Age- and sex-associated differences in the glycopatterns of human salivary glycoproteins and their roles against influenza A virus.

Recent studies have elucidated that expression of certain glycoproteins in human saliva is increased or decreased according to age; meanwhile, human saliva may inhibit viral infection and prevent viral transmission. However, little is known about the age- and sex-associated differences in the glycopatterns of human salivary glycoproteins and their significant roles against influenza A virus (IVA). Here, we investigate the glycopatterns of human salivary glycoproteins with 180 healthy saliva samples divided into six age/sex groups using lectin microarrays and fabricate saliva microarrays to validate the terminal carbohydrate moieties of glycoproteins in individual saliva samples. Furthermore, we assess the inhibiting and neutralizing activity of saliva against two strains of influenza A (H9N2) virus. We find that seven lectins (e.g., MAL-II and SNA) show significant age differences in both females and males, and seven lectins (e.g., WFA and STL) show significant sex differences in children, adults and elderly people. Interestingly, we observe that elderly individuals have strongest resistance to IVA partly by presenting more terminal α2-3/6-linked sialic acid residues in their saliva, which bind with the influenza viral hemagglutinations. We conclude that age- and sex-associated differences in the glycopatterns of human salivary glycoproteins may provide pivotal information to help understand some age related diseases and physiological phenomena.

Analysis of glycan-related genes expression and glycan profiles in mice with liver fibrosis.

Protein glycosylation plays an important role in the pathogenesis and progression of various liver diseases. However, little is known about the precise alterations in protein glycosylation or the potential correlation between glycan-related genes expression and glycan profiles in liver fibrosis. The aim of the study was to investigate potential associations between glycan-related genes expression and glycan profiles to evaluate liver fibrosis in a mouse model. Analyses of glycan-related genes expression and glycan profiles were performed using oligonucleotide microarrays and lectin microarrays, respectively. Real-time PCR and Western blot were used to confirm any altered glycan-related genes expression levels and protein levels. Moreover, altered glycan patterns on the surface of hepatocytes were verified by lectin histochemistry. These results revealed that the mRNA levels of 10 glycan-related genes were significantly altered in fibrotic liver. Furthermore, we observed an increase in multivalent sialic acid, poly-LacNAc, sialyl-T-antigen, Fucoseα-1,3/6GlcNAc, and GalNAcα1-3Gal in fibrotic liver specimens, whereas GlcNAc oligomers was decreased in fibrotic liver. Our findings indicated that the synthetic pathway of "Tn antigen → T antigen (core-1) → sialyl-T antigen" was activated for O-glycan during the process of liver fibrosis.

Detection of hepatitis B virus A1762T/G1764A mutant by amplification refractory mutation system.

OBJECTIVE: To study the role of hepatitis B virus with A1762T/G1764A double mutation in liver cirrhosis and hepatocellular carcinoma, and create a sensitive, fast, accurate assay for detection of A1762T/G1764A double mutation. METHODS: We developed an accurate and fast real-time amplification refractory mutation system to detect A1762T/G1764A double mutation. Cloned hepatitis B virus genome was used as a control. Assay sensitivity was determined by serial dilution and mixed template experiments. Specificity was determined by cross experiments with wild and mutant hepatitis B virus. Fifty clinical samples were tested by the real-time amplification refractory mutation system and the results were compared with sequencing. RESULTS: The real-time amplification refractory mutation system had a sensitivity of 100 copies of virus with these mutations, and 0.1% weak population virus with double mutation could be found in mixtures. A total of 50 randomly collected clinical samples were detected by real-time amplification refractory mutation system, and the results were consistent with those by DNA sequencing. Hepatitis B virus genotype C was more prevalent in 39 of 50 samples than genotype B (11 samples), and about 75% of genotype C carried a double mutation compared to 45% of genotype B. However, the percentage of A1762T/G1764A double mutation in hepatitis B e antigen-negative (58.3%) samples was almost the same as in hepatitis B e antigen-positive (61%) samples. CONCLUSION: The real-time amplification refractory mutation system is sensitive and specific for detection of hepatitis B virus double mutation.

