (GLUT9, OAT3, and ABCG2) in HN rats. TE prevented HN-induced liver and kidney damage and attenuated renal fibrosis. Moreover, it upregulated the abundance of SCFA-producing bacteria (Phascolarctobacterium, Alloprevotella, and Butyricicoccus) in the gut and reversed the amino acid-related metabolism disorder caused by HN. TE also decreased the circulating LPS and d-lactate levels and increased the fecal SCFA levels. This study supported the preliminary and indicative effect of coffee leaf tea in the prevention of hyperuricemia and HN.

The arachidonic acid metabolism protein-protein interaction network and its expression pattern in esophageal diseases.

Arachidonic acid (AA) and its metabolites are involved in the development and progression of inflammation and tumors in various tissues. We investigated the protein-protein interaction network (PPIN) of key enzymes in AA metabolism and their interacting proteins, as well as their expression patterns in different types of esophageal disease, involving esophagitis, Barrett's esophagus, adenocarcinoma and squamous cell carcinoma. PPINs were constructed to illustrate the key enzymes and their interacting proteins along the metabolic cascade. The network also showed key enzymes that could connect or cross-talk with at least one partner protein. The inflammation-related gene RELA (NF-kB) was found to interact with both PLA2G4A and ALOX5. Expression levels of the PPIN proteins, as well as their expression correlations, in different esophageal diseases were analyzed and integrated into the PPIN to illustrate a dynamic change. At least six significant pairs of expression relationships were identified across different esophageal diseases. The expression levels of eight enzymes (ALOX5, ALOX5AP, CYP2C8, CYP4F11, LTA4H, PLA2G4A, CYP2D6, PTGES2) correlated with the survival time of ESCC patients. In summary, we constructed an AA metabolic PPIN to explore AA metabolism-related gene expression patterns in esophageal diseases, showing their dynamic change and potential for therapeutic targeting from inflammation to cancer.

Network analyses elucidate the role of SMYD3 in esophageal squamous cell carcinoma.

SMYD3 is a member of the SET and myeloid-Nervy-DEAF-1 (MYND) domain-containing protein family of methyltransferases, which are known to play critical roles in carcinogenesis. Expression of SMYD3 is elevated in various cancers, including esophageal squamous cell carcinoma (ESCC), and is correlated with the survival time of patients with ESCC. Here, we dissect gene expression data, from a previously described KYSE150 ESCC cell line in which SMYD3 had been knocked down, by integration with the protein-protein interaction (PPI) network, to find the new potential biological roles of SMYD3 and subsequent target genes. By construction of a specific PPI network, differentially expressed genes (DEGs), following SMYD3 knockdown, were identified as interacting with thousands of neighboring proteins. Enrichment analyses from the DAVID Functional Annotation Chart found significant Gene Ontology (GO) terms associated with transcription activities, which were closely related to SMYD3 function. For example, YAP1 and GATA3 might be a target gene for SMYD3 to regulate transcription. Enrichment annotation of the total DEG PPI network by GO 'Biological Process' generated a connected functional map and found 532 significant terms, including known and potential biological roles of SMYD3 protein, such as expression regulation, signal transduction, cell cycle, cell metastasis, and invasion. Subcellular localization analyses found that DEGs and their interacting proteins were distributed in multiple layers, which might reflect the intricate biological processes at the spatial level. Our analysis of the PPI network has provided important clues for future detection of the biological roles and mechanisms, as well as the target genes of SMYD3.

Quantitative proteomics reveals the downregulation of GRB2 as a prominent node of F806-targeted cell proliferation network.

High-throughput proteomics has successfully identified thousands of proteins as potential therapeutic targets during investigations into mechanisms of drug action. A novel macrolide analog, denoted F806, is a potential antitumor drug. Here, using the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture (SILAC) coupled to high-resolution mass spectrometry (MS), we characterize the F806-regulating protein profiles and identify the potential target molecules or pathways of F806 in esophageal squamous cell carcinoma (ESCC) cells. From a total of 1931 quantified proteins, 181 proteins were found to be down-regulated (FDR p-value<0.1, H/L ratio<0.738), and 119 proteins were up-regulated (FDR p-value<0.1, H/L ratio>1.156). Among the down-regulated proteins, we uncovered the over- and under-represented protein clusters in biological process and molecular function respectively by Gene Ontology analysis. Furthermore, down-regulated and up-regulated proteins were significantly enriched in 37 pathways and 60 sub-pathways by bioinformatic analysis (FDR p-value<0.1), while a down-regulated molecule growth factor receptor-bound protein 2 (GRB2) was a prominent node in fourteen cell proliferation-related sub-pathways. We concluded that GRB2 downregulation would be a potential target of F806 in ESCC cells. BIOLOGICAL SIGNIFICANCE: This study used SILAC-based quantitative proteomics screen to systematically characterize molecular changes induced by a novel macrolide analog F806 in esophageal squamous cell carcinoma (ESCC) cells. Followed by bioinformatic analyses, signal pathway networks generated from the quantified proteins, would facilitate future investigation into the further mechanisms of F806 in ESCC cells. Notably, it provided information that growth factor receptor-bound protein 2 (GRB2) would be a prominent node in the F806-targeted cell proliferation network.

Exploration of potential roles of a new LOXL2 splicing variant using network knowledge in esophageal squamous cell carcinoma.

LOXL2 (lysyl oxidase-like 2), an enzyme that catalyzes oxidative deamination of lysine residue, is upregulated in esophageal squamous cell carcinoma (ESCC). A LOXL2 splice variant LOXL2-e13 and its wild type were overexpressed in ESCC cells followed by microarray analyses. In this study, we explored the potential role and molecular mechanism of LOXL2-e13 based on known protein-protein interactions (PPIs), following microarray analysis of KYSE150 ESCC cells overexpressing a LOXL2 splice variant, denoted by LOXL2-e13, or its wild-type counterpart. The differentially expressed genes (DEGs) of LOXL2-WT and LOXL2-e13 were applied to generate individual PPI subnetworks in which hundreds of DEGs interacted with thousands of other proteins. These two DEG groups were annotated by Functional Annotation Chart analysis in the DAVID bioinformatics database and compared. These results found many specific annotations indicating the potential specific role or mechanism for LOXL2-e13. The DEGs of LOXL2-e13, comparing to its wild type, were prioritized by the Random Walk with Restart algorithm. Several tumor-related genes such as ERO1L, ITGA3, and MAPK8 were found closest to LOXL2-e13. These results provide helpful information for subsequent experimental identification of the specific biological roles and molecular mechanisms of LOXL2-e13. Our study also provides a work flow to identify potential roles of splice variants with large scale data.

Functional analysis of the mRNA profile of neutrophil gelatinase­associated lipocalin overexpression in esophageal squamous cell carcinoma using multiple bioinformatic tools.

Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily; dysregulated expression of NGAL has been observed in several benign and malignant diseases. In the present study, differentially expressed genes, in comparison with those of control cells, in the mRNA expression profile of EC109 esophageal squamous cell carcinoma (ESCC) cells following NGAL overexpression were analyzed by multiple bioinformatic tools for a comprehensive understanding. A total of 29 gene ontology (GO) terms associated with immune function, chromatin structure and gene transcription were identified among the differentially expressed genes (DEGs) in NGAL overexpressing cells. In addition to the detected GO categories, the results from the functional annotation chart revealed that the differentially expressed genes were also associated with 101 functional annotation category terms. A total of 59 subpathways associated locally with the differentially expressed genes were identified by subpathway analysis, a markedly greater total that detected by traditional pathway enrichment analysis only. Promoter analysis indicated that the potential transcription factors Snail, deltaEF1, Mycn, Arnt, MNB1A, PBF, E74A, Ubx, SPI1 and GATA2 were unique to the downregulated DEG promoters, while bZIP910, ZNF42 and SOX9 were unique for the upregulated DEG promoters. In conclusion, the understanding of the role of NGAL overexpression in ESCC has been improved through the present bioinformatic analysis.

Desmocollin­2 affects the adhesive strength and cytoskeletal arrangement in esophageal squamous cell carcinoma cells.