Glycoproteomic analysis of tissues from patients with colon cancer using lectin microarrays and nanoLC-MS/MS.

The current study evaluated the glycoproteomic profile of tissues from colon cancer patients. The lectin microarray was first performed to compare the glycoprotein profiles between colon cancer and matched normal tissues. Level of N-acetylglucosamine (GlcNAc) that Solanum tuberosum lectin (STL) bound was found to be elevated in colon cancer, which was verified through lectin histochemistry. The subsequent glycoproteomic analysis based on STL enrichment of glycoproteins followed by label-free quantitative nano liquid chromatography-mass spectrometry/mass spectrometry (nanoLC-MS/MS) analysis identified 72 proteins in high confidence. Among these proteins, 17 were exclusively detected in cancer tissues, and 14 were significantly upregulated in tumor tissues. Annexin A1 and HSP90ß were chosen for further investigation by immunoprecipitation coupled with lectin blots, western blots and tissue microarrays. Both Annexin A1 and HSP90ß were GlcNAcylated, and their protein expressions were elevated in colon cancer, compared to normal tissues. Moreover, specific changes of GlcNAc abundances in Annexin A1 and HSP90ß suggested that tumor-specific glycan patterns could serve as candidate biomarkers of colon cancer for distinguishing cancer patients from healthy individuals.

Alteration of protein glycosylation in human hepatic stellate cells activated with transforming growth factor-ß1.

Although aberrant glycosylation of human glycoproteins is related to liver fibrosis that results from chronic damage to the liver in conjunction with the activation of hepatic stellate cells (HSCs), little is known about the precision alteration of protein glycosylation referred to the activation of HSCs by transforming growth factor-ß1 (TGF-ß1). The human HSCs, LX-2 were activated by TGF-ß1. The lectin microarrays were used to probe the alteration of protein glycosylation in the activated HSCs compared with the quiescent HSCs. Lectin histochemistry was used to further validate the lectin binding profiles and assess the distribution of glycosidic residues in cells. As a result, 14 lectins (e. g. AAL, PHA-E, ECA and ConA) showed increased signal while 7 lectins (e. g. UEA-I and GNA) showed decreased signal in the activated LX-2 compared with the quiescent LX-2. Meanwhile, AAL, PHA-E and ECA staining showed moderate binding to the cytoplasma membrane in the quiescent LX-2, and the binding intensified in the same regions of the activated LX-2. In conclusion, the precision alteration of protein glycosylation related to the activation of the HSCs may provide useful information to find new molecular mechanism of HSC activation and antifibrotic therapeutic strategies.

Detection of hepatitis B virus A1762T/G1764A mutant by amplification refractory mutation system

OBJECTIVE: To study the role of hepatitis B virus with A1762T/G1764A double mutation in liver cirrhosis and hepatocellular carcinoma, and create a sensitive, fast, accurate assay for detection of A1762T/G1764A double mutation. METHODS: We developed an accurate and fast real-time amplification refractory mutation system to detect A1762T/G1764A double mutation. Cloned hepatitis B virus genome was used as a control. Assay sensitivity was determined by serial dilution and mixed template experiments. Specificity was determined by cross experiments with wild and mutant hepatitis B virus. Fifty clinical samples were tested by the real-time amplification refractory mutation system and the results were compared with sequencing. RESULTS: The real-time amplification refractory mutation system had a sensitivity of 100 copies of virus with these mutations, and 0.1% weak population virus with double mutation could be found in mixtures. A total of 50 randomly collected clinical samples were detected by real-time amplification refractory mutation system, and the results were consistent with those by DNA sequencing. Hepatitis B virus genotype C was more prevalent in 39 of 50 samples than genotype B (11 samples), and about 75% of genotype C carried a double mutation compared to 45% of genotype B. However, the percentage of A1762T/G1764A double mutation in hepatitis B e antigen-negative (58.3%) samples was almost the same as in hepatitis B e antigen-positive (61%) samples. CONCLUSION: The real-time amplification refractory mutation system is sensitive and specific for detection of hepatitis B virus double mutation. .