Desmocollin­2 (DSC2), a transmembrane glycoprotein belonging to the desmosomal cadherin family, has been found to be differentially expressed in several types of cancer and to be involved in tumor progression. The tumor metastasis suppressing property of DSC2 in esophageal squamous cell carcinoma (ESCC) has been described, however, its contribution to cell cohesion in ESCC remains to be elucidated. In the present study, using RNA interference (RNAi), the expression of DSC2 was silenced in SHEEC and KYSE510 cells. Hanging drop and fragmentation assays were performed to investigate the role of DSC2 in cell­cell adhesion. Western blot analysis and confocal microscopy were used to analyze the expression and localization of cell adhesion molecules and cytoskeletal arrangement. The results demonstrated that DSC2 knock down by RNAi caused defects in cell­cell adhesion and a concomitant reduction in desmosomal protein expression and adherens junction molecule distribution. A decrease in the expression of DSC2 caused an increase in free γ­catenin levels, thus promoting its recruitment to the adherens junction complex. In addition, the RNAi­mediated inhibition of DSC2 led to keratin intermediate filament retraction and filamentous­actin cytoskeleton rearrangement. Taken together, these data support our previous findings and the proposal that DSC2 may be involved in the regulation of the invasive behavior of cells by a mechanism that controls cell­cell attachment and cytoskeleton rearrangement.

PPI network analysis of mRNA expression profile of ezrin knockdown in esophageal squamous cell carcinoma.

Ezrin, coding protein EZR which cross-links actin filaments, overexpresses and involves invasion, metastasis, and poor prognosis in various cancers including esophageal squamous cell carcinoma (ESCC). In our previous study, Ezrin was knock down and analyzed by mRNA expression profile which has not been fully mined. In this study, we applied protein-protein interactions (PPI) network knowledge and methods to explore our understanding of these differentially expressed genes (DEGs). PPI subnetworks showed that hundreds of DEGs interact with thousands of other proteins. Subcellular localization analyses found that the DEGs and their directly or indirectly interacting proteins distribute in multiple layers, which was applied to analyze the shortest paths between EZR and other DEGs. Gene ontology annotation generated a functional annotation map and found hundreds of significant terms, especially those associated with cytoskeleton organization of Ezrin protein, such as "cytoskeleton organization," "regulation of actin filament-based process," and "regulation of actin cytoskeleton organization." The algorithm of Random Walk with Restart was applied to prioritize the DEGs and identified several cancer related DEGs ranked closest to EZR. These analyses based on PPI network have greatly expanded our comprehension of the mRNA expression profile of Ezrin knockdown for future examination of the roles and mechanisms of Ezrin.

Network based analyses of gene expression profile of LCN2 overexpression in esophageal squamous cell carcinoma.

LCN2 (lipocalin 2) is a member of the lipocalin family of proteins that transport small, hydrophobic ligands. LCN2 is elevated in various cancers including esophageal squamous cell carcinoma (ESCC). In this study, LCN2 was overexpressed in the EC109 ESCC cell line and we applied integrated analyses of the gene expression data to identify protein-protein interactions (PPI) network to enhance our understanding of the role of LCN2 in ESCC. Through further mining of PPI sub-networks, hundreds of differentially expressed genes (DEGs) were identified to interact with thousands of other proteins. Subcellular localization analyses found the DEGs and their directly or indirectly interacting proteins distributed in multiple layers, which was applied to analyze the possible paths between two DEGs. Gene Ontology annotation generated a functional annotation map and found hundreds of significant terms, especially those associated with the known and potential roles of LCN2 protein. The algorithm of Random Walk with Restart was applied to prioritize the DEGs and identified several cancer-related DEGs ranked closest to LCN2 protein. These analyses based on PPI network have greatly expanded our understanding of the mRNA expression profile of LCN2 overexpression for future examination of the roles and mechanisms of LCN2.

Protein-protein interaction network analyses for elucidating the roles of LOXL2-delta72 in esophageal squamous cell carcinoma.

Lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase (LOX) family, is a copper-dependent enzyme that catalyzes oxidative deamination of lysine residues on protein substrates. LOXL2 was found to be overexpressed in esophageal squamous cell carcinoma (ESCC) in our previous research. We later identified a LOXL2 splicing variant LOXL2-delta72 and we overexpressed LOXL2-delta72 and its wild type counterpart in ESCC cells following microarray analyses. First, the differentially expressed genes (DEGs) of LOXL2 and LOXL2-delta72 compared to empty plasmid were applied to generate protein-protein interaction (PPI) sub-networks. Comparison of these two sub-networks showed hundreds of different proteins. To reveal the potential specific roles of LOXL2- delta72 compared to its wild type, the DEGs of LOXL2-delta72 vs LOXL2 were also applied to construct a PPI sub-network which was annotated by Gene Ontology. The functional annotation map indicated the third PPI sub-network involved hundreds of GO terms, such as "cell cycle arrest", "G1/S transition of mitotic cell cycle", "interphase", "cell-matrix adhesion" and "cell-substrate adhesion", as well as significant "immunity" related terms, such as "innate immune response", "regulation of defense response" and "Toll signaling pathway". These results provide important clues for experimental identification of the specific biological roles and molecular mechanisms of LOXL2-delta72. This study also provided a work flow to test the different roles of a splicing variant with high-throughput data.

Dissection of miRNA-miRNA interaction in esophageal squamous cell carcinoma.

The relationships between miRNAs and their regulatory influences in esophageal carcinoma remain largely unknown. Accumulated evidence suggests that delineation of subpathways within an entire pathway can underlie complex diseases. To analyze the regulation of differentially expressed miRNAs in subpathways of esophageal squamous cell carcinoma (ESCC), we constructed bipartite miRNA and subpathway networks to determine miRNA regulatory influences on subpathways. The miRNA-subpathway network indicated that miRNAs regulate numerous subpathways. Two principal biological networks were derived from the miRNA-subpathway network by the hypergeometric test. This miRNA-miRNA network revealed the co-regulation of subpathways between the upregulated and downregulated miRNAs. Subpathway-subpathway networks characterized scale free, small world, and modular architecture. K-clique analysis revealed co-regulation of subpathways between certain downregulated and upregulated miRNAs. When ESCC patients were grouped according to their expression levels of paired upregulation of miR-31 and downregulation of miR-338-3p, survival time analysis revealed a significant difference based on miR-31-miR-338-3p interaction. These findings can facilitate the understanding of the biological meaning of miRNA-miRNA interactions with either the same or opposite expression trend.

Comprehensive bioinformation analysis of the mRNA profile of fascin knockdown in esophageal squamous cell carcinoma.

BACKGROUND: Fascin, an actin-bundling protein forming actin bundles including filopodia and stress fibers, is overexpressed in multiple human epithelial cancers including esophageal squamous cell carcinoma (ESCC). Previously we conducted a microarray experiment to analyze fascin knockdown by RNAi in ESCC. METHOD: In this study, the differentially expressed genes from mRNA expression profilomg of fascin knockdown were analyzed by multiple bioinformatics methods for a comprehensive understanding of the role of fascin. RESULTS: Gene Ontology enrichment found terms associated with cytoskeleton organization, including cell adhesion, actin filament binding and actin cytoskeleton, which might be related to fascin function. Except GO categories, the differentially expressed genes were annotated by 45 functional categories from the Functional Annotation Chart of DAVID. Subpathway analysis showed thirty-nine pathways were disturbed by the differentially expressed genes, providing more detailed information than traditional pathway enrichment analysis. Two subpathways derivated from regulation of the actin cytoskeleton were shown. Promoter analysis results indicated distinguishing sequence patterns and transcription factors in response to the co-expression of downregulated or upregulated differentially expressed genes. MNB1A, c-ETS, GATA2 and Prrx2 potentially regulate the transcription of the downregulated gene set, while Arnt-Ahr, ZNF42, Ubx and TCF11-MafG might co-regulate the upregulated genes. CONCLUSIONS: This multiple bioinformatic analysis helps provide a comprehensive understanding of the roles of fascin after its knockdown in ESCC.

Regulation of neutrophil gelatinase-associated lipocalin expression by C/EBPß in lung carcinoma cells.

Neutrophil gelatinase-associated lipocalin (NGAL), a member of the lipocalin family, has been found to be overexpressed in a variety of tumors, including lung adenocarcinomas. However, the mechanism by which NGAL expression is regulated in lung carcinoma needs further evaluation. In this study, immunohistochemistry was employed to analyze the expression of NGAL in lung carcinoma tissue samples, including lung squamous carcinomas, adenocarcinomas, adenosquamous carcinomas and bronchial alveolar cell carcinomas. The results showed that NGAL was expressed in 82.61% (19/23) of the samples. RT-PCR and immunofluorescent staining showed that NGAL was localized to the cytoplasm in lung carcinoma cell lines. To explore the transcriptional regulation mechanism of NGAL basal expression in lung carcinoma, a 1515-bp fragment (-1431 to +84) of the NGAL promoter region was cloned and a series of deletion and mutation constructs were generated. These constructs were analyzed using the luciferase reporter assay. The results indicated that the cis-acting elements important for the basal activity of NGAL transcription were likely located between -152 and -141. Further analysis using site-directed mutagenesis and the luciferase reporter assay suggested that the C/EBP binding sites were responsible for the activity of the NGAL promoter. Finally, the binding ability and specificity of the transcription factors were determined by electrophoretic mobility-shift assay (EMSA). The results showed that C/EBPß was able to bind to the -152 and -141 segments. Taken together, these findings suggest that NGAL is expressed in lung carcinomas and that NGAL expression is mediated by the binding of C/EBPß to the -152 and -141 segment of the NGAL promoter.

Expression of NGAL and NGALR in human embryonic, fetal and normal adult tissues.

This study investigated the distribution of neutrophil gelatinase-associated lipocalin (NGAL) and neutrophil gelatinase-associated lipocalin receptor (NGALR) in human embryonic, fetal and normal adult tissues. Tissue microarray technology was used to perform immunohistochemical examination on human embryos, fetuses at 4-22 weeks of gestation and adult tissue specimens. Results demonstrated that during the development of the nervous system, NGALR was prevalent in the neural tube and cerebrum, and NGAL was only detected in the stellate cells of the cerebrum and the Purkinje cells of the cerebellum. NGAL and NGALR were expressed in the lung alveolar epithelium and in the gastrointestinal tract in embryos, but were almost undetectable in later developmental stages. In the embryonic adrenal glands, the two proteins demonstrated moderate positivity in the cortex and the medulla. In adults, NGAL was particularly present in the cells of the inner and outer layers of the cortex and was absent in the medulla, while NGALR exhibited strong positivity in the cortex and the medulla. Evident expression of NGAL and NGALR was observed throughout development in the neutrophil-rich sites, the renal tubule epithelium and certain gland epithelia of the gastrointestinal tract, but was undetectable in the heart, liver and thyroid gland. Taken together, these results demonstrated that the expression of NGAL and NGALR was time-specific and highly tissue­specific. Correlations between their expression in embryogenesis and carcinogenesis should be examined.

Determinants of career aspirations of medical students in southern China.

BACKGROUND: With recent changes in both the Chinese medical system and compensation of medical doctors, the career aspirations of Chinese medical students have become more diverse. Shantou University Medical College has conducted evaluations and instituted programs to enhance student preparedness to enter a variety of medical careers. METHODS: A survey was conducted with 85 students to evaluate medical career aspirations and their association with family background, personal skills, English language proficiency, and interest in biomedical research, which were considered as possible factors affecting their career interest. RESULTS: Chinese students aspire to traditional as well as nontraditional medical careers. A significant minority of students are now interested in nontraditional careers such as medical teaching or research. However, poor proficiency in the English language and lack of computer skills may limit their academic and career opportunities. CONCLUSION: Career aspirations have changed among medical undergraduates. Although many wish to pursue a traditional clinical doctor career, many are interested in research and teaching careers. Factors such as family background, personal characteristics, school mentoring, and extracurricular support may play a role.